cancer cell research studies

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• Published: 10 September 2021

Cell cycle control in cancer

• Helen K. Matthews 1 , 2 ,
• Cosetta Bertoli   ORCID: orcid.org/0000-0001-5684-4630 1 &
• Robertus A. M. de Bruin   ORCID: orcid.org/0000-0001-9957-1409 1 , 3  

Nature Reviews Molecular Cell Biology volume  23 ,  pages 74–88 ( 2022 ) Cite this article

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• Cell division
• Checkpoints
• DNA damage checkpoints
• Tumour-suppressor proteins

Cancer is a group of diseases in which cells divide continuously and excessively. Cell division is tightly regulated by multiple evolutionarily conserved cell cycle control mechanisms, to ensure the production of two genetically identical cells. Cell cycle checkpoints operate as DNA surveillance mechanisms that prevent the accumulation and propagation of genetic errors during cell division. Checkpoints can delay cell cycle progression or, in response to irreparable DNA damage, induce cell cycle exit or cell death. Cancer-associated mutations that perturb cell cycle control allow continuous cell division chiefly by compromising the ability of cells to exit the cell cycle. Continuous rounds of division, however, create increased reliance on other cell cycle control mechanisms to prevent catastrophic levels of damage and maintain cell viability. New detailed insights into cell cycle control mechanisms and their role in cancer reveal how these dependencies can be best exploited in cancer treatment.

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Acknowledgements

R.A.M.d.B. and C.B. are supported by core funding from the MRC–UCL University Unit (reference MC_EX_G0800785) and R.A.M.d.B.’s Cancer Research UK Programme Foundation Award. H.K.M received funding from a CRUK–EPSRC Multidisciplinary Project Award (C1529/A23335). The authors thank J. Pines and J. Downs for helpful discussions and A. Barr and the peer reviewers for critical reading of the manuscript. They apologize to colleagues whose work could be cited only indirectly.

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(CDKs). CDKs depend on cyclins for their kinase activity. The levels of cyclins increase during the cell cycle and cyclin–CDK complex activity drives cell cycle progression by phosphorylating protein targets.

A reversible cellular state, outside G1 phase, from which cells can re-enter the cell cycle.

(APC/C). Ubiquitin ligase complex activity that is restricted to mitosis and G1 phase and is required to initiate exit from mitosis and indirectly for DNA replication.

Removal of nucleotides by exonucleases involved in repairing DNA, exposing tracks of single-stranded DNA at sites of double-strand break repair. It is required for homologous recombination.

DNA double-strand break repair mechanism based on the juxtaposition of two pieces of DNA.

Mechanism of DNA double-strand break repair requiring the presence of duplicated chromatids, occurring only in S and G2 phases. It requires the resection of DNA ends at the break site.

A non-reversible cellular state, outside G1 phase, from which cells cannot re-enter the cell cycle.

Stochastic acquisition of genetic change over many cell divisions that can result in mutations and chromosomal rearrangements and aneuploidy.

(CIN). Type of genome instability involving structural and/or numerical chromosome aberrations.

Abnormal number of chromosomes in a cell.

Compounds that selectively kill cells that are in a state of senescence.

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Matthews, H.K., Bertoli, C. & de Bruin, R.A.M. Cell cycle control in cancer. Nat Rev Mol Cell Biol 23 , 74–88 (2022). https://doi.org/10.1038/s41580-021-00404-3

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• Published: 14 January 2024

Scaffold-based 3D cell culture models in cancer research

• Waad H. Abuwatfa 1 , 2 ,
• William G. Pitt 3 &
• Ghaleb A. Husseini   ORCID: orcid.org/0000-0002-7244-3105 1 , 2  

Journal of Biomedical Science volume  31 , Article number:  7 ( 2024 ) Cite this article

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Three-dimensional (3D) cell cultures have emerged as valuable tools in cancer research, offering significant advantages over traditional two-dimensional (2D) cell culture systems. In 3D cell cultures, cancer cells are grown in an environment that more closely mimics the 3D architecture and complexity of in vivo tumors. This approach has revolutionized cancer research by providing a more accurate representation of the tumor microenvironment (TME) and enabling the study of tumor behavior and response to therapies in a more physiologically relevant context. One of the key benefits of 3D cell culture in cancer research is the ability to recapitulate the complex interactions between cancer cells and their surrounding stroma. Tumors consist not only of cancer cells but also various other cell types, including stromal cells, immune cells, and blood vessels. These models bridge traditional 2D cell cultures and animal models, offering a cost-effective, scalable, and ethical alternative for preclinical research. As the field advances, 3D cell cultures are poised to play a pivotal role in understanding cancer biology and accelerating the development of effective anticancer therapies. This review article highlights the key advantages of 3D cell cultures, progress in the most common scaffold-based culturing techniques, pertinent literature on their applications in cancer research, and the ongoing challenges.

Graphical Abstract

Introduction

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells in the body. It is one of the leading causes of death worldwide, with millions of new cases and deaths each year [ 1 ]. Despite significant advances in cancer research and treatment over the years, the disease remains a major public health challenge and a substantial burden on patients, families, and society. Cancer research is crucial to develop new and effective treatments, improve patient outcomes, and find cures for this disease. As understanding of cancer biology and genetics continues to evolve, so do the approaches used to diagnose, treat, and prevent the disease. However, there is still much to learn about the complex mechanisms underlying cancer development and progression and the unique challenges posed by different types of cancers [ 2 ]. In addition, there is a need to develop more personalized and targeted therapies that can improve patient outcomes and minimize side effects. As such, cancer research must continue to innovate and advance to keep pace with the evolving understanding of the disease. This includes exploring new treatment modalities, developing more sophisticated diagnostic tools, and understanding the genetic and molecular mechanisms involved in its development and progression [ 3 ].

Two-dimensional (2D) cell culture is a commonly used technique to grow and maintain cells in the laboratory. Cancer research extensively uses it to study cells under controlled conditions, where they are grown on a flat surface supplied with a nutrient-rich liquid medium that provides the necessary nutrients for cell growth and survival. The growth medium used in cell culture varies depending on the type of cancer being studied and the desired goals of the study. One of the most critical aspects of cell culture for cancer research is maintaining cell viability and function, as cancer cells are highly susceptible to environmental changes [ 4 ]. Another challenge facing cell culture for cancer research is the ability to accurately model the complexity of human tumors. These are typically highly heterogeneous, comprising different cell types, including cancer, stromal, and immune cells. Understandably, 2D cell culture does not accurately mimic tumors’ three-dimensional (3D) environment [ 5 ]. The architecture and organization of cells in a 3D environment differ from those in a 2D environment, which can affect cell behavior and drug response. Recreating this complexity in a laboratory setting is difficult, as it requires the development of culture conditions that promote the growth and interaction of multiple cell types in a multifaceted environment [ 6 ]. Therefore, 3D cell culture models were developed as they offer sophisticated platforms that mirror the structural and functional complexities of in vivo tissues, providing valuable insights for cancer research and drug development. This review article highlights the key advantages of 3D cell cultures, the most common scaffold-based 3D culturing techniques, pertinent literature about applications in cancer research, and the challenges associated with these culturing techniques. Due to the topic’s vastness, this paper focuses on examining scaffold-based models of 3D cell cultures.

Physiological relevance of 3D cell cultures to the ECM

Tumors are complex structures composed of cancer cells, non-cancerous cells (i.e., immune cells, fibroblasts, endothelial cells, etc.), and various extracellular matrix (ECM) components. The ECM plays a crucial role and contributes to the hallmarks of cancer in tumor progression, metastasis, and response to therapy [ 7 , 8 ]. The ECM can (1) secrete growth factors and cytokines that promote cell proliferation and survival [ 9 ], (2) modulate the expression of genes involved in cell cycle regulation and apoptosis [ 10 ], (3) control the expression of telomerase, an enzyme that extends the telomeres of chromosomes, (4) secrete angiogenic factors that promote the formation of new blood vessels, thereby providing the tumor with the nutrients and oxygen it needs to grow [ 11 ], (5) promote the epithelial-to-mesenchymal transition (EMT), a process by which epithelial cells acquire the ability to migrate and invade other tissues [ 12 ], and (6) temper the immune response by influencing the recruitment and function of immune cells in the TME [ 13 ]. Romero-López and colleagues [ 14 ] tested how the ECM derived from normal and tumor tissues impacted blood vessels and tumor growth using reconstituted ECM. Tumor tissue obtained from liver metastases of colon tumors was subjected to hematoxylin and eosin (H&E) staining to confirm the successful decellularization of both colon and tumor tissues. Subsequently, significantly distinct protein composition and stiffness were observed among the reconstituted matrices, leading to notable variations in vascular network formation and tumor growth in both in vitro and in vivo. Fluorescence Lifetime Imaging Microscopy was employed to evaluate the free/bound ratios of the nicotinamide adenine dinucleotide (NADH) cofactor in tumor and endothelial cells to indicate cellular metabolic state. Notably, cells seeded in tumor ECM exhibited elevated levels of free NADH, indicating an increased glycolytic rate compared to those seeded in normal ECM. These findings underscore the substantial influence of ECM on cancer cell growth and the accompanying vasculature (e.g., increased vessel length, increased vascular heterogeneity). Alterations in the composition of tumor ECM, such as augmented deposition and crosslinking of collagen fibers, can be attributed to communication between tumor cells and tumor-associated stromal cells.

Every tissue type has a distinct ECM composition, topology, and organization [ 15 ]. These factors play a significant role in controlling cell function, behavior, and interactions with the microenvironment, as they generate spatial gradients of biochemicals and metabolites that, in turn, may elicit distinctive cell-mediated responses (e.g., differentiation, migration) [ 16 ]. Langhans [ 17 ] analyzed the chemical components of ECM and reported that it contains water, carbohydrates, and proteins, such as fibrous matrix proteins, glycoproteins, proteoglycans, glycosaminoglycans, growth factors, protease inhibitors, and proteolytic enzymes. Thus, ECM organization can influence cell genotypes and phenotypes, where such effects can be explored through 3D cell cultures [ 16 , 18 ]. For example, variations in the gene and protein expression and activity of the epidermal growth factor receptors (EFGR), phosphorylated protein kinase B (phospho-AKT), and p42/44 mitogen-activated protein kinases (phospho-MAPK) in colorectal cancer cell lines (e.g., HT-29, CACO-2, DLD-1) affected the genotype and phenotype of cells in 3D cultures, as compared to 2D monolayers [ 19 , 20 ]. Moreover, the ECM can influence cell morphology and expression of chemokine receptors. Kiss et al. [ 21 ] showed that 3D cultured prostate cancer cells (e.g., LNCaP, PC3) exhibited a high level of interaction between the cells and ECM, which resulted in the upregulation and overexpression of the CXCR7 and CXCR4 chemokine receptors. While 2D cell culture has been the mainstay of laboratory cancer research, it has become increasingly clear that this approach is inadequate in replicating the in vivo conditions that cells experience in the human body. As a result, researchers have been turning to 3D cell culture as a more physiologically relevant model for studying cellular processes and disease. A key advantage of 3D models for cancer research is that they can better mimic the complex microenvironment of tumors, including tumor morphology and topography, upregulation of pro-angiogenic proteins, dispersion of biological and chemical factors, cell–cell and cell–matrix interactions, gradients of oxygen and nutrients, and a more realistic ECM composition [ 6 , 22 , 23 ]. Necrotic, hypoxic, quiescent, apoptotic, and proliferative cells are often found in spheroid cell clusters at different phases of development [ 24 ]. Since the outer layer of the spheroid has greater exposure to the nutrient-supported medium, it contains a higher number of proliferating cells. Cells in the spheroid core are hypoxic and often quiescent as they receive less oxygen, growth agents, and nutrients from the media. This results in more physiologically relevant gradients in tissue composition that can better inform drug discovery and development [ 24 ]. Furthermore, 3D cell culture accurately depicts the cellular response to drugs and other therapeutic agents. Such a model’s spatial and physical characteristics influence the transmission of signals between cells, which alters gene expression and cell behavior [ 25 ]. Loessner et al. [ 26 ] demonstrated a flexible 3D culture method where a synthetic hydrogel matrix with crucial biomimetic properties provided a system for studying cell–matrix dynamics related to tumorigenesis. The 3D cultured cells overexpressed mRNA for receptors on their surface (e.g., protease, α3, α5, β1 integrins) compared to 2D cultured cells. Moreover, spheroid progression and proliferation depended on the cells’ ability to proteolytically transform their ECM and cell-integrin interactions. Consequently, the 3D spheroids showed higher survival rates in contrast to 2D monolayers after exposure to the chemotherapeutic agent paclitaxel, which indicates that it better stimulates in vivo chemosensitivity and pathophysiological events. Table 1 below summarizes studies using different 3D models to investigate different types of cancers.

Figure  1 summarizes the main characteristics of 2D and 3D cell cultures. The shift to 3D cell culture is a significant advancement in laboratory research, as it provides a more physiologically relevant model for studying cellular processes and disease. While some challenges remain to be addressed, the advantages of 3D culture outweigh the limitations of 2D culture. As technology continues to evolve, 3D culture is likely to become an increasingly crucial tool in cancer research and other fields of biomedical science.

Characteristics of 2D and 3D cell cultures

Table 2 below provides a comprehensive overview of 2D, 3D, and other model systems employed in cancer research. Besides 2D and 3D cell cultures, tissues and organs present structural and functional intricacies, capturing organ-specific responses but posing challenges in maintenance and accessibility. Furthermore, model animals mimic in vivo systemic responses, yet ethical concerns, high costs, and species differences limit their utility. While clinically relevant, patient-derived samples present challenges in experimental control and sample heterogeneity [ 43 ]. It is noteworthy to highlight the difference between spheroids and organoids as both are commonly used terms within the scope of 3D cell cultures [ 44 ]. Organoids and spheroids are different 3D cell culture models that can be cultured with different techniques. Organoids, characterized by intricate structures replicating real organs or tissues, are composed of multiple cell types that self-organize to mirror tissue-like architecture, deriving from stem cells or tissue-specific progenitors. Due to their high biological relevance, they find applications in disease modeling, drug testing, and understanding organ development. Beyond organoids, tumoroids (i.e., tumor-like organoids), derived from patient cancer tissues containing tumor and stroma cells of the TME, are becoming advanced 3D culture platforms for personalized drug evaluation and development. In contrast, spheroids are simpler spherical cellular aggregates lacking the distinct organ-like structures of organoids. Comprising one or multiple cell types, spheroids are used to study fundamental cellular behaviors and drug responses in a 3D environment. While both contribute to 3D cell culture studies, organoids closely resemble real organs compared to the simpler cellular aggregates represented by spheroids [ 44 ]. Patient models are valuable tools that aim to replicate the complexities of human tumors, providing insights into disease mechanisms, therapeutic responses, and personalized treatment strategies. They can be utilized in Patient-Derived Xenografts (PDX), organoids and 3D cultures, patient-derived cell lines, liquid biopsies, and clinical trials [ 45 ].

Cell sources and 3D culture heterogeneity

In 3D cell culture, achieving an optimal balance between homogeneity and heterogeneity is intricately linked to the cellular source, considering stem cells, induced Pluripotent Stem Cells (iPSCs), or mixed primary cells derived from tissues [ 46 ]. Stem cells in in vitro cell culture encompass embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult or somatic stem cells. Embryonic stem cells exhibit high pluripotency, capable of differentiating into any cell type, but their use raises ethical concerns due to their origin from embryos. iPSCs are generated from somatic cells (e.g., skin or blood cells) through reprogramming, reverting them to an embryonic-like pluripotent state, but face reprogramming efficiency and potential tumorigenicity challenges. This transformation creates an extensive and diverse reservoir of human cells, capable of developing into any cell type required for therapeutic applications. Human-induced pluripotent Stem Cells (HiPSCs) are particularly relevant in cancer research (Table  3 ) [ 47 ]. Thus, the reprogramming process pioneered by Shinya Yamanaka has opened new avenues for advancing cancer biology, drug discovery, and regenerative medicine in cancer treatment. Lastly, adult or somatic stem cells are tissue-specific, mirroring the characteristics of their origin, and present fewer ethical concerns as they are derived from adult tissues. However, they have limited differentiation potential and a finite lifespan in culture. The selection of the cell source significantly influences the composition and behavior of the 3D culture. Stem cells and iPSCs, known for their pluripotency, introduce an inherent heterogeneity due to their ability to differentiate into various cell types [ 45 , 46 ].

Furthermore, primary cells, derived directly from living organisms, possess unique characteristics that make them invaluable for in vitro studies. Maintaining biological relevance, these cells closely mimic the tissue or organ from which they are isolated, reflecting the intricacies of in vivo conditions. With donor-specific variability, primary cells allow researchers to explore genetic diversity’s impact on cell behavior, disease susceptibility, and drug responses. Retaining tissue-specific functions, differentiated primary cells are crucial for studying specific physiological processes and diseases associated with particular tissues [ 46 ]. However, these cells have challenges, including a limited lifespan and sensitivity to culture conditions. The finite replicative capacity and sensitivity contribute to the heterogeneity observed in 3D cell cultures, emphasizing the importance of carefully considering culture conditions and donor-specific variations to accurately represent in vivo scenarios. Despite these challenges, primary cells are vital in advancing our understanding of cell biology, disease mechanisms, and therapeutic development. Similarly, using mixed primary cells derived from tissues can contribute to a more heterogeneous cellular composition, resembling the complexity found in native tissues. Striking the right balance is crucial, as an excessive degree of heterogeneity may obscure specific responses, while too much homogeneity might oversimplify the representation of the tissue microenvironment. Therefore, a nuanced understanding of the cellular source is essential for tailoring 3D cell culture models to accurately reflect the intricacies of actual tissues and organs.

Scaffold-based techniques for 3D cell culture

As explained above, developing 3D cell culture techniques that more accurately model the TME is a major area of focus in cancer research [ 6 , 59 ]. Different approaches for 3D cell cultures exist and can be generally divided into scaffold-based and scaffold-free methods. Scaffold-free 3D cell culture refers to a cell culture technique in which cells are cultured and assembled into 3D structures without external scaffold material. Instead of being embedded within a supportive matrix, the cells self-assemble and interact with neighboring cells to form 3D tissue-like structures. Such cultures allow for more accurate cell–cell interactions, spatial organization, and physiological responses, making them valuable tools for various applications, including drug testing. They also usually have higher cell densities than scaffold-based models, which can influence cellular behavior, gene expression, and cellular functions. Lastly, non-scaffold models offer versatility and customizability in terms of cell types, culture conditions, and experimental designs. However, it is essential to consider that scaffold-free approaches might have limitations in providing mechanical support, shape control, and reproducibility compared to scaffold-based 3D cell culture methods [ 60 ]. As such, researchers often select the appropriate 3D cell culture method based on their specific research goals and the tissue or organ system they aim to model or engineer. Due to the topic’s vastness, the paper’s purviews' are limited to the examination of scaffold-based models of 3D cell cultures. Scaffolds are essential components in 3D cell culture systems, as they provide a 3D environment for cells to grow and interact with each other and their surroundings [ 61 , 62 ]. Biomaterials employed in such models can be categorized into the following primary groups: polymer scaffolds, hydrogels, decellularized tissue scaffolds, and hybrid scaffolds (e.g., incorporating microfluidic devices). Tables 4 and 5 summarize the advantages and limitations of commonly used scaffold-free and scaffold-based 3D cell culture techniques, respectively.

Polymer-based scaffolds

Polymer scaffolds revolutionize 3D cell culture by providing a biomimetic environment imitating the natural ECM, fostering cell proliferation and differentiation, often with remarkable efficiency and precision. These scaffolds offer a versatile platform for studying complex cell behaviors and hold immense promise in cancer research applications. They can be generally classified as natural or synthetic-derived (see Fig.  2 ). Natural polymer scaffolds are made from naturally occurring polymers. They can be processed into various forms, including fibers, films, or porous structures. They can be further classified into two main categories: protein-based and polysaccharide-based scaffolds. Protein-based scaffolds are derived from large molecules composed of amino acids (e.g., collagen, silk, gelatin, fibronectin [ 93 , 94 ]). Due to their bioactive properties, these scaffolds provide cell adhesion sites and can regulate cell behavior and tissue development. A 3D cell culture platform using collagen scaffolds was developed to investigate the tumorigenicity of cancer stem cells (CSCs) in breast cancer [ 95 ]. The study revealed that the 3D cell culture system demonstrated increased expression of pro-angiogenic growth factors, indicating a potential role in promoting blood vessel formation. Moreover, the overexpression of CSC markers such as OCT4A and SOX2, as well as breast cancer stem cell markers including SOX4 and JAG1, was observed in the 3D scaffolds, suggesting that the 3D model successfully replicated the molecular characteristics associated with CSCs. In terms of behavior, the 3D model more closely mimicked the characteristics of CSCs compared to an in vivo model, indicating its effectiveness in capturing the tumorigenic properties of CSCs. Therefore, the collagen scaffold-based 3D cell culture platform provided a valuable tool for studying CSC tumorigenicity in breast cancer, demonstrating the upregulation of pro-angiogenic growth factors, the overexpression of CSC and breast cancer stem cell markers, and a close resemblance to CSC behavior when compared to an in vivo model. Another study by McGrath et al. [ 96 ] used a 3D collagen matrix (GELFOAM™) to create an endosteal bone niche (EN) model, referred to as 3D-EN, for studying breast cancer cells’ quiescence and dormancy behaviors. The 3D-EN model effectively facilitated the identification of several genes associated with dormancy-reactivation processes, where among the tested cell lines, only MDA-MB-231 cells exhibited dormancy behavior, suggesting that they have a propensity for entering a dormant state in the simulated physiological conditions.

Classification of polymers used for fabricating polymer-based 3D cell culture scaffolds

On the other hand, polysaccharide-based scaffolds are composed of long chains of sugar molecules (e.g., chitosan and hyaluronic acid). They are biocompatible, biodegradable, and can often be modified to adjust their physical and biological properties. Arya et al. [ 97 ] developed a 3D cell culture model using a chitosan scaffold, a natural polymer derived from chitin, to study breast cancer behavior. The scaffold was cross-linked with genipin, a natural cross-linker, to enhance its stability. The study found that the chitosan–gelatin (GC) scaffold provided a suitable environment for the growth of MCF-7 breast cancer cells, with the cells showing good adhesion and proliferation. The scaffold also supported the formation of cell clusters, which are more representative of in vivo tumor conditions compared to 2D cultures. The study concluded that the chitosan/gelatin scaffold could be useful for studying breast cancer in vitro, providing a more physiologically relevant model than traditional 2D cultures. GC scaffolds have been shown to support the formation of tumoroids that mimic tumors grown in vivo , making them an improved in vitro tumor model. These scaffolds have been successfully used to study lung cancer, as well as other types of cancer, such as breast, cervix, and bone [ 98 ]. These scaffolds have demonstrated gene-expression profiles similar to tumors grown in vivo, indicating their potential for studying cancer progression and drug screening for solid tumors [ 99 ]. The GC scaffolds have also been shown to improve the predictivity of preclinical studies and enhance the clinical translation of therapies [ 100 ]. Overall, the GC scaffolds provide a valuable tool for studying tumor development and evaluating the efficacy of anti-cancer drugs in an in vitro setting.

Synthetic polymer scaffolds (e.g., polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL) can be tailored to have specific mechanical and biochemical properties. However, they can be less biocompatible than natural polymers and may require surface modifications to promote cell attachment and growth [ 60 ]. Palomeras et al. [ 101 ] tested the efficiency of 3D-printed PCL scaffolds for the culture of MCF7 breast cancer cells. The researchers found that the scaffold’s design, specifically the deposition angle, significantly influenced cell attachment and growth. Scaffolds with a deposition angle of 60° showed the highest cell counting after treatment with trypsin. Furthermore, the study found that the 3D culture in PCL scaffolds enriched the cancer stem cell (CSC) population compared to 2D culture control, increasing their Mammosphere Forming Index (MFI). The study concluded that 3D PCL scaffold culture could spur MCF7 cells to generate a cell population with CSC properties. This suggests its potential for studying CSC properties and screening new therapeutic agents targeting CSC populations. These efforts highlight the potential of natural polymer scaffolds in creating more physiologically relevant 3D cell culture models for cancer research. Using these scaffolds can enhance the understanding of cancer cell behavior and potentially lead to the discovery of more effective therapeutic strategies. Similarly, Rijal et al. [ 88 ] utilized modified gas foaming-based synthetic polymer scaffolds from poly(lactic-co-glycolic) acid (PLGA) and PCL for conducting 3D tissue cultures and animal models in breast cancer research. The research group investigated the response of MDA-MB-231 cells to anticancer drugs, their viability, morphology, proliferation, receptor expression, and ability to develop in vivo tumors using the 3D scaffolds. MDA-MB-231 cells were cultured on PLGA-coated 2D microscopic glass slides and in 3D-porous PLGA scaffolds to examine cancer cells’ survival on the polymeric substrata. The number of dead cells detected on the PLGA-coated glass slides and PLGA 3D scaffolds was negligible on Day 1. However, a significant increase in the number of dead cells was observed on the PLGA-coated glass slides compared to the 3D scaffolds on day 14. Additionally, the expression of ECM proteins and cell surface receptors on the synthetic polymers was investigated, where strong staining signals of type I collagen and integrin α2 were detected in both cell types using immunofluorescence (IF) microscopy. It is worth noting that integrin α2β1, which acts as a primary receptor for type I collagen, displayed a basal expression level in the 3D model. This expression pattern may promote breast cancer cell migration and tumor growth, as high levels of the integrin receptor tend to inhibit cancer cell migration. Notably, integrin α2 receptors showed a prominent colocalization with type I collagen, particularly around the cell edges, suggesting local deposition of type I collagen and subsequent binding of integrin α2 receptors, facilitating cell attachment and migration. Lastly, to evaluate the tumor formation capabilities of the polymeric porous scaffolds in mice, MDA-MB-231 cells were coated onto porous PLGA scaffolds and implanted into the mammary fat pads of NOD/SCID mice. Blank scaffolds without cells served as the negative control. As anticipated, the proliferating cell nuclear antigen biomarker Ki-67 was not detected in the blank scaffold implants. At the same time, its expression was significantly high within the tumors derived from the MDA-MB-231 cell-laden PLGA scaffolds. This finding suggested that the cancer cell population within the scaffolds exhibited rapid proliferation when embedded in the native breast tissues.

Hydrogel scaffolds

Hydrogels are 3D networks of hydrophilic polymers (can be natural, synthetic, or hybrid), that can absorb large amounts of water or biological fluids while maintaining their structural integrity [ 102 ]. Figure  3 shows common techniques for culturing with hydrogel scaffolds. In the dome technique (see Fig.  3 A), cells are mixed with temperature-sensitive hydrogels and then seeded as droplets within a cell culture vessel. This technique relies on careful temperature control to allow the hydrogel to polymerize and form a dome structure. Once the hydrogel has polymerized and the cell-hydrogel droplet is stabilized, it is delicately covered with cell culture media. This allows for a localized 3D cell culture in a larger vessel and can create multiple individual cell clusters or spheroids in a single plate. However, the maintenance of dome integrity can be challenging over time and might be affected by changes in temperature or physical disturbance. Also, it may not be suitable for long-term culture or cells requiring complex structural support due to the relatively simple and isolated 3D structure. Figure  3 B illustrates the insert wells technique, which consists of porous inserts to hold the cell-hydrogel mixture while cell culture media is added to the well surrounding the insert. This separation creates a differential environment, allowing for nutrient exchange while maintaining a distinct 3D culture within the insert. Heterogeneous spheroids will eventually form on the insert bottom due to gravitational pull and cell–cell interactions. Such a model can be used to study cell invasion or migration by placing the cell-hydrogel mixture on one side of a permeable membrane and chemo-attractants on the other. The gel-bottom support method (see Fig.  3 C) involves creating a thick layer of hydrogel at the bottom of a culture well, on top of which the cell suspension is placed. For instance, this method can be used for embedding cells within macroporous hydrogel scaffolds, such as AlgiMatrix ® (Thermo Fisher Scientific/Life Technologies, Carlsbad, USA)—an ionically gelled and dried scaffold that is conveniently provided in sterile pre-loaded disc format in standard cell culture well plates [ 103 , 104 ]. To initiate the cell culture, a concentrated cell suspension in culture media is seeded on top of the hydrogel, where it is subsequently absorbed, resulting in the entrapment of the cells within the porous structure of the hydrogel. Lastly, in the embedding technique (see Fig.  3 D), the cells are mixed with a hydrogel and directly placed at the bottom of a culture vessel, followed by a layer of culture media, allowing the cells to grow within the matrix of the hydrogel, thereby more accurately mimicking the in vivo 3D environment. This technique is beneficial for studying cell–cell and cell–matrix interactions, invasion, migration, and drug responses. However, it can be more technically challenging to embed cells evenly throughout the hydrogel; retrieving cells from the matrix for downstream analysis can be challenging. The permeability of the hydrogel to nutrients, gases, and wastes may need careful optimization to avoid creating a hypoxic environment or nutrient deprivation for cells located in the interior of the gel. Each of these methods must be selected based on the needs of the specific experiment and the type of cells being cultured. Additionally, the hydrogel composition and mechanical properties should be tuned according to the native ECM properties of the cell type of interest.

Common methods of hydrogel 3D cultures: A the dome technique: cells are mixed with temperature-sensitive hydrogels then seeded as droplets in the cell culture vessel, then carefully covered with media, B insert wells: media is added in the well whereas cell suspension (cell in hydrogel mix) is placed in the insert, then covered with another layer of media. Heterogeneous spheroids will form on the insert bottom, C gel-bottom support: the bottom of the well is covered with a thick layer of hydrogel, on top of which the cell suspension is placed, and D embedding technique: cells mixed with hydrogel are placed on the bottom and then covered with a layer of media to support spheroid growth in the matrix

Due to their adjustable properties, synthetic hydrogels offer notable benefits in 3D cell culture. The RADA16-I peptide is a self-assembling peptide derived from a segment of Zuotin, a left-handed Z-DNA-binding protein originally discovered in yeast. This peptide has emerged as a novel nano-biomaterial due to its ability to form nanofiber scaffolds. Consequently, these scaffolds provide a supportive framework that promotes cell growth and fosters a conducive 3D milieu for cell culture. The peptide sequence can be modified to incorporate specific functional groups, thus fine-tuning the mechanical, chemical, and biological attributes of the resultant scaffold. This remarkable flexibility enables customization to align precisely with the unique demands of the cultured cells or the intended experimental objectives. These scaffolds, which are about 10 nm in diameter, are driven by positively and negatively charged residues through complementary ionic interactions. When dissolved in water, the RADA16-I peptide forms a stable hydrogel (nanofiber networks with pore sizes of about 5–200 nm) with extremely high water content at concentrations of 1–5 mg/mL, which closely mimics the porosity and gross structure of ECMs, making it suitable for the fabrication of artificial cell niches for applications in tumor biology. Yang and Zhao [ 105 ] prepared a RADA16-I peptide hydrogel that provided an elaborate 3D microenvironment for ovarian cancer cells in response to the surrounding topography. The 3D cell cultures exhibited a two to five-fold increase in drug resistance (paclitaxel, curcumin, and fluorouracil) compared to the 2D monolayers, which showed a good representation of the primary tumor and were likely to simulate the in vivo biological characteristics of ovarian cancer cells. Similarly, Song et al. [ 106 ] also proved that RADA16-I hydrogels can provide prominent and dynamic nanofiber frameworks to sustain robust cell growth and vitality. HO-8910PM cells, metastatic human ovarian cancer cells, were cultured in three hydrogel biomaterials, namely RADA16-I hydrogel, Matrigel, and collagen I. The specially designed RADA16-I peptide exhibited a well-defined nanofiber network structure within the hydrogel, providing a nanofiber-based cellular microenvironment similar to Matrigel and collagen I. Notably, the HO-8910PM cells exhibited distinctive growth patterns within the three matrices, including cell aggregates, colonies, clusters, strips, and multicellular tumor spheroids (MCTS). Moreover, the molecular expression of integrin β1, E-cadherin, and N-cadherin in 3D-cultured MCTS of HO-8910PM cells was elevated, and their chemosensitivity was reduced to cisplatin and paclitaxel in comparison to the 2D cell culture, evidenced by IC 50 values and inhibition rates.

Furthermore, polyvalent hyaluronic acid (HA) hydrogels are considered synthetic, as they are typically created through chemical modification of HA molecules, introducing crosslinking agents or functional groups that enable the formation of a gel-like structure. This modification allows for control over the physical and mechanical properties of the hydrogel, such as its stiffness, degradation rate, and bioactivity. Suo et al. [ 107 ] engineered an ECM-mimicking hydrogel scaffold to replicate the native breast cancer microenvironment to provide an effective in vitro model for studying breast cancer progression. HA hydrogels from polyvalent HA derivatives were prepared through an innovative dual crosslinking process involving hydrazone and photo-crosslinking reactions. Hydrazone crosslinking is a versatile, reversible process that allows for rapid gelation, while photo-crosslinking stabilizes the formed hydrogel. Using this approach, they could efficiently produce HA hydrogels in under 120 s. It was found that the developed HA hydrogels closely resembled the topography and mechanical properties of breast tumors, and their characteristics (i.e., rigidity and porosity) could be fine-tuned by adjusting the amount of aldehyde-HA in the hydrogel formulation. This ability to modulate the mechanical properties of the hydrogels opens up possibilities for modeling different stages of tumor progression or different types of tumors. Moreover, a critical feature of the developed HA hydrogels was their dual-responsive degradation behavior, which was found to be responsive to glutathione and hyaluronidase. The glutathione responsiveness allows for degradation in response to the redox environment, which is often disturbed in cancer cells. Meanwhile, responsiveness of hyaluronidase makes the hydrogels sensitive to an enzyme that is typically upregulated in invasive cancer cells. Significantly, the HA hydrogel-cultured MCF-7 cells displayed upregulated expression of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) compared to their 2D cultured counterparts. These molecules are key mediators of angiogenesis and inflammation in cancer, suggesting that the HA hydrogel environment better replicates the conditions that promote these processes in tumors. Besides, the hydrogel-cultured cells exhibited enhanced migration and invasion abilities, which are key hallmarks of aggressive cancer cells. In vivo studies supported these results and confirmed the superior tumorigenic capacity of the MCF-7 cells cultured in HA hydrogels compared to those cultured in 2D. The outcomes of this research are anticipated to have far-reaching implications for both the in vitro study of breast cancer and the development of effective therapeutic strategies.

Another investigation by Wang et al. [ 108 ] supported that the level of methacrylation significantly influenced the hydrogel’s microstructure, mechanical characteristics, and capacity for liquid absorption and degradation. The refined hydrogel, synthesized through the photocrosslinking of methacrylated HA, displayed a highly porous structure, a high equilibrium swelling ratio, appropriate mechanical properties, and a degradation process responsive to hyaluronidase. It was found that the HA hydrogel promoted the growth and proliferation of MCF-7 cells, which formed aggregates within the hydrogel. In addition, 3D-cultured MCF-7 cells showed an increased expression of VEGF, bFGF, and interleukin-8, and enhanced invasion and tumorigenesis capabilities compared to their 2D-cultured counterparts. As such, the HA hydrogel has proven to be a dependable alternative for constructing tumor models. Gelatin methacryloyl (GelMA) is another commonly used natural biomaterial for 3D hydrogel scaffolds in cancer research. GelMA is derived from gelatin, a natural protein obtained from collagen-rich sources. It is modified by adding methacryloyl groups that enable it to undergo photocrosslinking when exposed to ultraviolet (UV) light. This property allows GelMA to form stable hydrogel networks, making it suitable for creating 3D scaffolds that mimic the tumor microenvironment (TME). The tunable mechanical and biochemical properties of GelMA hydrogels, biocompatibility, and ability to support cell growth make them valuable tools for studying cancer cell behavior, tumor invasion, drug screening, and other aspects of cancer research. Kim et al. [ 109 ] developed a 3D cell culture model for the bladder by employing a novel acellular matrix and bioreactor. GelMA was utilized as a 3D scaffold for the bladder cancer cell culture, with an optimal scaffold height of 0.08 mm and a crosslinking time of 120 s [ 110 ]. Subsequently, 5637 and T24 cells were cultured in 2D and 3D environments and subjected to rapamycin and Bacillus Calmette-Guérin (BCG) drug treatments. It was found that the 3D bladder cancer cell culture model exhibited a faster establishment process and greater stability when compared to the 2D model. Moreover, the 3D-cultured cancer cells demonstrated heightened drug resistance and reduced sensitivity compared to the 2D-cultured cells. Additionally, the researchers observed cell-to-cell interaction and basal activity in the 3D model, closely resembling the in vivo environment.

Along the same lines, Arya et al. [ 111 ] investigated the suitability of GelMA hydrogels as in vitro 3D culture systems for modeling key characteristics of metastatic progression in breast cancer, specifically invasiveness and chemo-responsiveness. The mechanical and morphological properties of the hydrogels were tuned by varying the percentage of GelMA used. Compression testing revealed that the stiffness of 10% GelMA hydrogels was within the range reported for breast tissue, making them suitable matrices for mimicking the breast viscoelasticity in vitro, as cells cultured on 10% GelMA hydrogels exhibited a higher proliferation rate compared to 15% GelMA in both cell lines tested, making them robust systems for long-term cell culture. Furthermore, proliferation studies showed that the GelMA hydrogels could sustain breast cancer cells longer than 2D cultures. Overexpression of genes associated with invasiveness was also observed in 3D cultured breast cancer cells, suggesting potential changes important for metastatic progression. The response to chemotherapeutic drugs was evaluated, and it was observed that 3D spheroids of breast cancer cells cultured on GelMA hydrogels exhibited decreased sensitivity to taxane drugs like paclitaxel. The study highlighted the importance of an adequate matrix pore size for cell penetration, migration, proliferation, exchanging oxygen, nutrients, and waste materials in and out of the 3D culture scaffolds. Significantly, these studies emphasized the importance of the 3D cancer cell culture model in establishing a patient-like model. Utilizing such models can achieve a more precise evaluation of drug responses, potentially leading to advancements in cancer treatment and other diseases.

Cells are known to respond to their mechanical environment in a process known as mechano-transduction, where they transmute mechanical stimuli into biochemical signals, subsequently prompting alterations in cellular behavior and functional operations. Curtis et al. [ 112 ] investigated the influence of mechanical stimuli on the cell proliferation, growth, and protein expression of 4T1 breast cancer cells, serving as a model for cells that metastasize to bone. The researchers used 4T1 breast cancer cells and implanted them in gelatin-mTGase hydrogels that mimicked the mechanical properties of bone marrow. The hydrogels had different moduli of either 1 or 2.7 kPa. The cells were cultured under different conditions, including static culture, perfusion of media through the hydrogel, and combined perfusion with cyclic mechanical compression for 1 h per day for 4 days. Control samples were cultured under free-swelling conditions. Immunostaining techniques were used to analyze the protein expression within the cell spheroids formed during the culture. The study found that mechanical stimuli significantly influenced the behavior of the 4T1 breast cancer cells. The cells formed spheroids during the culture period, with larger spheroids observed in statically cultured constructs than those exposed to perfusion or compression. In the stiffer gelatin, compressed constructs resulted in smaller spheroids compared to perfusion alone, while compression had no significant effect in the softer gelatin. The immunostaining revealed the expression of proteins associated with bone metastasis within the spheroids, including osteopontin, parathyroid hormone-related protein, and fibronectin. The proliferative marker Ki67 was present in all spheroids on day 4. The intensity of Ki67 staining varied depending on the culture conditions and gelatin stiffness. It highlighted the mechanical sensitivity of 4T1 breast cancer cells and demonstrated how mechanical stimuli can impact their proliferation and protein expression within soft materials that mimic the mechanical properties of bone marrow. The findings emphasized the role of the mechanical environment in the bone for both in vivo and in vitro models of cancer metastasis.

Understanding the influence of mechanical factors on cancer cell behavior is crucial for developing effective strategies to prevent and treat metastasis to bone, potentially leading to improved clinical outcomes for patients with advanced cancer. Similarly, Cavo et al. [ 113 ] investigated the impact of substrate elasticity on breast adenocarcinoma cell activity using mechanically tuned alginate hydrogels. The study evaluated the viability, proliferation rates, and cluster organization of MCF-7 breast cancer cells in 3D alginate hydrogels compared to standard 2D environments. The elastic moduli of the different alginate hydrogels were measured using atomic force microscopy (AFM). The results demonstrated that substrate stiffness directly influenced cell fate in 2D and 3D environments. In the 3D hydrogels with an elastic modulus of 150–200 kPa, the MCF-7 cells exhibited uninhibited proliferation, forming cell clusters with 100 μm and 300 μm diameters after 1 and 2 weeks, respectively. This unimpeded cell growth observed in softer hydrogels mimicked the initial stages of solid tumor pre-vascularization and growth. Furthermore, the multicellular, cluster-like conformation observed in the 3D hydrogels closely resembled the in vivo organization of solid tumors, demonstrating the advantage of 3D cancer models for replicating cell–cell and cell–matrix interactions. The study also highlighted the influence of microenvironment dimensionality on cellular morphology, as cells displayed a flat shape in 2D cultures while adopting a round shape in the 3D environment. Cell proliferation in the 3D setting depended highly on substrate stiffness, which impacted nutrient diffusion and intracellular signaling through a mechano-transduction mechanism. The findings underscore the importance of considering substrate stiffness in the design of 3D cancer models, as it directly affects cell viability, proliferation, and organization. By understanding the relationship between substrate stiffness and cellular behavior, researchers can develop more realistic in vitro models that better mimic the microenvironment of solid tumors. These models can advance our understanding of cancer development and aid in the development of targeted therapies by allowing for the investigation of cell–cell and cell–matrix interactions in a more accurate setting.

Decellularized tissue scaffolds

Decellularized tissues have had their cellular components removed, leaving behind the ECM. Decellularized tissues can be used as scaffolds for 3D cell culture, providing a natural environment for cells to grow and interact [ 114 ]. The use of decellularized tissues as 3D cell culture scaffolds offers several advantages. Firstly, they retain the intricate ECM composition, including structural proteins, growth factors, and signaling molecules, which play critical roles in cell behavior and tissue organization. This enables cancer cells to interact with the ECM more akin to in vivo conditions, influencing their adhesion, migration, invasion, and differentiation. Moreover, decellularized tissues offer spatial organization and architectural cues that guide cellular behavior. Preserving tissue-specific topography, such as vasculature, allows for studying angiogenesis and vascularization processes in cancer progression. These scaffolds also provide mechanical support and stiffness that influence cellular mechanotransduction, impacting cell morphology, proliferation, and gene expression patterns. They can be derived from various sources, including solid organs, such as the liver or lung, or specific tissue compartments, such as the ECM-rich decellularized basement membrane (see Fig.  4 ).

Preparation methods, characterization techniques, and sources of decellularized tissues used as scaffolds for 3D cell culture. SEM: scanning electron microscopy; AFM: atomic force microscopy; FTIR: Fourier-transform infrared spectroscopy

Landberg et al. [ 115 ] hypothesized that using a pre-clinical platform based on decellularized patient-derived scaffolds as growth substrates to account for hidden clinically relevant information and aid in modeling the individualized properties of microenvironments could be optimized for personalized treatment planning. Different decellularization techniques, such as chemical, physical, or enzymatic methods, remove cellular components while preserving the ECM integrity (see Table  6 ) [ 116 ]. The choice of decellularization method depends on the tissue type, desired scaffold characteristics, and the specific requirements of the study. Combinations of different techniques may also be employed to achieve optimal decellularization outcomes. However, challenges remain in the field. The immunogenicity and biocompatibility of decellularized tissues must be carefully considered to prevent adverse reactions when introducing foreign matrices into cell culture systems. Standardization and reproducibility of decellularization protocols are also crucial to ensure consistency across studies and facilitate comparison of results. Integration with advanced technologies, such as microfluidics or organ-on-a-chip systems, can further enhance the functionality and relevance of decellularized tissue models.

D’Angelo et al. [ 117 ] developed a more representative 3D model of colorectal cancer liver metastasis using patient-derived scaffolds. These scaffolds, created by decellularizing tissue-specific ECM, retain the metastatic microenvironment’s biological properties and structural characteristics. The HT-29 CRC cell line was cultured within these scaffolds, obtained explicitly from cancer patients. The study observed increased cell proliferation and migration in the cancer-derived scaffolds, highlighting their ability to provide a more conducive environment for tumor cell growth and spreading. Furthermore, the 3D culture system demonstrated a reduced response to chemotherapy. HT-29 cells cultured in the cancer-specific 3D microenvironments showed decreased sensitivity to treatment with 5-fluorouracil and a combination of 5-fluorouracil with Irinotecan, when used at standard IC50 concentrations. The use of patient-derived scaffolds allows for the study of colorectal cancer metastasis progression and the assessment of their response to chemotherapy agents, to develop new therapeutic strategies and personalized treatments. Additionally, it provides an opportunity to identify potential prognostic biomarkers and therapeutic targets specific to peritoneal metastasis. Varinelli et al. [ 118 ] conducted a study that employed a tissue-engineered model for investigating peritoneal metastases (PM) in vitro, yielding similar conclusions. The model involved seeding PM-derived organoids onto decellularized extracellular matrices (dECMs) sourced from the peritoneum, enabling the exploration of intricate interactions between neoplastic cells and the ECM in the PM system. Both neoplastic peritoneum and corresponding normal peritoneum tissues were utilized to generate 3D-dECMs. Utilizing confocal reflection and polarized light microscopy techniques, the study observed disparities in tissue texture and the distribution and integrity of individual collagen fibers between normal and neoplastic-derived tissues obtained from three distinct PM patients. The results demonstrated that 3D-dECMs derived from neoplastic peritoneum exhibited a notably higher proportion of Ki-67-positive cells after 5 and 12 days. Furthermore, expression levels of specific genes critical for tissue architecture, stiffness, ECM remodeling, fibril generation, epithelial cell differentiation, resistance to compression, and regulation of angiogenesis were found to be elevated in 3D-dECMs generated from neoplastic tissue compared to those from normal tissue or Matrigel-based models. In summary, by utilizing patient-derived scaffolds and cutting-edge techniques, the researchers successfully developed more physiologically relevant models that significantly contribute to our comprehension of colorectal cancer and PM biology. These models, alongside others [ 119 , 120 , 121 , 122 ], offer valuable insights into the intricate interplay between tumor cells and the ECM, paving the way for the potential discovery of novel therapeutic targets and the development of personalized treatment strategies for peritoneal metastases.

Furthermore, decellularized tissue scaffolds provide an efficient platform to study the interactions between different components abundantly found in the ECM, like macrophages and endothelial cells. Macrophages and endothelial cells are known for their involvement in cancer progression in the context of the ECM within solid tumors, as they are often found in large numbers [ 123 ]. Macrophages within the tumor (often referred to as tumor-associated macrophages or TAMs) can be “hijacked” by cancer cells and reprogrammed to support tumor growth and progression. For example, they can promote cancer cell proliferation, enhance blood vessel formation (angiogenesis), assist in tissue remodeling, and suppress the immune response against the tumor. Pinto et al. [ 123 ] investigated how human colorectal tumor matrices influence macrophage polarization and their subsequent role in cancer cell invasion. To facilitate this, a novel 3D-organotypic model was utilized using decellularized tissues from surgical resections of colorectal cancer patients. This model preserved native tissue characteristics, including major ECM components, architecture, and mechanical properties, while removing DNA and other cellular components. The study found that macrophages within tumor matrices displayed an M2-like anti-inflammatory phenotype, characterized by higher expression of IL-10, TGF-β, and CCL18, and lower expression of CCR7 and TNF. Furthermore, it was observed that tumor ECM-educated macrophages effectively promoted cancer cell invasion through a mechanism involving CCL18, as demonstrated by Matrigel invasion assays. The high expression of CCL18 at the invasive front of human colorectal tumors correlates with advanced tumor staging, underscoring its clinical significance. The findings highlight the potential of using tumor-decellularized matrices as exceptional scaffolds for recreating complex microenvironments, thereby enabling a more comprehensive understanding of cancer progression mechanisms and therapeutic resistance.

Besides TAMs, endothelial cells express various adhesion molecules and chemokines, such as selectins, integrins, and members of the immunoglobulin superfamily, which can interact with ligands on cancer cells, facilitating their adhesion to the endothelial cell layer. This adhesion is a critical step in the extravasation process, where cancer cells exit the bloodstream and invade surrounding tissues to form metastases. Moreover, endothelial cells can signal and recruit macrophages and other immune cells to the tumor site. Once there, macrophages can be “educated” by the tumor to adopt a pro-tumor phenotype, suppressing the immune response and promoting tumor growth. Therefore, decellularized matrices are suitable for studying such interactions as they closely resemble the natural tumor environment, including native adhesion sites, signaling molecules, and mechanical cues. Helal-Neto et al. [ 124 ] examined the influence of dECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and their intracellular cell differentiation signaling pathways. The researchers studied the differences in the ultrastructural organization and composition of melanocyte-derived ECM (NGM-ECM) and melanoma-derived (MV3-ECM). Higher levels of tenascin-C and laminin and lower fibronectin expression were detected in MV3-ECM. Moreover, endothelial cells cultured in the MV3-ECM underwent morphological transformations and exhibited increased adhesion, mobility, growth, and tubulogenesis. The interaction between the endothelial cells and decellularized matrix induced integrin signaling activation, resulting in focal adhesion kinase (FAK) phosphorylation and its association with Src (a non-receptor tyrosine kinase protein). Src activation, in turn, stimulated the activation of vascular endothelial growth factor receptor 2 (VEGFR2), enhancing the receptor’s response to VEGF. The activation of VEGF and the association between FAK and Src was inhibited by blocking the αvβ3 integrin, which reduced tubulogenesis. In conclusion, the findings suggested that the interaction of endothelial cells with melanoma-ECM triggered integrin-dependent signaling, which led to the activation of the Src pathway that sequentially potentiated VEGFR2 activation and enhanced angiogenesis. Thus, progress in cancer biology relies on understanding the specific cellular responses influenced by the matrix signals within the ECM, as its nature inherently imposes spatial variations on cellular signaling, composition, topography, and biochemical factors. Table 7 summarizes some studies using hydrogel and decellularized tissue scaffolds for 3D cell cultures.

Hybrid scaffolds

Integrating multiple scaffold types offers the potential to create 3D cell culture systems that closely mimic the physiological conditions of living tissues. This approach enables researchers to develop more accurate and biologically relevant models for studying cellular behavior, disease progression, and therapeutic responses. By combining different scaffold materials, such as natural and synthetic polymers or hydrogels, researchers can replicate the complexity and heterogeneity of the native tissue microenvironment. These hybrid scaffolds can provide a range of physical, chemical, and mechanical cues that influence cell behavior, including cell adhesion, migration, proliferation, and differentiation. Additionally, the combination of scaffolds can enhance the functionality of the 3D cell culture systems by incorporating specific features, such as the controlled release of growth factors or the inclusion of microvascular networks. Utilizing diverse scaffold types in 3D cell culture offers an innovative and promising approach for advancing our understanding of tissue biology, disease mechanisms, and developing more effective therapies. Bassi et al. [ 98 ] addressed the limitations of conventional therapies for osteosarcoma, a type of bone cancer, by introducing two innovative approaches in tumor engineering that aim to improve therapy outcomes. The study utilized hydroxyapatite-based scaffolds that mimic the in vivo TME, specifically emphasizing the CSC niche. Two types of scaffolds were employed: a biomimetic hybrid composite scaffold obtained through biomineralization, involving the direct nucleation of magnesium-doped hydroxyapatite (MgHA) on self-assembling collagen fibers (MgHA/Coll), and porous hydroxyapatite scaffolds (HA) produced by a direct foaming process. These scaffolds provided a framework for the subsequent investigation of the biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched CSCs within these complex 3D cell culture models. Immunofluorescence and other characterization techniques were employed to evaluate the response of the osteosarcoma cell lines and CSCs to the biomimetic scaffolds. The results demonstrated the successful formation of sarcospheres, which are stable spheroids enriched with CSCs, with a minimum diameter of 50 µm. Comparing the advanced 3D cell culture models with conventional 2D culture systems, the study revealed the former’s superiority in mimicking the osteosarcoma stem cell niche and enhancing the predictivity of preclinical studies. The findings underscore the significance of the TME and emphasize the potential of combining CSCs with biomimetic scaffolds as a promising approach to developing novel therapeutic strategies for osteosarcoma. Further efforts can be focused on developing more sophisticated 3D models that accurately replicate the heterogeneity of the osteosarcoma microenvironment, incorporating patient-derived cells and elements such as immune cells and vasculature. Additionally, the advanced 3D cell culture models can serve as valuable tools for drug screening and personalized medicine approaches, further contributing to advancing osteosarcoma research and treatment strategies.

A unique cell culture technique known as “sequential culture” was used to establish a biomimetic bone microenvironment that facilitated the EMT of metastasized prostate cancer cells [ 141 ]. The approach involved incorporating bioactive factors from the osteogenic induction of human mesenchymal stem cells (MSCs) within porous 3D scaffolds, specifically polymer–clay composite (PCN) scaffolds, by incorporating hydroxyapatite (HAP) clay into PCL. The researchers also modified sodium clay Montmorillonite (Na-MMT) clay using 5-amino valeric acid to create HAPclay through in situ hydroxyapatite biomineralization into the intercalated nano clay. They performed RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) analysis to investigate gene expression changes. Additionally, they conducted a comparative analysis of bone metastasis between the low and high metastatic cell lines, providing insights into their differential responses to the bone microenvironment. It was shown that both, the highly metastatic prostate cancer cell line PC-3 and the non-metastatic cell line MDAPCa2b, underwent MET transition when exposed to the biomimetic bone microenvironment in the 3D scaffold model. However, notable differences were observed in their morphological characteristics and cell–cell adhesion, suggesting distinct responses to the microenvironment. Additionally, quantitative variations in gene expression were observed between tumors generated using the two cell lines in the bone microenvironment. These findings are essential for developing targeted therapeutic strategies against prostate cancer bone metastasis. Bai et al. [ 142 ] conducted a study in which they incorporated graphene oxide (GO) onto a copolymer of polyacrylic acid-g-polylactic acid (PAA-g-PLLA) to create a stimuli-responsive scaffold. This scaffold, combined with PCL and gambogic acid (GA), exhibited a selective response towards tumors and demonstrated a significant accumulation of GO/GA in vitro breast tumor cells (MCF-7 cells) under acidic conditions (pH 6.8), while showing minimal impact on normal cells (MCF-10A cells) at physiological pH (pH 7.4). The study further revealed that the synergistic use of pH-responsive photo-thermal conversion was more effective in inhibiting tumor growth than independent treatments. In vivo experiments showed remarkable tumor suppression (99% reduction within 21 days) through tumor tissue disintegration, degeneration, and overall tumor suppression when treated with GO-GA scaffolds combined with photo-thermal therapy, in comparison to control groups or those treated with either GO-GA scaffolds or near-infrared (NIR) irradiation alone.

Microfluidics provide a versatile platform for 3D cell culture, offering both scaffold-based and scaffold-free approaches. Researchers can tailor the platform to suit the specific requirements of their experiments, whether involving cell-laden scaffolds or the aggregation of cells to form spheroids or organoids. The microfluidic setup allows for precise control over the microenvironment, including the flow of nutrients and oxygen, as well as the ability to introduce gradients of specific molecules. Lee et al. [ 143 ] utilized soft lithography to fabricate a 7-channel microchannel plate using poly-dimethylsiloxane (PDMS). Within separate channels, PANC-1 pancreatic cancer cells and pancreatic stellate cells (PSCs) were cultured within a collagen I matrix. The study observed the formation of 3D tumor spheroids by PANC-1 cells within five days. Intriguingly, the presence of co-cultured PSCs resulted in an increased number of spheroids, suggesting a potential influence of PSCs on tumor growth. In the co-culture setup, PSCs exhibited heightened expression of α-smooth muscle actin (α-SMA), a marker associated with fibroblast activation, as well as various EMT-related markers, including vimentin, transforming growth factor-beta (TGF-β), TIMP1, and IL-8. These findings indicated that PSCs may induce an EMT-like phenotype in PANC-1 cells, potentially promoting tumor invasiveness, chemoresistance, and metastasis. Upon treating the co-culture with gemcitabine, the survival of the spheroids did not exhibit significant changes. However, when combined with paclitaxel, the tumor spheroids demonstrated a notable inhibitory effect on growth. The model revealed a complex interplay between PANC-1 cells and PSCs within the TME. Nonetheless, the combination of gemcitabine and paclitaxel showed promise to overcome resistance and inhibit tumor growth. The implications of these findings are significant for understanding the complex interplay between tumor cells and the surrounding stromal cells within the TME. Tumor-stroma interactions play a critical role in cancer progression and therapy response. Using microfluidic-based 3D co-culture models allows researchers to better recapitulate the in vivo conditions, providing a more accurate representation of tumor behavior and therapeutic responses.

Likewise, Chen et al. [ 144 ] developed a microchannel plate-based co-culture model to recreate the in vivo TME by combining Hepa1-6 tumor spheroids with JS-1 stellate cells (liver cancer)—the novel model aimed to mimic key aspects of EMT and chemoresistance observed in tumors. The integration of these cell types in 3D concave microwells allowed for the formation of 3D tumor spheroids in 3 days. The experimental setup was optimized to ensure optimal culture proliferation conditions and appropriate interactions between Hepa1-6 and JS-1 cells. Co-cultured JS-1 cells displayed noticeable changes in cellular morphology, including an increase in the expression of α-SMA. In contrast, the co-cultured Hepa1-6 spheroids exhibited higher expression levels of TGF-β1 than those cultured alone. These findings suggested that JS-1 stellate cells induced an EMT-like phenotype in the Hepa1-6 cells, potentially contributing to increased invasiveness and resistance to chemotherapy. Jeong et al. [ 145 ] conducted a similar study involving the formation of 3D spheroids composed of human colorectal carcinoma cells (HT-29) using a microfluidic chip. They reported a notable enhancement in HT-29 growth when co-cultured with fibroblasts (see Fig.  5 ). This enhancement was demonstrated by a 1.5-fold increase in the percentage change in spheroid diameter over 5 days. Furthermore, after 6 days of culture, the co-cultured spheroids exhibited reduced expression of Ki-67, a marker associated with proliferation, while showing increased fibronectin expression. These findings indicated altered cellular behavior compared to the spheroid monocultures. The presence of fibroblasts in the co-culture environment also led to their activation, as evidenced by an upregulation in the expression of α-smooth muscle actin (α-SMA) and an increase in migratory activity. This reciprocal interaction between the spheroids and fibroblasts within a microfluidic chip established a dynamic relationship. Additionally, when exposed to paclitaxel, the co-culture displayed a survival advantage over 2D monoculture, suggesting the potential role of fibroblasts in conferring drug resistance. Integrating the 3D tumor spheres and CAFs within a collagen matrix-incorporated microfluidic chip provided a valuable tool for studying the TME and evaluating drug screening and efficacy. This approach allowed for the replication of essential interactions between tumor cells and stromal components, which are known to influence cancer progression and therapeutic response. By utilizing the proposed microfluidic chip-based model, researchers can delve into the intricate dynamics of the TME and explore novel therapeutic approaches. The ability to control and better mimic the in vivo conditions within the chip provides a valuable platform for investigating drug responses and evaluating the effectiveness of anticancer treatments. Further exploration and refinement of this model could lead to significant advancements in our understanding of tumor biology and the development of targeted therapies for improved patient outcomes. Table 8 summarizes some studies using microfluidic-based systems to develop 3D cell cultures.

illustration of the microfluidic chip used in 3D co-culture of human colorectal cancer cells (HT-29) and normal colorectal fibroblasts (CCD-18Co) in a collagen matrix. The chip comprised 4 units, each featuring 7 channels for cell loading or media fill. Cancer and fibroblast cells were loaded into channels 4 and 2 in the co-culture, while channels 1 and 3 were designated for media fill. A cell loading channel’s detailed structure and dimensions are illustrated at the bottom left. Figure adapted from [ 145 ]

Challenges and future prospectives

While 3D cell culture offers many advantages over traditional 2D culture, it also presents some unique challenges that must be addressed to realize its potential for advancing research fully. One significant challenge is maintaining a stable and reproducible culture system. 3D cell culture systems often require specialized equipment, such as bioreactors and microfluidic devices, which can be expensive and difficult to use. These systems can be more challenging to reproduce compared to 2D systems due to the increased complexity and high heterogeneity of the culture environment, as cells are often embedded in matrices or scaffolds, making it difficult to control factors such as temperature, pH, and the presence of growth factors and/or other signaling molecules [ 149 ]. In addition, there is often a high degree of variability between different batches of cells and between experiments, making it difficult to draw statistically supported conclusions. Considering 3D cell cultures, adhering to Good Manufacturing Practices (GMP) principles is essential for translating these advanced models from research to clinical and commercial applications. However, several challenges and considerations arise when implementing GMP standards, including standardization of culture conditions, scalability, quality control, raw materials and biologics sourcing, regulatory compliance, data integrity, and documentation. GMP-compliant manufacturing processes require high reproducibility and control over critical parameters such as cell sourcing, culture media, culture supplements, and environmental conditions [ 150 , 151 ]. As mentioned above, achieving this consistency can be challenging, given the inherent biological variability of primary cells and the sensitivity of 3D cultures to slight changes in culture conditions. Furthermore, meeting regulatory requirements is a paramount challenge in translating 3D spheroid cultures to clinical applications. Regulatory bodies, such as the Food and Drug Administration (FDA) in the United States and the European Medicines Agency (EMA) in Europe, have specific guidelines for the use of cell-based therapies and products [ 152 ]. GMP compliance is necessary to navigate these regulatory pathways and obtain approval for clinical trials and commercialization.

Moreover, oxygen accessibility is a critical consideration in 3D cell culture methods, and its heterogeneity within these environments poses a significant challenge in replicating physiological conditions and obtaining accurate experimental results. Cells located in the interior of 3D structures, such as spheroids, often encounter limited oxygen availability due to microenvironmental factors (i.e., tumor spheroids naturally develop hypoxic regions due to irregular vascularization in tumors) and diffusion barriers (e.g., densely packed cells, ECM, scaffolding matrices) [ 153 ]. As cells proliferate and form 3D structures, the demand for oxygen increases due to the larger volume that oxygen must traverse. Oxygen diffusion from the surrounding culture medium becomes progressively hindered as the distance from the culture surface to the interior of the 3D structure increases. This results in an oxygen gradient, where cells near the periphery have sufficient oxygen, but those in the core encounter oxygen deficiency, leading to hypoxia. Hypoxic core cells often exhibit altered gene expression, reduced proliferation, and changes in metabolic pathways as they enter a dormant state and cease cycling when deprived of oxygen and nutrients. This reduced activity renders them relatively resistant to cytostatic drugs that predominantly target actively dividing cells, leading to increased drug resistance, as is often observed in solid tumors [ 154 , 155 ]. Confocal microscopy can be used to visualize dormant cells by labeling them with a nucleoside analog, allowing for their quantification and distinction from actively proliferating cells. This analog gets diluted in actively dividing cells. Still, it remains retained in quiescent, non-dividing cancer cells, thus providing a valuable tool for distinguishing them from the surrounding actively proliferating cells [ 156 ]. Leveraging this characteristic of 3D spheroids, they offer potential avenues for developing novel therapeutics targeting cancer cells resistant to cytostatic anticancer drugs. Wenzel et al. [ 157 ] cultivated T47D breast cancer cells in 3D cultures and used confocal imaging to differentiate cells within the inner core from those in the surrounding outer core. Cells in the inner core, experiencing limited access to oxygen and nutrients, exhibited reduced metabolic activity compared to their counterparts in the outer core. Through screening small molecule libraries against these 3D cultures, the authors identified nine compounds that selectively targeted and killed the inner core cancer cells while sparing the more actively proliferating outer cells. The identified drugs primarily affected the respiratory chain pathway, aligning with the altered metabolic activity of oxygen-deprived cells transitioning from aerobic to anaerobic metabolism. Hence, compounds selectively targeting dormant cancer cells significantly improved the effectiveness of commonly employed cytostatic anticancer drugs. Alternatively, the use of microfluidic devices that enable the creation of controlled oxygen gradients within cultures, the incorporation of oxygen-permeable materials, and the addition of oxygen-releasing compounds to provide a more uniform distribution of oxygen in vitro. However, it is important to acknowledge that these strategies may not fully replicate the complexity of oxygen gradients in real tissues [ 158 ]. Boyce et al. [ 159 ] presented the design and characterization of a modular device that capitalized on the gas-permeable properties of silicone to create oxygen gradients within cell-containing regions. The microfabricated device was constructed by stacking laser-cut acrylic and silicone rubber sheets, where the silicone not only facilitated oxygen gradient formation but also served as a barrier, separating the flowing gases from the cell culture medium to prevent evaporation or bubble formation during extended incubation periods. The acrylic components provided structural stability, ensuring a sterile culture environment. Using oxygen-sensing films, gradients with varying ranges and steepness in the microdevice can be achieved by adjusting the composition of gases flowing through the silicone elements. Furthermore, a cell-based reporter assay illustrated that cellular responses to hypoxia were directly proportional to the oxygen tension established within the system, proving efficacy.

Another practical challenge in 3D cultures arises from the intricacy of extracting cells from biomaterial-based 3D constructs. Typically, the construction of degradable hydrogel scaffolds involves integrating breakable crosslinks and/or cleavable components into the polymer structure or incorporating naturally biodegradable ECM constituents such as hyaluronic acid, laminin, fibronectin, and collagen [ 160 ]. Yet, traditional dissociation techniques prove to be notably inefficient and are influenced by the inherent structural complexities of the culture system. Enzymatic degradation, for example by collagenase, is a widely employed method for retrieving cells from 3D cell culture collagen-based scaffolds. The enzyme is selected to match the specific collagen type in the scaffold. During incubation, collagenase enzymatically cleaves the collagen fibers, releasing cells that were embedded or adhered to these fibers. Once the collagen has been broken down, the cells are collected as a suspension in the culture medium [ 161 ]. Cell viability and functionality assessments are typically performed to maintain the cells’ health and functionality. While using enzymatic degradation for 3D cell culture scaffolds is common, it remains an intricate approach associated with several limitations. It is important not to underestimate the impact of collagenase or other enzymes on cell viability and functionality. Careful optimization of digestion time and enzyme concentration is essential to balance efficient scaffold degradation and preserving cell quality [ 162 ]. Additionally, potential changes in cell phenotype during digestion are a significant concern, necessitating diligent monitoring of digestion parameters. In complex 3D scaffolds, particularly those with intricate structures, enzymatic digestion may be less effective, prompting researchers to explore alternative retrieval methods or adapt the digestion process. Ethical considerations also come into play, especially when working with human or animal-derived cells, raising concerns about using enzymes like collagenase. Adherence to ethical guidelines and institutional regulations is crucial for maintaining responsible and ethical research practices.

Hence, extensive research efforts have been directed toward developing improved techniques for cell retrieval from scaffold-based 3D cell cultures without compromising the cells’ integrity. For instance, Kyykallio et al. [ 163 ] developed an innovative pipeline for extracting extracellular vesicles (EVs) from 3D cancer spheroids using nanofibrillar cellulose (NFC) scaffolds as a cell culture matrix. This pipeline encompassed two distinct approaches: a batch method optimized for maximal EV yield at the conclusion of the culture period, and a harvesting method designed to facilitate time-dependent EV collection, allowing integration of EV profiling with spheroid development. Both approaches provided convenient setup, quick execution, and reliably produced a significant number of electric vehicles (EVs). Compared to scaffold-free 3D spheroid cultures on ultra-low affinity plates, the NFC-based approach demonstrated similar EV production per cell, offering scalability, preserved cell phenotype and integrity, and greater operational simplicity, ultimately leading to higher EV yields. Another approach is based on cell-mediated degradation of hydrogel scaffolds, where living cells actively break down the hydrogel structure [ 164 ]. This degradation mechanism is particularly relevant in tissue engineering and regenerative medicine. When cells are encapsulated within a hydrogel scaffold, they can secrete enzymes and other molecules that interact with its components, leading to its gradual breakdown. As cells proliferate and remodel their microenvironment, they may alter the scaffold’s properties and eventually facilitate its degradation. This dynamic process allows for the controlled release of cells, growth factors, and other bioactive substances within the hydrogel, making it a valuable technique for drug delivery applications.

While synthetic degradable polymer scaffolds are significant for developing 3D cell culture models, a concern regarding their in vitro and in vivo biocompatibility pertains to the presence of potentially toxic elements and chemicals utilized during the polymerization of synthetic hydrogels or the crosslinking of natural polymer hydrogel precursors, especially when the reaction conversion is less than 100%. These substances release unreacted monomers, stabilizers, initiators, organic solvents, and emulsifiers. These are integral to the hydrogel preparation process but may pose harm if they seep into the seeded cells or tissues [ 165 , 166 ]. For instance, widely employed free radical photo-initiators (e.g., Irgacure) have been observed to diminish cell viability, even at minimal concentrations [ 167 , 168 ]. Consequently, hydrogel scaffolds intended for embedding cells in 3D cultures typically require purification (e.g., by dialysis or solvent washing) to eliminate any residual hazardous chemicals before seeding. However, in certain scenarios, the purification of hydrogel scaffolds is more challenging or unfeasible, particularly when dealing with hydrogels generated through in situ gelation. In such cases, cells are introduced to the reactants necessary for hydrogel synthesis while still in a pre-polymer solution. As a result, when employing in situ gelation techniques, utmost caution must be exercised to ensure that all components are non-toxic and safe.

Furthermore, another challenge associated with 3D cell culture is the difficulty characterizing the cellular response to drugs and other therapeutic agents. In 2D cell culture, cells are typically analyzed using a range of standard assays that are well-established and easy to interpret. However, in 3D cell culture, there is often a lack of such standardized assays and protocols. Fang and Eglen [ 169 ] highlighted that the cultures’ complex morphology, functionality, and architecture hampered the application of some well-developed biochemical assays to 3D systems. Cells tend to aggregate into dense and/or large clusters over time, even in macroporous scaffolds, causing diffusional limitations when carrying out in situ characterization assays. Limitations arise due to the impeded diffusion and confinement of gases, nutrients, waste, and reagents within the system, compounded by challenges when quantifying and normalizing data between different biomimetic cultures [ 170 , 171 , 172 ]. For instance, Totti et al. [ 173 ] demonstrated that assessing a culture of pancreatic cancer cells in macroporous polyurethane foam-type scaffolds with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay showed minimal differences between various scaffold conditions (e.g., ECM coatings on the scaffolds). However, sectioning, immunostaining, and imaging revealed clearer cell proliferation, morphology, and growth distinctions between the conditions. Likewise, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay failed in capturing the differences in pancreatic cells’ viability cultured in polyurethane scaffolds after drug and irradiation screening, which were realized using advanced microscopy and imaging [ 174 ]. Hence, it is crucial for researchers to carefully consider the appropriate analytical approach that aligns with their study objectives before commencing the analysis of any 3D cultures. Also, they must be aware that some of the classical gold-standard approaches used in 2D cultures may not be directly applicable in 3D settings, as Hamdi et al. [ 175 ] showed that it is unfeasible to extract cells from spheroids for colony formation assays, which are used for developing post-treatment survival curves. Consequently, the researchers suggested in situ characterization readouts, which are novel and/or different from the existing 2D culture protocols.

Using stem cells and differentiated markers is crucial for characterizing and monitoring the cellular composition and differentiation status within 3D spheroids. These markers can help researchers achieve specific goals and outcomes, such as assessing the differentiation potential of stem cells, tracking the progression of differentiation, and studying the dynamics of cell populations in the spheroids [ 176 , 177 ]. However, using such markers in 3D spheroid cultures presents certain challenges that need to be addressed for accurate and meaningful results. One primary challenge is the heterogeneity of stem cells within spheroids. Spheroids often comprise a mixture of stem cells and differentiated cells, so the stem cell markers may not exclusively identify and isolate the stem cell population, leading to difficulty in studying the specific behavior of stem cells within the spheroid. Another challenge is the variability in the expression of stem cell markers. These markers’ expression can fluctuate spatially and temporally within the spheroid, making it complex to track and interpret changes in marker expression over time. Additionally, in larger spheroids, stem cell markers may not effectively penetrate the core of the spheroid, limiting the ability to assess the stem cell population in the inner regions [ 176 , 177 ]. Researchers can employ several strategies to overcome these challenges and effectively use stem cell markers in 3D spheroid cultures [ 178 , 179 ]. An alternative method involves combining stem cells and other cellular markers to better understand the cellular composition within the spheroid. This multi-marker approach can help mitigate the issues related to marker heterogeneity. Moreover, live imaging techniques, such as confocal microscopy, can provide real-time insights into the dynamics of marker expression within spheroids. Controlling the size of spheroids is another strategy to enhance marker penetration and access to the innermost cells. Utilizing microfluidic techniques allows for the accurate regulation of spheroid size, ensuring effective penetration of markers throughout all regions of the spheroid [ 178 , 179 ]. Additionally, single-cell analysis methods, such as single-cell RNA sequencing and proteomic analysis, enable the characterization of individual cells within spheroids. This approach can identify unique gene or protein expression patterns and shed light on the behavior of stem cell populations. Another valuable strategy is creating spheroids with genetically encoded stem cell reporters, which produce fluorescent or luminescent signals in stem cells, making them more visible and trackable. Lastly, mimicking the stem cell niche or microenvironment within 3D culture conditions can help maintain stemness and marker expression in spheroids [ 179 ].

Although imaging provides valuable information about cell distribution and binding, quantitative measurements using image analysis in 3D cultures are often lacking because they require cell count consistency across samples [ 180 ]. The challenge lies in the inability to visualize the whole-cell population, leading to difficulties obtaining accurate and reliable data from the entire culture. This is due to the hampered diffusion of fluorescent markers, primarily due to their large size, governed by the inherent heterogeneity of 3D cultures. One potential solution is to measure cell number from imageable cross-sections; however, Sirenko et al. [ 181 ] noted that light interferences and dye diffusion limitations resulted in unreliable results, as the number of cells counted substantially differed from the number of cells seeded. In addition, technical limitations such as prohibitive costs and limited scalability must also be considered [ 149 ]. Implementing 3D culture systems may incur higher costs compared to 2D culture systems, attributed to the requirement for specialized equipment, materials, and expertise [ 182 , 183 ]. Similarly, scaling up 3D culture systems for industrial or clinical applications can be challenging due to the increased complexity of the culture environment and the need for specialized equipment [ 184 ]. This can limit the potential for the widespread adoption of 3D culture techniques in these settings.

Significant strides have been made in creating dynamic scaffolds that can respond to or guide resident cells [ 185 ]. For example, thermoresponsive hydrogels like poly-N-isopropylacrylamide (pNIPAm) have been proven effective for cell population harvesting [ 186 , 187 ]. Moreover, the fusion of microscale technologies for cell culture with adaptable hydrogel designs has facilitated various investigations. These include investigating cell migration within microfluidic hydrogels and establishing high-throughput screening platforms to explore interactions between cells and materials [ 188 ]. Notably, the mechanobiology field is intrigued by various mechanically dynamic hydrogels that can either stiffen, soften, or reversibly transition between these states to examine cellular responses. These dynamic substrates offer a means to scrutinize how mechanical cues influence cell behavior, similar to the study of soluble factors over decades [ 189 ]. Techniques for introducing heterogeneity and multiple cell types within 3D constructs are also advancing. This includes innovative methods where hydrogels serve as bio-inks to print cells, either layer-by-layer from a 2D base or directly within a 3D space enclosed by another hydrogel. As these platforms progress, they are expected to become more widely accessible [ 190 , 191 ]. In the interim, it remains crucial to maintain an open and collaborative dialogue between cell biologists, materials scientists, and engineers. This collaborative effort will ensure that the next generation of scaffold-based 3D cell culturing systems is well-equipped to address the significant challenges posed by the increasing biological and technical complexities.

To conclude, scaffold-based 3D cell culture has emerged as a valuable tool in cancer research, providing a more physiologically relevant environment for studying tumor behavior, drug responses, and interactions between cancer cells and the surrounding microenvironment. Various scaffold materials, including polymers, decellularized tissue, hydrogels, and hybrids with microfluidics, have been explored to create complex and biomimetic 3D models. Polymer-based scaffolds offer tunable mechanical properties and are relatively easy to fabricate, making them versatile for 3D cell culture. The choice of polymers can influence cell behavior, proliferation, and migration, allowing researchers to study cancer progression and metastasis in a more realistic context. Additionally, incorporating bioactive molecules into polymer scaffolds can enable the controlled release of drugs and growth factors, facilitating drug screening and targeted therapy development. Furthermore, hydrogels offer high biocompatibility and can be functionalized with bioactive signals to direct cell behavior and tissue formation. In cancer research, hydrogels provide a platform to investigate the effect of mechanical cues on tumor growth, immune cell infiltration, and angiogenesis. Additionally, the ease of incorporating multiple cell types within hydrogels enables the study of tumor-stroma interactions. Likewise, decellularized tissue scaffolds retain native ECM composition, topography, and mechanical properties, closely mimicking the natural tumor microenvironment. As a result, cancer cells cultured in decellularized tissue scaffolds can exhibit more accurate tumor behaviors, including invasion and angiogenesis. Moreover, these scaffolds can be derived from patient-specific tissues, enabling personalized medicine approaches and improving the predictability of drug responses. Lastly, hybrid scaffolds that integrate microfluidic channels offer unique advantages for cancer research. By combining 3D cell culture with microfluidics, researchers can study tumor angiogenesis, metastasis, and drug penetration in a more physiologically relevant manner. Furthermore, microfluidics can facilitate high-throughput screening of anticancer drugs, enabling rapid and cost-effective testing of potential therapies.

Data availability

Not applicable.

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Acknowledgements

The authors would like to acknowledge the financial support of the American University of Sharjah Faculty Research Grants, the Al-Jalila Foundation [AJF 2015555], the Al Qasimi Foundation, the Patient’s Friends Committee-Sharjah, the Biosciences and Bioengineering Research Institute [BBRI18-CEN-11], GCC Co-Fund Program [IRF17-003], the Takamul Program [P OC-00028-18], the Technology Innovation Pioneer (T IP) Healthcare Awards, Sheikh Hamdan Award for Medical Sciences [MRG-57-2019-2020], and the Dana Gas Endowed Chair for Chemical Engineering. We also would like to acknowledge student funding from the Material Science and Engineering Ph.D. program at AUS.

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Abuwatfa, W.H., Pitt, W.G. & Husseini, G.A. Scaffold-based 3D cell culture models in cancer research. J Biomed Sci 31 , 7 (2024). https://doi.org/10.1186/s12929-024-00994-y

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Cancer Cell Lines Are Useful Model Systems for Medical Research

Affiliations.

• 1 IRCCS SDN, 80143 Naples, Italy. [email protected].
• 2 IRCCS SDN, 80143 Naples, Italy.
• PMID: 31374935
• PMCID: PMC6721418
• DOI: 10.3390/cancers11081098

Cell lines are in vitro model systems that are widely used in different fields of medical research, especially basic cancer research and drug discovery. Their usefulness is primarily linked to their ability to provide an indefinite source of biological material for experimental purposes. Under the right conditions and with appropriate controls, authenticated cancer cell lines retain most of the genetic properties of the cancer of origin. During the last few years, comparing genomic data of most cancer cell lines has corroborated this statement and those that were observed studying the tumoral tissue equivalents included in the The Cancer Genome Atlas (TCGA) database. We are at the disposal of comprehensive open access cell line datasets describing their molecular and cellular alterations at an unprecedented level of accuracy. This aspect, in association with the possibility of setting up accurate culture conditions that mimic the in vivo microenvironment (e.g., three-dimensional (3D) coculture), has strengthened the importance of cancer cell lines for continuing to sustain medical research fields. However, it is important to consider that the appropriate use of cell lines needs to follow established guidelines for guaranteed data reproducibility and quality, and to prevent the occurrence of detrimental events (i.e., those that are linked to cross-contamination and mycoplasma contamination).

Keywords: biological samples; cell lines; leukemia; medical research; solid cancer.

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List of major breakthroughs in…

List of major breakthroughs in the historical progress of cancer cell lines.

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MSK Research Highlights, July 31, 2024

Wednesday, July 31, 2024

New research from Memorial Sloan Kettering Cancer Center (MSK) showed giving chemotherapy shortly after a stem cell or bone marrow transplant from a less than perfectly matched donor greatly reduces the chances that the patient will develop graft-versus-host disease (GVHD); and sheds new light on cell state changes in prostate cancer.

Improving transplant success in patients without fully matched blood stem cell donors

Stem cell and bone marrow transplants (BMTs) provide the possibility of a cure for certain types of blood cancer, including many cases of  leukemia and  lymphoma . But for those transplants that require blood stem cells from a donor — known as  allogeneic transplants — it can be hard to find an exact match. This is especially true for patients with certain genetic ancestries, especially African, Latin American, and Asian. Their tissues, or HLA types, are more diverse.

In a new retrospective study, MSK  bone marrow transplant specialist and cellular therapist Brian Shaffer, MD ,  and a multicenter team of investigators demonstrated how to improve transplants in patients who are not able to find fully matched donors. The study was overseen and funded by the  National Marrow Donor Program (NMDP). The research confirmed that for patients receiving only partially matched cells, giving chemotherapy shortly after the transplant greatly reduces the chances that the patient will develop  graft-versus-host disease (GVHD) , a potentially fatal side effect of BMTs that occurs when the donor’s immune system attacks the recipient’s healthy tissues.

The analysis included data from more than 10,000 BMT recipients, nearly half of whom received cyclophosphamide chemotherapy after their transplant. (The rest received anti-rejection drugs called calcineurin inhibitors, or CNIs.) Patients who received chemotherapy after their transplant had better outcomes than those who received CNIs. In fact, outcomes for patients with partially matched donors who received chemotherapy were almost as good as for those who had fully matched donors. An  ongoing clinical trial at MSK is now looking at whether lower doses of chemotherapy are as effective as higher doses at preventing GVHD and whether they reduce the side effects of chemotherapy.

Read more in the  Journal of Clinical Oncology .

Single cell analysis sheds light on cell state changes in prostate cancer

Cancer can resist treatment by  changing its identity , a transformation known as lineage plasticity. A notable example is when prostate adenocarcinoma, the most common type, transforms into neuroendocrine prostate cancer (NEPC), a more aggressive form. It is important to understand how this plasticity affects the expression of cell surface antigens – especially because promising new drugs are being tested that  target specific surface antigens . A team led by MSK  physician-scientist Charles Sawyers, MD , and  Michael Haffner, MD, PhD , of the Fred Hutchinson Cancer Center used immunohistochemistry and single-cell analysis of treatment-resistant prostate cancer and found a surprisingly high variety of cell states. This heterogeneity is not captured by conventional histology-based methods. They further showed that the cell states could be identified by gene regulatory networks that could improve diagnostic precision and help in the selection of patients who will respond to therapies aimed at specific cell surface antigens. Read more in the  Proceedings of the National Academies of Sciences .

National Cancer Institute - Cancer.gov

Dietary glutamine may be linked to B-cell lymphomas in abdominal lymph nodes

B cells migrating outside of the center of the B cell follicle in mice without GNA13 . Image provided by Jagan Muppidi, M.D., Ph.D.

Researchers have discovered that dietary glutamine, an amino acid found in many foods, may be linked to the development of certain kinds of B-cell lymphomas in abdominal structures called mesenteric lymph nodes in mouse models. The study appeared July 18, 2024, in Nature Immunology .

B-cell lymphoma is a common form of cancer of the lymphatic system. It can originate from B cells at different stages of activation. These diverse origins may contribute to how patients respond to therapies.

“One of the field’s bigger efforts over the last 20 years has been trying to define what targeted therapies might be effective in different forms of this common lymphoma,” said Jagan R. Muppidi, M.D., Ph.D. , Stadtman Investigator in the Lymphoid Malignancies Branch , and the lead investigator of the study.

The investigators focused on germinal center B cells. These cells are responsible for making antibodies in germinal centers, which are clusters of immune cells that form in lymph nodes. Patients with cancers that are derived from germinal center B cells commonly have loss of function mutations in a gene called GNA13 , which encodes the protein G-alpha-13. This protein typically inhibits migration of germinal center B cells.

The researchers used mouse models that lacked the G-alpha-13 protein in B cells and found that these mice tended to develop lymphoma from germinal centers within mesenteric lymph nodes in the abdomen. Additionally, they found gene expression studies showed the absence of G-alpha-13   led to increased activity of a protein called mTOR and increased expression of the protein MYC, both of which are involved in cell growth and proliferation, specifically in germinal center B cells in mesenteric lymph nodes.

“Part of the novelty of our study is that it provides evidence for different anatomic locations being linked to different biology in lymphoma,” said Muppidi. “At present, there are no comprehensive databases that link mutations in lymphoma to the sites of disease, so it isn’t clear whether people with tumors that have GNA13 mutations are more likely to develop disease in the mesenteric lymph nodes as the mice did.”

The researchers next explored possible mechanisms that could explain why the mice developed cancer in this particular location. Within the gut, the lymphatic system transports nutrients such as fats, proteins and vitamins from the intestines to the mesenteric lymph node. Since the G-alpha-13 protein typically confines germinal center B cells to the center of the B cell follicle, researchers hypothesized that these B cells in mice without G-alpha-13 could be migrating outside of the center of the cells and be exposed to substances in the lymphatic system that originated in the gut.

The researchers did discover that this was the case, and specifically found that diet-derived glutamine supported increased levels of MYC and mTOR activity in G-alpha-13-deficient germinal center B cells, leading to increased proliferation.

“We are interested in more completely defining the molecular pathway by which dietary glutamine and potentially other nutrients support the expression of MYC  in G-alpha-13-deficient cells,” Muppidi said. These findings are only applicable in mouse models today, but if extended into future human studies, this research could eventually identify therapeutic targets for G-alpha13-deficient lymphoma. Next, the researchers hope to learn whether alteration of dietary glutamine or other dietary modifications can suppress the progression of existing lymphoma in animal models. 

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New approaches and procedures for cancer treatment: Current perspectives

Dejene tolossa debela.

1 Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia

Seke GY Muzazu

2 Enteric Diseases and Vaccines Research Unit, Centre for Infectious Disease Research in Zambia (CIDRZ), Lusaka, Zambia

Kidist Digamo Heraro

3 Wachemo University, Hossana, Ethiopia

Maureen Tayamika Ndalama

Betelhiem woldemedhin mesele.

4 Kotebe Metropolitan University, Addis Ababa, Ethiopia

Dagimawi Chilot Haile

5 University of Gondar, Gondar, Ethiopia

Sophia Khalayi Kitui

Tsegahun manyazewal.

Cancer is a global health problem responsible for one in six deaths worldwide. Treating cancer has been a highly complex process. Conventional treatment approaches, such as surgery, chemotherapy, and radiotherapy, have been in use, while significant advances are being made in recent times, including stem cell therapy, targeted therapy, ablation therapy, nanoparticles, natural antioxidants, radionics, chemodynamic therapy, sonodynamic therapy, and ferroptosis-based therapy. Current methods in oncology focus on the development of safe and efficient cancer nanomedicines. Stem cell therapy has brought promising efficacy in regenerating and repairing diseased or damaged tissues by targeting both primary and metastatic cancer foci, and nanoparticles brought new diagnostic and therapeutic options. Targeted therapy possessed breakthrough potential inhibiting the growth and spread of specific cancer cells, causing less damage to healthy cells. Ablation therapy has emerged as a minimally invasive procedure that burns or freezes cancers without the need for open surgery. Natural antioxidants demonstrated potential tracking down free radicals and neutralizing their harmful effects thereby treating or preventing cancer. Several new technologies are currently under research in clinical trials, and some of them have already been approved. This review presented an update on recent advances and breakthroughs in cancer therapies.

Introduction

Cancer is a global health problem responsible for one in six deaths worldwide. In 2020, there were an estimated 19.3 million new cancer cases and about 10 million cancer deaths globally. Cancer is a very complicated sequence of disease conditions progressing gradually with a generalized loss of growth control. 1 – 3 There were only a few options of cancer treatment for patients for many decades which include surgery, radiation therapy, and chemotherapy as single treatments or in combination. 4 , 5 But recently, many pathways involved in cancer therapy progression and how they can be targeted has improved dramatically, with combinatorial strategies, involving multiple targeted therapies or “traditional” chemotherapeutics, such as the taxanes and platinum compounds, being found to have a synergistic effect. 6 New approaches, such as drugs, biological molecules, and immune-mediated therapies, are being used for treatment even if the excepted therapy level has not reached that resists the mortality rate and decreases the prolonged survival time for metastatic cancer.

The creation of a new revolution in neoplastic cancer or targeting drugs depends on the pathways and characteristics of different tumor entities. 7 Chemotherapy is considered the most effective and widely used modality in treating cancers as used alone or in combination with radiotherapy. Genotoxicity is how chemotherapy drugs target the tumor cells mainly producing reactive oxygen species that largely destroy tumor cells. 8 Hormonal treatments are also widely used for cancer malignancies and considered as cytostatic because it restricts tumor development by limiting the hormonal growth factors acting through the direction of hypothalamic–pituitary–gonadal axis (HPGA), hormone receptor blockage, and limiting of adrenal steroid synthesis. 9

In this narrative review, a general overview of the most advanced and novel cancer therapies was provided. In addition, also new strategies currently under investigation at the research stage that should overwhelm the drawbacks of standard therapies; different strategies to cancer diagnosis and therapy; and their current status in the clinical context, underlining their impact as innovative anti-cancer approaches.

Cancer treatment modalities

We can see cancer treatment modalities by dividing them into conventional (traditional) and advanced or novel or modern categories. In this era worldwide, over half of all ongoing medical treatment trials are focusing on cancer treatments. 7 Entities, such as the type of cancer, its site, and severity, guide to select treatment options and its progress. The most widely used traditional treatment methods are surgery, chemotherapy, and radiotherapy, while modern modalities include hormone therapy, anti-angiogenic, stem cell therapies, immunotherapy, and dendritic cell-based immunotherapy. 10

Conventional cancer therapies

The most recommended conventional cancer treatment strategies include surgical resection of the tumors followed by radiotherapy with x-rays and/or chemotherapy. 11 Of these modalities, surgery is most effective at an early stage of disease progression. Radiation therapy can damage healthy cells, organs, and tissues. Although chemotherapy has reduced morbidity and mortality, virtually all chemotherapeutic agents damage healthy cells, especially rapidly dividing and growing cells. 12 Drug resistance, a major problem with chemotherapy, is a phenomenon wherein cancer cells that initially were suppressed by an anti-cancer drug develop resistance to the drug. This is caused primarily by reduced drug uptake and increased drug efflux. 13 Limitations of conventional chemotherapeutic modality, such as dosage selection difficulty, lack of specificity, rapid drug metabolism, and mainly harmful side effects. 14

Advanced and innovative cancer therapies

Among the obstacles of cancer, drug resistance and its delivery systems are the most problem in cancer cure and decreasing signs and symptoms; but currently, there are many approved treatment approaches and drugs. The efficiency of conventional cancer is reduced due to tumor pathology and architectural abnormality of tumor tissue blood vessels. 15 The following are the advanced and innovative cancer therapy types with their benefits and challenges.

Stem cells therapy

Stem cells are undifferentiated cells present in the bone marrow (BM) with an ability to differentiate into any type of body cell. Stem cell therapeutic strategy is also one of the treatment options for cancer which are considered to be safe and effective. Application of stem cell is yet in the experimental clinical trial; for example, their use in the regeneration of other damaged tissue is being explored. Mesenchymal stem cells (MSCs) are currently being used in trials that are delivered from the BM, fat tissues, and connective tissues. 16

Pluripotent stem cells

Embryonic stem cells (ESCs) isolated from the uniform inner mass cells of the embryo possess the flexibility to administer rise to any or all kinds of cells except those within the placenta. In 2006, the invention of Yamanaka factors to induce pluripotent stem cells (iPSCs) from physical cells in a culture marked a breakthrough in cell biology. 17 Avoiding ethical issues from embryo destruction, iPSCs and ESCs have the same characteristics. Hematopoietic embryonic stem cells (hESCs) and iPSCs are currently used for the induction of effector T cells and natural killer (NK) cells, 18 and anti-tumor vaccine preparation. 19

Adult stem cells

Adult stem cells (ASCs) groups often used in tumor therapy include hematopoietic stem cells (HSCs), MSCs, and neural stem cells (NSCs). HSCs, located in BM, can form all mature blood cells in the body. Currently, only approved by the Food and Drug Administration (FDA) is the infusion of HSCs derived from cord blood to treat multiple myeloma and leukemia. 20 MSCs are found in many tissues and organs, playing important roles in tissue repair and regeneration into cells, such as osteocytes, adipocytes, and chondrocytes. MSCs have special biological characteristics and are used as complimentary with other approaches in treating tumors. 21 NSCs can self-renew and generate new neurons and glial cells and are used for treating both primary and metastatic breast and other tumors. 22

Cancer stem cells

Cancer stem cells (CSCs) are generated in normal stem cells or precursor/progenitor cells by the epigenetic mutations process. Their role in tumor treatment includes cancer growth, metastasis, and recurrence, so that it could give promise in the treatment of solid tumors. 23 Stem cells have several action mechanisms in treating the tumor. The homing process is one mechanism which is a rapid migration of HSCs into defined stem cell niches in BM after that the transplants undergo the engraftment process before giving rise to specialized blood cells. This mechanism is dependent on the active interaction between stem cell CXCR4 receptors and requires their interaction with endothelial cells through LFA-1, VLA-4/5, CD44, and the secretion of matrix degradable enzyme MMP-2/9. 22 The second mechanism is the tumor-tropic effect in which the migration of MSCs toward tumor microenvironment (TM) after attraction by CXCL16, SDF-1, CCL-25, and IL-6 secreted by tumor cells and differentiation of MSCs within the tumor cells which contributes to tumor stromal development. 24 Stem cells also act by paracrine factor secretion, including extracellular vesicles (EVs) and soluble materials, 25 and their differentiation capacity, such as transplanted HSCs, can give rise to all blood cell types. 26

Generally, cancer treatment using stem cell therapy by various strategies, including transplantation of HSC, 27 MSC infusion, 28 therapeutic carriers, 29 generation of immune effector cells, 30 and vaccine production. 31 The stem cell cancer therapy approach confronted the following side effects: (1) tumorigenesis, (2) adverse events in allogeneic HSC transplantation, (3) drug toxicity and drug resistance, (4) increased immune responses and autoimmunity, and (5) viral infection. 22 Despite several successes, there are challenges, such as therapeutic dose control, low cell targeting, and retention in tumor sites, that should be investigated and overcome in the future. In addition, existing results from stem cell technologies are highly encouraging for tumor treatment but it still needs further efforts to improve the safety and efficacy before they could enter clinical trials. Table 1 summarized the licensed list of stem cell therapies.

Licensed stem cell therapies.

AML: acute myelogenous leukemia; FDA: The US Food and Drug Administration; iPSC: induced pluripotent stem cell; MSC-INβ: mesenchymal stem cells with interferon beta.

Targeted drug therapy

Targeted cancer therapies are drugs or other substances which are sometimes interchangeably used as “molecularly targeted drugs,” “molecularly targeted therapies,” and “precision medicines.” Those drugs’ mechanism of action is by interfering with growth molecules which leads to blocking the growth and spreading of cancer. 34 Tumor initiation and progression are determined by the TM of an atypical tumor which comprises endothelial cells, pericytes, smooth muscle cells, fibroblasts, various inflammatory cells, dendritic cells, and CSCs. There are various signaling mechanisms and pathways that TM-forming cells dynamically interact with the cancerous cells which are suitable for sustaining a reasonably high cellular proliferation. So, it is the area of research interest using TM conditions to mediate effective targeting measures for cancer therapy. 35

Selectively treating cancer cells with conventional chemotherapy is difficult since it is similar to normal cells. So those problems are intervened by cellular mechanisms, such as cell cycle arrest, apoptosis induction, proliferation prevention, and interfering with metabolic reprogramming by targeted drug therapy agents. 36 Modifying TM and targeting TM for drug delivery for effective treatment are two strategies that can be used for the treatment of cancer. 37 Targeted therapy drugs do work in different ways from standard chemotherapy drugs treatment like attacking cancer cells while doing less damage to normal cells which is a programming that sets them apart from normal, healthy cells. 38

Using targeted therapy markedly increased the survival rate for some diseases, for example, from 17% to 24% in patients with advanced pancreatic cancer, the addition of erlotinib to standard chemotherapy. Imatinib has had a dramatic effect on chronic myeloid leukemia, and rituximab, sunitinib, and trastuzumab have revolutionized the treatment of renal cell carcinoma and breast cancer, respectively. 39

We can classify the targeted cell agents based on the mechanism of their work or their target site. Some enzymes serve as signals for cancer cells to grow. Some targeted therapies inhibit enzymes that are signals for cancer cells to grow. These drugs are called enzyme inhibitors. Blocking these cell signals can inhibit cancer from getting bigger and spreading. 40

Some targeted therapies are called apoptosis-inducing drugs because they are aimed right at the parts of the cell that control whether cells live or die. The examples are serine/threonine kinase, protein kinase B (PKB/Akt), which promotes cell survival, and inhibitors of this protein are in the preclinical phase. 41

These agents stop the tumors from making new blood vessels which helps cut off the tumors’ blood supply so that tumors cannot grow. In addition, they arrest tumor growth that involves by curtailing blood supply to the tumor by inhibiting angiogenic factors, such as vascular endothelial growth factor (VEGF) or its receptors. The study showed the survival of patients with advanced colorectal carcinoma extended by months after the use of Avastin (bevacizumab) in combination with 5-fluorouracil-based chemotherapy. 42

Types of target agents

Monoclonal antibodies.

Antibody drugs are man-made versions of immune system proteins administered intravenously to attack certain targets on cancer cells. They contain a more proportion of human components than murine components. 43 Their attack mechanisms of action are recruiting host immune functions to attack the target cell, binding to ligands or receptors thereby interrupting essential cancer cell processes, and carrying a lethal payload, such as radioisotope or toxin, to the target cell. 44 Gemtuzumab is an example of a CD-33-specific monoclonal antibody currently used for AML treatment by conjugating with calicheamicin. 45 In addition, ibritumomab tiuxetan is an anti-CD20, a 90Y metal isotope-based is developed in clinical therapy. 46 Delivery of active therapeutics, prodrug activation enzymes, and chemotherapy toxins are also another use of target agents of monoclonal antibodies. 47

Small molecule inhibitors

These are smaller protein in size (⩽500 Da) than monoclonal antibodies, so that they can simply translocate through plasma membranes and can be taken orally. Their main function is interrupting cellular processes by interfering with the intracellular signaling of tyrosine kinases which leads to the inhibition of tyrosine kinase signaling and initiates a molecular cascade that can lead to the inhibition of cell growth, proliferation, migration, and angiogenesis in malignant tissues. 48 Examples of small molecule inhibitors are gefitinib and erlotinib which inhibit epidermal growth factor receptor (EGFR) kinase and EGFR in non-small cell lung cancer (NSCLC) patients, respectively. There are also lapatinib and sorafenib which act on the inhibition of EGFR/Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2) for ERBB2-positive breast cancer and VEGFR kinase, in renal cancer. 49

Ablation cancer therapy

Ablation is a treatment technique that destroys tumors without removing them mostly indicated for small-size tumors of less than 3 cm and the surgical option is contraindicated. Ablation is also used with embolization for larger tumors. However, this technique might not be indicated for treating tumors near major blood vessels, the diaphragm, or major bile ducts due to destroying some of the normal tissue around the tumor. 50

Thermal ablation

This technique uses extreme hyperthermia or hypothermia to destruct tumor tissue concentrating on a focal zone in and around the tumor. Similar to surgery, thermal ablation removes the tumor and a 5–10 mm thick margin of seemingly normal tissue but the tissue is killed in situ and then absorbed by the body later. The procedure is similar to surgery using an open, laparoscopic, or endoscopic approach but is commonly applied using a percutaneous or non-invasive approach. The type of tumor, site, physician’s choice, and health status determine the approach. 51

Radiofrequency ablation (RFA), microwave ablation, high-intensity focused ultrasound, and cryoablation are currently being used in the clinical setting. Cryoablation uses a hypothermic modality to induce tissue damage by a freeze-thaw process against others. All these treatments operate on the principle of hyperthermia except cryoablation. Of all the ablation techniques, cryoablation demonstrated the highest potential to elicit a post-ablative immunogenic response. 52

Recent studies showed additional to tissue disruption RFA and cryoablation can modulate the immune system that they were applied as therapy on TM and in the systematic circulation. Evidence has shown that ablation procedures affect carcinogenesis due to its local inflammatory response leading to an immunogenic gene signature. 53

The advantage of this procedure over surgery is that it provides a minimal (e.g. percutaneously or laparoscopically) or non-invasive approach to cancer therapy and gets attention as an alternative to standard surgical therapies. 54

Cryoablation

Cryoablation is one of the ablation techniques which ablates the extensive tissue by freezing to lethal temperatures followed by liquid formation, causing extensive tissue. Benign and malignant primary tumors are mostly treated by this therapy. 55 James Arnott reported that the freezing temperatures can impair cancer cell viability after he attempted the usage of cold temperatures by salt and ice solutions for the generation of local numbness before surgical operations in the nineteenth century. He suggested cryoablation as an attractive therapeutic option and increased a patient’s survival. 56

Cryoablation techniques are based on the principle of the Joule–Thomson effect which was studied in the 1930s by many researchers and concluded using liquid CO 2 under high pressure, liquid air, and liquid oxygen to achieve the cooling effect and the subsequent formation of ice crystals so employed to treat lesions, warts, and keratosis. However, after 1950, Allington replaced liquid N 2 for the treatment of various skin lesion disorders. 47

RFA therapy

RFA is a minimally invasive procedure and an image-guided technique using hyperthermic (high-frequency electrical currents) conditions to destroy cancer cells. Imaging techniques, such as ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI), guide needle electrodes into a tumor cell. Generally, RFA is the most effective approach for treating small-size tumors of less than 3 cm in diameter. RFA can be used in combination with other conventional cancer treatment options. 57 After starting the use of deployable devices or multiple-electrode systems, RFA can treat medium tumors (up to 5 cm diameter). 58

Gene therapy

Gene therapy is the insertion of a normal copy of a defective gene in the genome to cure a specific disorder. The first application dates back to 1990 when a retroviral vector was exploited to deliver the adenosine deaminase (ADA) gene to T cells in patients with severe combined immunodeficiency (SCID). Approximately, about 2900 gene therapy clinical trials are currently ongoing, two-third of which are related to cancer. Strategies, such as expression of proapoptotic and chemosensitizing genes, expression of wild-type tumor suppressor genes, expression of genes able to solicit specific anti-tumor immune responses, and targeted silencing of oncogenes, are under evaluation for cancer gene therapy. 47

Thymidine kinase (TK) gene delivery is effective for the administration of prodrug ganciclovir to activate its expression and induce specific cytotoxicity. 59 The p53 tumor suppressor gene which is vectors carrying has been assessed for the clinical purpose very recently. ONYX-015 has been tested in NSCLC patients and gave a high response rate when given alone or combined with chemotherapy. 60 Gendicine, a recombinant adenovirus carrying wild-type p53-induced complete disease regression in head and neck squamous cell cancer had similar success when combined with radiotherapy. 61

Some challenges that have been faced with gene therapy are the selection of the right conditions and the choice of the best delivery mechanism. Identified drawbacks of this therapy are genome integration, limited efficacy in specific subsets of patients, and high chances of being neutralized by the immune system. Basic research and medical translation used RNA interference (RNAi) as an efficient technology that able to produce targeted gene silencing. 62 RNA-induced silencing complex (RISC) mediates the targeted gene silencing process by cleaving the messenger RNA (mRNA) and interference with protein synthesis. 63 A siRNAs can be designed to block desired targets, involving cell proliferation and metastatic invasion; hence, precise molecular mechanisms are a triggering factor for tumor formation. This method relies on siRNA-mediated gene silencing of anti-apoptotic proteins, transcription factors (i.e. c-myc gene), 64 , 65 or cancer mutated genes (i.e. K-RAS). 66

Advantages of siRNA-based drugs are safety, high efficacy, specificity, few side effects, and low costs of production. 67 However, occasionally, they can induce off-target effects or elicit innate immune responses, followed by specific inflammation. 68 Delivery methods currently under study are chemical modification (insertion of a phosphorothioate at 3’ end, introduction of a 2’ O-methyl group, and modification by 2,4-dinitrophenol) and lipid encapsulation, or conjugation with organic molecules (polymers, peptides, lipids, antibodies, small molecules) efficiently target to spontaneously cross cell membranes of naked siRNAs. 69 Interaction of cationic liposomes with negatively charged nucleic acids facilitates easy transfection by simple electrostatic interactions. 70 They can be constituted by 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and N-[1-(2,3-dioleoyloxy) propyl]-N, Ntrimethylammonium methyl sulfate (DOTMA). 71 Currently, a Phase I clinical trial is recruiting patients for evaluating the safety of Eph receptor A2 (EphA2) targeting 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) encapsulated siRNA (siRNA-EphA2- DOPC) in patients with advanced and recurrent cancer. 72 siRNAs can be concentrated in cationic polymers, such as chitosan, cyclodextrin, and polyethyleneimine (PEI). 73 CALAA-01 is one of the cyclodextrin polymers conjugated with human transferrin is being entered a Phase I clinical trial. PEI has been used as an anti-cancer by forming small cationic nanoparticles and loading with human epidermal growth factor receptor 2 (HER-2 receptor)-specific siRNA. 74 Phase II clinical trial has been started to evaluate Local Drug EluteR (siG12D LODER) directed to mutated Kirsten rat sarcoma (K-RAS) oncogene for the treatment of advanced pancreatic cancer. Conjugating to peptides, antibodies, and aptamers improves stability during circulation and enhances cellular uptake of siRNAs. 75 The introduction of nanocarriers has largely improved siRNAs stability, pharmacokinetics and biodistribution properties, and targeting specificity. Polyallylamine phosphate nanocarriers have been developed to release siRNAs in the cytoplasm after disassembly at low endosomal pH. 76

Dose correction and variabilities between individuals and different stages of disease are challenging issues on clinical translation of the siRNA-based approach. In the future, the needed research is on setting up the best-personalized therapy and toward controlled release to reach only specific targets on treating the tumor. Table 2 summarizes the gene therapy drugs based on their mechanism of action and induction.

Summary of gene therapy approaches.

Natural antioxidants

Day to day, the anatomy undergoes many exogenous insults, such as ultraviolet (UV) rays, pollution, and tobacco smoke, that end in the assembly of reactive species, particularly oxidants and free radicals, liable for the onset of many diseases, together with cancer. These molecules can even be made as a consequence of clinical administration of medication; however, they are additionally naturally created within our cells and tissues by mitochondria and peroxisomes, and from macrophages metabolism, throughout traditional physiological aerobic processes. 47

Oxidative stress and radical oxygen species can significantly change the regulation of transcription factors by damaging the DNA and other bio-macromolecule. 77

Vitamins, polyphenols, and plant-derived bioactive compounds are natural antioxidants used as preventive and therapeutic drugs against these molecules that damage the body due to their anti-inflammatory and antioxidant properties. 78 Studies added to cancer therapy after appreciating their anti-proliferative and proapoptotic properties. Compounds, such as vitamins, alkaloids, flavonoids, carotenoids, curcumin, berberine, quercetin, and others, are examples of natural antioxidants screened in vitro and in vivo. 79

Limited bioavailability and/ or toxicity is one of the challenges of natural drugs while their translation into clinical practice. 47 Curcumin has cytotoxic effects in different kinds of tumors, such as the brain, lung, leukemia, pancreatic, and hepatocellular carcinoma, 80 while sparing normal cells at effective therapeutic doses. The curcumin’s biological properties, treatment duration and efficient therapeutic doses are under study. 80 This day, about 27 clinical trials are done, while 40 are under study on curcumin.

Berberine is an alkaloid compound that has been studied to be effective against different cancers as a chemopreventive agent, modulating many signaling pathways. Different nanotechnological strategies have been developed to facilitate its delivery across cell membranes due to their poorly soluble in water. 81 Six clinical trials are under study and two have been completed.

Quercetin is another natural plant origin agent that is effective alone and also in combination with chemotherapeutic agents in treating many cancers, such as lung, prostate, liver, colon, and breast cancers. 82 Quercetin’s mechanism of action is by binding to cellular receptors and the interference of several signaling pathways. 83 Currently, six clinical trials are under study and seven studies have been completed.

Current clinical trials

In recent years, analysis of cancer medication has taken outstanding steps toward more practical, precise, and fewer invasive cancer treatments in the research of clinical trials ( Figure 1 ).

Current status of clinical trials of innovative and novel strategies of cancer therapy.

Currently, the most frequent entries focusing on cancer therapies in the database of clinical trials ( www.clinicalTrials.gov ) include the terminologies stem cell, targeted therapy, immunotherapy, and gene therapy because they are very promising and effective. Table 3 summarizes the potential advantages and disadvantages of the new treatment approaches.

Comparison of advantageous and disadvantageous of new cancer therapies.

MRI: magnetic resonance imaging.

Table 4 summarizes the approaches to advanced cancer therapies and their respective delivery systems with examples.

Advanced therapy approaches and delivery systems.

Current methods in oncology focus on the development of safe and efficient cancer nanomedicines. Targeted medical care helped rising the biodistribution of recent or already tested chemotherapeutical agents around the specific tissue to be treated; different methods, such as sequence medical care, siRNAs delivery, therapy, and inhibitor molecules, supply new potentialities to cancer patients. Gene therapy acts by direct in situ insertion of exogenous genes into benign tumors. Noticeably, stem cells can be used as regenerative medicine, therapeutic carriers, drug targeting, and generation of immune cells because of having unique biological actions on other cells. 22 On the opposite hand, thermal ablation and magnetic hyperthermia are promising alternatives to the growth surgical process. Finally, radionics and pathomics approaches facilitate the management of huge knowledge sets from cancer patients to enhance prognosis and outcomes. Much progress has been made, but many others are likely to come soon, producing more and more ad hoc personalized therapies. Further development and refinement of drug delivery systems are essential for improving therapeutic outcomes.

Acknowledgments

The authors thank the Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), Addis Ababa University, for the support rendered.

Author contributions: D.T.D. is a major contributor in writing the manuscript. All others reviewed and approved the manuscript.

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.

• Open access
• Published: 29 July 2024

Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma

• Ying Chen 1 , 2 , 3 ,
• Zhiyong Zhang 1 , 2 , 3 ,
• Fan Pan 3 , 6 ,
• Pengfei Li 1 , 2 , 3 ,
• Weiping Yao 3 , 6 ,
• Yuxi Chen 1 , 2 , 3 ,
• Lei Xiong 5 ,
• Tingting Wang 1 , 2 , 3 ,
• Yan Li 4 &
• Guichun Huang 6 , 7  

Journal of Experimental & Clinical Cancer Research volume  43 , Article number:  210 ( 2024 ) Cite this article

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Metrics details

It has been proposed that anti-angiogenesis therapy could induce tumor "vascular normalization" and further enhance the efficacy of chemotherapy, radiotherapy, target therapy, and immunotherapy for nearly twenty years. However, the detailed molecular mechanism of this phenomenon is still obscure.

Overexpression and knockout of CCL28 in human lung adenocarcinoma cell line A549 and murine lung adenocarcinoma cell line LLC, respectively, were utilized to establish mouse models. Single-cell sequencing was performed to analyze the proportion of different cell clusters and metabolic changes in the tumor microenvironment (TME). Immunofluorescence and multiplex immunohistochemistry were conducted in murine tumor tissues and clinical biopsy samples to assess the percentage of pericytes coverage. Primary pericytes were isolated from lung adenocarcinoma tumor tissues using magnetic-activated cell sorting (MACS). These pericytes were then treated with recombinant human CCL28 protein, followed by transwell migration assays and RNA sequencing analysis. Changes in the secretome and metabolome were examined, and verification of retinoic acid metabolism alterations in pericytes was conducted using quantitative real-time PCR, western blotting, and LC–MS technology. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to validate the transcriptional regulatory ability and affinity of RXRα to specific sites at the ANGPT1 promoter.

Our study showed that after undergoing anti-angiogenesis treatment, the tumor exhibited a state of ischemia and hypoxia, leading to an upregulation in the expression of CCL28 in hypoxic lung adenocarcinoma cells by the hypoxia-sensitive transcription factor CEBPB. Increased CCL28 could promote tumor vascular normalization through recruiting and metabolic reprogramming pericytes in the tumor microenvironment. Mechanistically, CCL28 modified the retinoic acid (RA) metabolism and increased ANGPT1 expression via RXRα in pericytes, thereby enhancing the stability of endothelial cells.

We reported the details of the molecular mechanisms of "vascular normalization" after anti-angiogenesis therapy for the first time. Our work might provide a prospective molecular marker for guiding the clinical arrangement of combination therapy between anti-angiogenesis treatment and other therapies.

Introduction

Lung cancer stands as the foremost cause of mortality among patients with malignant tumors, with lung adenocarcinoma being a predominant pathological subtype [ 1 , 2 ]. Angiogenesis is a crucial hallmark of lung adenocarcinoma, and significant strides have been made in applying anti-angiogenesis therapy for its treatment [ 3 ]. Extensive research data indicates that anti-angiogenic tumor therapy exhibits the potential to augment the effectiveness of diverse therapeutic modalities, including chemotherapy, radiotherapy, tyrosine kinase inhibitors (TKIs), and immunotherapy. Nevertheless, a subset of patients fails to derive benefits from the combined approach of anti-angiogenic therapy and other anti-tumor treatments. Unraveling the synergistic mechanisms underlying anti-angiogenic therapy holds substantial clinical implications for optimizing combination therapies.

Since the 2000s, there has been a fundamental shift in the understanding of anti-angiogenic therapy for tumors, transitioning from the initial concept of solely inhibiting angiogenesis to inducing vascular normalization in tumors [ 4 ]. Presently, there is a consensus that optimizing the effectiveness of additional anti-tumor treatments through anti-angiogenic therapy is imperative. This notion is central to anti-angiogenic drugs' capability to induce transient vascular normalization in tumor blood vessels [ 5 ]. Preliminary investigations undertaken by our research group propose that anti-angiogenic drugs, employing diverse mechanisms of action, can prompt vascular normalization in lung adenocarcinoma [ 6 , 7 , 8 , 9 ]. The balance between pro-angiogenesis and anti-angiogenesis factors was postulated as the key molecular base of vascular normalization [ 10 ]. However, uncertainties persist regarding the timing and the limited duration of vascular normalization post-treatment. Addressing this issue is critical, as the temporal aspects of vascular normalization and its short-lived nature need clarification [ 7 , 8 , 9 , 11 , 12 ]. The pressing clinical challenge involves establishing protocols to monitor and regulate vascular normalization in lung adenocarcinoma, offering valuable guidance for developing combined anti-angiogenic therapies with other anti-tumor treatment strategies. A comprehensive understanding of the molecular mechanisms underpinning anti-angiogenic therapy-induced vascular normalization in lung adenocarcinoma forms the foundational basis for resolving this intricate clinical dilemma.

Pericytes, one of the structural cells found in capillary vessels, envelop the basal membrane of endothelial cells and interact with vascular endothelial cells to ensure the stability and functionality of microvessels [ 13 , 14 ]. Commonly used markers for identifying pericytes include platelet-derived growth factor receptor β (PDGFRβ), chondroitin sulfate proteoglycan 4 (CSPG4, also known as NG2), and alpha-smooth muscle actin (α-SMA). Several reports suggest that platelet-derived growth factor B (PDGFB) can promote vascular normalization in colon cancer and malignant melanoma by recruiting pericytes [ 15 ]. However, PDGFB does not perform this function in pericytes in lung adenocarcinoma [ 16 ]. The mechanism of pericyte recruitment in lung adenocarcinoma following anti-angiogenesis therapy remains unclear.

Human CC motif chemokine ligands 28 (CCL28) can recruit various cell types, including lymphatic endothelial cells [ 13 ], T cells, and plasma cells, through its receptors CCR3 or CCR10. Apart from its chemotactic properties, CCL28 has been implicated in promoting tumor development in ovarian cancer [ 17 ], gastric cancer [ 18 ], liver cancer, and lung adenocarcinoma [ 19 ]. Under hypoxic conditions, tumor cells mobilize various cell types using different chemokines to induce angiogenesis in response to nutritional and oxygen requirements. Notably, hypoxia induces the expression of CCL28 in ovarian cancer, which, in turn, enhances angiogenesis by recruiting regulatory T cells (Tregs) [ 17 ]. In contrast, CCL28 has been identified as a negative regulator of tumor growth and bone invasion in oral squamous cell carcinoma [ 20 ]. These diverse findings suggest that CCL28 may play distinct roles in various tumors. While one study discovered that CCL28 increased the proliferation, migration, and secretion of IL-6 and HGF in oral fibroblasts [ 21 ], the functional role of CCL28 in pericytes in lung adenocarcinoma has not been elucidated. Furthermore, previous studies have reported that CCL28 was involved in angiogenesis in various diseases, including tumors, skin wound healing [ 22 ], and rheumatoid arthritis [ 23 ]. However, little is known about the mechanism of CCL28 in tumor vascular normalization.

Our previous report highlighted that CCL28 can moderately enhance the angiogenesis in lung adenocarcinoma [ 19 ]. Interestingly, we found a normalized vasculature in CCL28 highly expressed tumors. The current study delves into the previously undisclosed connection between pericytes and CCL28 in lung adenocarcinoma. Both in vivo and in vitro experiments collectively illustrate that CCL28 derived from tumor cells is pivotal in advancing vascular normalization by mobilizing and reprogramming pericytes.

Materials and methods

Cancer cell lines.

The human lung adenocarcinoma cell line (A549, SPC-A1, and H1975) and mouse Lewis lung cancer cell line (LLC) were purchased from the Shanghai Institute of Biochemistry and Cell Biology (SIBCB) and maintained in our lab. Lung cancer cells were cultured in RPMI-1640, or DMEM medium (Gibco, LifeTech, USA) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (100U/ml penicillin and 100 μg/ml streptomycin) at 37℃ in a humidified 5% CO 2 atmosphere.

Isolation and identification of pericytes

Pericytes were isolated and cultured as we previously described [ 24 ]. Fresh lung cancer samples were collected from patients in Jinling Hospital (Nanjing, China). Tissue samples were cut into small blocks of approximately 1 ~ 2 mm in diameter, digested with trypsin and 0.5% collagenase, and filtered through the cell strainer to obtain single-cell suspension. PDGFRβ + cells were isolated by PE-PDGFRβ antibody and anti-PE magnetic beads (Miltenyi Biotec,130–123-772). The separated cells were cultured in F12K medium (Gibco, LifeTech, USA) containing 10% fetal bovine serum (Gibco, Life Tech, USA) with 100 U/mL penicillin and 100 μg/mL streptomycin. After two or three passages, pericytes were identified by morphology and immunofluorescence staining for α-Smooth Muscle Actin (α-SMA, CST, #19,245), platelet-derived growth factor receptor β (PDGFRβ, Abcam, ab69506) and chondroitin sulfate proteoglycan 4 (NG2, Abcam, ab129051). Patients with incomplete data were excluded to evaluate the clinical effect. Written informed consent was obtained from all subjects before collecting the samples. All the methods followed the institutional guidelines and were approved by the Ethical Review Committee of Jinling Hospital, Nanjing, China(2022DZGZR-QH-005).

Hypoxic culture model and RNA sequence assay

As previously reported, the hypoxic cell culture model was established with hypoxic chambers in our lab [ 19 ]. Briefly, lung adenocarcinoma cells were cultured under two different oxygen concentrations, 1% and 20%, respectively. The model was testified by the expression changes of HIF-1α and its regulated genes, such as GLUT1 and VEGFA (Fig. 1 C). Pericytes were cultured under different stimulation. After culturing for 24 h, the cells were collected, and the total amount of RNA or protein was extracted for western blot or quantitative PCR. RNA sequence assay was applied to detect the gene expression differences of pericytes cultured with or without the stimulation of CCL28 (MCE, HY-P7250) under hypoxia.

CCL28 expression is upregulated after anti-angiogenesis therapy by hypoxia-sensitive transcription factor CEBPB in lung adenocarcinoma

RNA extraction and quantitative PCR

Total RNA was extracted from different cell lines using Trizol reagent (Invitrogen, USA). Subsequently, reverse transcription and quantitative PCR were performed using SYBR Green with an ABI StepOne Plus System (Applied Biosystems, Life Tech, USA). Relative gene expression was calculated by the ΔΔCt method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin levels. All reactions were run in triplicate.

Luciferase reporter assay

Wild-type (pGL3 wt, 5’-TGATTATGCAATGG-3') and mutant (pGL3 mut, 5’-ACTAATACGTTACC-3') promoters of the CCL28 gene were constructed into pGL3 firefly luciferase reporter plasmid vector purchased from Nanjing Realgene Bio-Technology Company (Nanjing China). The pRL-TK vector expressing renilla luciferase was used as an internal control. The reporter plasmid was co-transfected with pRL-TK vector and CEBPB expressing pcDNA3.1 vector or control vector into A549 cell. Dual-Luciferase Reporter Assay System (Promega, USA) was used to detect the luciferase activity after 48-h incubation.

Chromatin Immunoprecipitation (ChIP)

Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA. Subsequently, chromatin immunoprecipitation was conducted to enrich DNA fragments bound by chromatin proteins. After cross-link reversal and DNA purification, real-time quantitative PCR (qPCR) technology is employed to measure enriched DNA, determining the relative abundance at specific gene loci.

Liquid Chromatography-Mass Spectrometry (LC–MS)

Liquid chromatography-mass spectrometry was applied to measure retinoic acid (RA, HY-14649) in pericytes. Briefly, after treatment with or without CCL28, pericytes were subjected to enzymatic digestion, washed three times with PBS, flash-frozen in liquid nitrogen for one minute, and then stored at -80 °C. When performing the detection, begin by thawing the sample at 4 °C. Next, add 0.5 mL of methanol solution, followed by 10 min of sonication and 30 min of shaking for extraction. Subsequently, place the centrifuge tube in a low-temperature centrifuge and centrifuge for 10 min at 4 °C and 12,000 rpm. Collect 800μL of the supernatant, evaporate it, and then add 100μL of methanol solution. Finally, retain 80 μL of the supernatant for subsequent liquid chromatography-mass spectrometry analysis. A standard curve is generated to calculate the sample's content using a retinoic acid standard (yuanye Bio-Technology).

Single-cell RNA sequencing

Lewis lung cancer cells overexpressing CCL28 were inoculated in C57BL/6 mice subcutaneously (1 × 10 6 cells per mouse). When the tumor grew to about 100 mm 3 , fresh tumor samples were collected and dissociated into single cells. The tissue was washed with PBS, then chopped and incubated in an enzyme digestion solution for 40 min at 37 °C. After digestion, the solution was filtered through a 40 μm filter and centrifuged. Red blood cells were removed using a red blood cell lysis solution, followed by cell counting. The cell suspension was filtered using FACS tubes, centrifuged again, and washed twice. Finally, after microscopic examination and cell counting, the single-cell suspension was used for scRNA sequencing.

The scRNA sequencing analysis was carried out according to a single cell analysis workflow with BD Rhapsody™ Systems (BD Biosciences, USA), and RNA sequencing and data analysis were completed on the platform of NovaSeq 6000 system (Illumina, USA). The entired scRNA sequencing workflow was provided in the Supplementary document 1.

Animal model

Six-week-old female BALB/c nude mice and C57BL/6 female mice were used for tumor model establishment. LLC (1 × 10 6 per mouse) with or without overexpressing CCL28 were subcutaneously injected in C57BL/6 mice ( n  = 6, each group). Mice inoculated with CCL28 knock-out LLC cells and wild-type LLC were randomly divided into two groups, respectively, and treated with or without RA by gavage daily (n = 6, each group). Mice were observed, and the tumors' length and width were measured daily. When tumors grew to a specific size, subcutaneous tumors were collected and photographed. The A549 (CCL28 overexpression or CCL28 knock-out) tumor model followed the same procedure. All animal experiments were carried out following the institutional guidelines and approved by the Ethical Review Committee of Comparative Medicine, Jinling Hospital, Nanjing, China (2022DZGKDWLS-0091).

Western blot and ELISA

Proteins were extracted from the cultured cells by lysis buffer, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, USA). The filters were blocked in Tris-buffered saline containing 0.2% Tween plus 5% non-fat milk and incubated with primary antibodies overnight at 4 °C. Secondary antibodies were used for visualization through chemiluminescence (ECL, Amersham Pharmacia Biotech, UK). Primary antibodies against RXRα (CST,1:1000), RARα (CST,1:1000), RDH13 (Proteintech,1:1000), DHRS11 (Proteintech,1:1000), CCR10 (Abmart,1:1000), β-actin (Servicebio, 1:1000) and neutralizing antibody against CCR3 (R&D Systems, USA) were applied in the present study. Anti-rabbit IgG, HRP-linked Antibody (CST, 1:1000) and anti-mouse IgG, HRP-linked Antibody (CST, 1:1000) were utilized as the secondary antibodies. CCL28 in serum was detected by an ELISA kit (Abcam, USA) according to the manufacturer's instructions.

Immunofluorescence and multiplex Immunohistochemistry (mIHC)

Tumor samples were collected and fixed in 10% formalin before processing and paraffin embedding. Immunofluorescence was conducted on 5 µm sections. For culture cell staining, lung adenocarcinoma-associated pericytes were washed with 1 × PBS and fixed with acetone for 10 min on ice. Tumor microvascular endothelial cells and cancer pericytes were stained by CD31 antibody (Abcam, ab281583) and rat anti-human/mouse monoclonal NG2 antibody (Abcam, ab129051). At the same time, tumor cells were stained by mouse anti-human pan-cytokeratin (Pan-ck) monoclonal antibody (Abcam, ab215838). Goat anti-mouse IgG antibody (labeled with Alex Fluor 488), goat anti-rabbit IgG antibody (labeled with Alex Fluor 555 or Alex Fluor 488), and goat anti-rat IgG antibody (labeled with Alex Fluor 555 or Alex Fluor 488) (Abcam) were applied as secondary antibody in immunofluorescence staining analysis.

For multiplex immunohistochemistry, the slides were incubated with the primary antibodies (anti-CD31, anti-NG2, anti-Pan-CK antibody, and anti-CCL28 (Proteintech, 18,214–1-AP), respectively) and horseradish peroxidase-conjugated secondary antibody, and tyramine signal amplification (TSA) was performed following the pre-optimized antibody concentration and the order of staining. Antibody stripping and antigen retrieval were performed after each round of TSA. DAPI (Sigma-Aldrich, USA) was used for nuclei staining. A whole slide scan of the multiplex tissue sections produced multispectral fluorescent images visualized in SlideViewer software. A specialized pathologist chose representative regions of interest (ROI), and multiple fields of view were acquired at 20 × power for further analysis. The mean fluorescence intensity of CCL28 in each ROI was calculated by the ImageJ program ( https://imagej.nih.gov/ij/ ).

CRISPR-cas9 knock out

Briefly, three designed sgRNAs were synthesized and co-transfected with Cas9 nuclease/sgRNA in A549 lung adenocarcinoma cells. After 72 h, the cells were seeded in a 96-well plate for single-cell cloning by limiting dilution analysis (LDA). After 7 to 15 days, about ten single-cell clones were selected for PCR and ELISA. Mutant sites were further confirmed by DNA sequencing.

Knockdown of RDH13 and DHRS11

For RDH13 and DHRS11 gene silence, pericytes were infected with negative control siRNAs, RDH13-targeting, or DHRS11-targeting siRNAs (genepharma). Briefly, pericytes were seeded in 24-well plates, and the cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions.

Bioinformatic analysis

Transcription factor binding sites prediction in promotor of CCL28 gene was conducted in PROMO 3.0, virtual laboratory TFSEARCH (ver.1.3) and JASPAR ( https://alggen.lsi.upc.edu/rerecer/menu_recerca.html , https://diyhpl.us/~bryan/irc/protocol-online/protocol-cache/TFSEARCH.html , https://jaspar.elixir.no/ ). RNA sequencing dates of lung adenocarcinoma tumor samples or lung adenocarcinoma cell lines were extracted on the cBioPortal platform ( https://www.cbioportal.org/ ). The relationship between expression levels of CEBPB and CCL28, RXRα, and ANNGPT1 were calculated online, as well as the predictive value of CEBPB for the survival of lung adenocarcinoma patients.

Statistical analyses

Data were presented as mean ± SEM. Student's unpaired two-tailed tests were used for comparisons between two groups. Pearson or Spearman correlation was applied to analyze the relationship between the expression scores of two genes. Statistical analyses were performed on GraphPad Prism 8.0. Multiple comparisons were analyzed by one-way ANOVA using the LSD test. Statistical significance was confirmed when p  < 0.05.

CCL28 expression is up-regulated after anti-angiogenesis therapy by hypoxia-sensitive transcription factor CEBPB in lung adenocarcinoma

The GEO database ( https://www.ncbi.nlm.nih.gov/gds/ ) was retrieved to elucidate the impact of anti-angiogenesis therapy on the gene expression of cancer patients. Molecular expression profile data (GSE61676) of lung adenocarcinoma patients undergoing anti-angiogenesis treatment was reanalyzed. Twenty-three stage IV lung adenocarcinoma patients, treated with a combination of anti-VEGF monoclonal antibody (Bevacizumab) and TKI, were selected for analysis. The changes in mRNA profile (Affymetrix Human Exon 1.0 ST Arrays) were examined before and 24 h after Bevacizumab treatment. The findings revealed a significant increase in the hypoxia index (GLUT1, one of the primary target genes regulated by hypoxia-inducing factor HIF-1) and up-regulation of CCL28 expression in lung adenocarcinoma patients 24 h after VEGF monoclonal antibody treatment compared to baseline levels (Fig. 1 A, 1B). After treatment with bevacizumab, CCL28 expression showed a log2 fold change of 1.258 compared to baseline, with a corresponding p-value of 0.0152, as depicted in Fig. 1 A. After anti-angiogenesis therapy, tumor cells often experience a state of ischemia and hypoxia. To simulate this hypoxic state of tumor cells, nine cell lines of 4 different tumor types were culture in hypoxia and normoxia conditions, including two hepatoma cell lines (HEPG2 and SMMC), three lung adenocarcinoma cell lines (A549, SPC-A1, and H1975), two breast carcinoma cell lines (MCF7 and MDA-MB-231), and two colorectal cancer cell lines (HCT116 and SW480). The expression of GLUT1 and VEGFA, which was driven by the hypoxia-induced factor (HIF), was detected by qRT-PCR. VEGFA and GLUT1 were up-regulated in all nine cell lines in the hypoxic chamber, indicating that the hypoxic microenvironment was established (Fig. 1 C). We previously reported that high expression of CCL28 was induced in hypoxic lung adenocarcinoma cell lines, A549 and SPC-A1. To further confirm our study, we examined the expression of CCL28 under hypoxia conditions in the nine cell lines. CCL28 was up-regulated in all three lung adenocarcinoma cell lines, whereas others were not (Fig. 1 D). Then, we utilized PROMO 3.0 to predict the transcription factors binding sites to the CCL28 promoter region and intersected them with previously reported hypoxia-related transcription factors, revealing six transcription factors involved (Fig. 1 E). CEBPB and Sp1 can potentially regulate CCL28, while only CEBPB was highly expressed in hypoxic tumor cells (as listed in Supplementary Table 1). We then used a public integrative database (cBioPortal) to analyze the correlation of the expression of CEBPB with that of CCL28 in 44 lung adenocarcinoma cell lines (as listed in Supplementary Table 2). As expected, CEBPB expression strongly correlated with CCL28 (Fig. 1 F). The CEBPB binding motif was shown in Fig. 1 G. Thus, we speculated that hypoxia-induced high expression of CCL28 was mediated by CEBPB. To verify our prediction, we generated luciferase reporter plasmids harboring either wild-type (WT) or mutated (MUT) CEBPB binding sequencing within the promotor element of the CCL28 gene. Luciferase reporter analysis indicated that CEBPB could directly regulate the expression of the CCL28 gene in A549 cells (Fig. 1 H). In addition, disease-free survival and overall survival of lung adenocarcinoma patients with high CEBPB expression were significantly decreased ( p  = 0.0065 and p  = 0.032, respectively) (Fig. 1 I).

Tumor-derived CCL28 promotes vascular normalization and pericyte recruitment in the tumor microenvironment

To investigate the effects of CCL28 on tumor growth and vascular normalization, we established CCL28 overexpression and knock-out lung adenocarcinoma cells by lentivirus vectors and Cas9 nuclease/sgRNA (Supplementary Fig. 1), respectively. As we previously reported, in vivo studies indicated that CCL28 could promote tumor growth in A549 human lung adenocarcinoma (Fig. 2 A). Microvessels and pericytes were subsequently assessed by immunofluorescence staining using antibodies against CD31 (red) and NG2 (green) in tumor tissue in different groups. The percentage of pericyte coverage and microvessel density (MVD) in A549-CCL28 tumor tissue significantly increased compared to A549-NC, and this effect was reversed after CCL28 was knocked out (Fig. 2 B). These results suggested that CCL28 could promote vascular normalization and mobilize pericytes.

Tumor-derived CCL28 recruits pericytes to promote vascular normalization in the tumor microenvironment

Further, we isolated primary pericytes from lung cancer tissues to explore how CCL28 recruits pericytes in vitro. The characteristics of pericytes were identified by immunofluorescence staining α-SMA, PDGFRβ, and NG2 (Fig. 2 C). Cell viability remained unaffected by different concentrations of recombinant CCL28 in pericytes and A549 (Supplementary Fig. 2A,2B), but enhanced cell migration of A549 (Supplementary Fig. 2C). The transwell migration assay was performed to confirm the effect of CCL28 on the migration of pericytes by using different concentrations of human recombinant CCL28 protein (Fig. 2 D, left panel). The number of cells migrated to the lower chamber was calculated (Fig. 2 D, right panel), and we found that the optimal concentration of CCL28 for enhancing the migration efficiency of pericytes is 250 ng/ml. Thus, we selected a concentration of 250 ng/ml to conduct subsequent migration experiments. To further verify the recruitment effect of CCL28 on pericytes, cells were inoculated around matrigel mixed with or without CCL28 recombinant protein. The graphs visually illustrate cell migration distance and quantity (Fig. 2 E, left panel). CCL28 significantly increased the migration distance of pericytes compared to the control group (Fig. 2 E, right panel). Subsequently, we assessed the expression of two CCL28 receptors, CCR3 and CCR10, in pericytes with or without CCL28. Western blot experiments revealed that the expression of CCR3 was significantly higher than that of CCR10 in pericytes, suggesting that CCR3 likely plays a predominant role in mediating CCL28 signal transduction within pericytes (Fig. 2 F). Blocking CCR3 with neutralizing antibodies diminished the chemotactic effect of CCL28 on pericytes (Fig. 2 G). Further, we validated the relationship between CCL28 and pericytes coverage in biopsy tissue using multiplex immunofluorescence. CCL28 was mainly expressed in lung adenocarcinoma cells due to the fluorescence overlapping between CCL28 and PAN-CK (Fig. 2 H). We found a significant positive correlation between CCL28 expression levels and the percent of pericytes coverage (Fig. 2 I). The above findings indicate that CCL28 could recruit pericytes through the receptor CCR3, thereby promoting vascular normalization. Because the action of CCL28 leads to vascular normalization, making the vascular network healthier and more regular, it may also increase the density and functionality of tumor blood vessels, providing more nutrients and oxygen to promote tumor growth.

Tumor-derived CCL28 promotes expression of angiopoietin-1 via CCR3 in pericytes

Pericytes interact with vascular endothelial cells to promote the maturation of neovasculature [ 25 ]. However, the molecular mechanisms underlying pericytes-induced vascular normalization in lung adenocarcinoma after anti-angiogenesis therapy remain unclear. To clarify the exact role and potential mechanism of CCL28 in vascular normalization, we mimicked the hypoxic microenvironment in lung adenocarcinoma, cultured vascular pericytes, and conducted secretome analysis after CCL28 treatment. We detected augmented production of angiopoietin-1 in pericytes stimulated by CCL28 (Fig. 3 A). Compared to the control group, the expression of ANGPT1 changed by 5.415-fold under CCL28 stimulation, with a p-value of 0.033. RNA-seq results were confirmed by quantitative PCR in different concentrations of recombinant CCL28 protein under hypoxia conditions (Fig. 3 A right panel). Angiopoietin-1, a protein typically associated with angiogenesis and vascular normalization, maintains vascular stability and integrity by interacting with endothelial cells. Consistent with prior studies [ 24 , 26 ], western blot experiments showed that recombinant human angiopoietin-1 could up-regulate endothelial nitric oxide synthase (eNOS) expression in a dose-dependent manner in human umbilical vein endothelial cell (HUVEC) (Fig. 3 B). Likewise, recombinant human angiopoietin-1promoted cell survival by activating the PI3K-AKT signaling pathway in a Tie2-dependent manner in endothelial cells (Fig. 3 C). To further explore the regulatory mechanism of ANGPT1 expression, we identified a significant increase in the transcription factor RXRa and RARα after CCL28 stimulation in transcriptome data, verified by qPCR (Fig. 3 D and E ). However, protein level detected by western blot, CCL28 up-regulated RXRα and CCR3 but not RARα (Fig. 3 F). Also, the effect of CCL28 on RXRα up-regulation was reversed by adding a CCR3 neutralizing antibody (Fig. 3 G). Next, we confirmed the relation between transcription factor RXRα and expression of ANGPT1. Correlation analysis showed that RXRα expression positively correlates with ANGPT1 expression (Fig. 3 H). The binding motif of the RARα and RXRα complex (Fig. 3 I) and the transcription factor binding sites in the promotor area of the CCL28 gene (Fig. 3 J) were shown. CHIP-qPCR was performed to investigate the binding of transcription factor RXRα to the promoter region of the target gene ANGPT1 . Compared to the NC group, CCL28 enhanced the degree of enrichment, and this effect could be dampened after the CCR3 neutralization, suggesting a direct interaction between RXRα and the ANGPT1 promoter. The electrophoresis image displayed the specificity of the amplified DNA fragments (Fig. 3 K).

Tumor-derived CCL28 promotes the expression of angiopoietin-1 via CCR3 in pericytes

Furthermore, the mRNA level of ANGPT1 was increased by the addition of CCL28 but attenuated by the blockade of CCR3 (Fig. 3 L). RXRα is a subtype of nuclear receptor closely associated with vitamin A metabolism and retinoic acid signaling. These results led us to speculate whether retinoic acid metabolism was also involved in regulating vascular normalization by CCL28. Thus, we measured the retinoic acid content in pericytes using LC–MS technology. Interestingly, the retinoic acid production was notably increased (almost 2.5-fold change) compared to the control group in pericytes stimulated by CCL28 (Fig. 3 M). Additionally, the exogenous addition of retinoic acid in pericytes could promote the expression of ANGPT1 in a dose-dependent manner (Fig. 3 N). These results indicate that CCL28 can activate the nuclear transcription factor RXRα through the CCR3 receptor, promoting the expression of ANGPT1 in pericytes. In interaction with endothelial cells, pericytes-deriore, the mRNA level of ANGPT1 was increased by the addition of CCL28 but attenuated by the blockade ofon.

CCL28 activates retinoic acid signaling in pericytes through CCR3

We further analyzed the sequencing data to understand better the mechanism responsible for retinoic acid accumulation in pericytes after CCL28 stimulation. Enrichment analysis of gene pathways showed that RA signaling pathways were activated after CCL28 stimulation (Fig. 4 A). Two key enzymes involved in retinoic acid metabolism have changed, including RDH13 and DHRS11 (Fig.4B). RDH13 plays a role in converting retinol to retinaldehyde, a critical rate-limiting enzyme step in the retinoid metabolic pathway. However, DHRS11 reduces retinoic acid metabolites to related forms of retinol. In this way, the balance between RDH13 and DHRS11 can affect the level of retinoic acid in cells. The metabolic balance model diagram of retinoic acid shows the transformation process of the three substances (Fig. 4 C). In this metabolic process, RDH13 was up-regulated, but DHRS11 was decreased in pericytes after treatment of CCL28 (Fig. 4 B). These results were confirmed by qPCR (Fig. 4 D) and western blot (Fig. 4 E). The correlation analysis exhibited a positive relationship between RDH13 and CCL28 in lung adenocarcinoma (Fig. 4 F). Expression changes of RDH13 and DHRS11 in pericytes stimulated by CCL28 could be reversed by blocking CCR3 (Fig. 4 G). However, treating pericytes directly with retinoic acid did not result in changes in key molecules involved in RA metabolism (Supplementary Fig. 3 A). To further investigate whether the expression of two key enzymes in the retinoic acid synthesis process is correlated with the expression of ANGPT1, we knocked down the expression of RDH13 and DHRS11 using siRNA and then examined the expression of ANGPT1. After knockdown of RDH13 (Fig. 4 H), RXRα and ANGPT1 were significantly reduced (Fig. 4 I, J). However, DHRS11 did not exhibit this effect (Supplementary Fig. 3B, 3C). To further elucidate the relationship between CCL28 and its downstream molecules, immunofluorescence staining of key molecules—RDH13, DHRS11, and ANGPT1—was performed on tumor biopsy tissues (Fig. 4 K, left panel), followed by correlation analysis. In these samples, we observed a positive correlation between CCL28 expression and the levels of ANGPT1 and RDH13, while a negative correlation was found with DHRS11 (Fig. 4 K, right panel).All these results illuminated that CCL28 promoted the retinoic acid synthesis process by disturbing the balance between RDHs and DHRS, but the changes are temporary and dynamic.

Retinoic acid signaling is activated by CCL28 in pericytes through CCR3 A , Volcano plot of changes in metabolic pathways after CCL28 stimulation. B Volcano plot of the enrichment of gene expression after CCL28 stimulation. C Diagram of the metabolic conversion process in the retinoic acid metabolic signaling pathway. D and E Expression of RDH13 and DHRS11 detected by qPCR and western blot with or without exogenous supplement of CCL28. F Correlation of expression of CCL28 with RDH13 in lung adenocarcinoma. G The protein level of DHRS11 and RDH13 stimulated with or without CCL28 and CCR3 neutralizing antibody in pericytes (left) and gray value was calculated(right). H Knockdown efficiency of RDH13 was confirmed by qPCR. I and J Relative expression of RXRα and ANGPT1 after knockdown of RDH13 with or without stimulation of CCL28. K Representative immunofluorescence images of PAN-CK, NG2, CCL28 with DHRS11 or RDH13 or Angiopoietin-1 on biopsy tissues from lung cancer patients (left panel). Scale bar = 100 μm. The correlation between the expression of CCL28 and the levels of DHRS11, RDH13, and angiopoietin-1 (right panel). Data with error bars are shown as mean ± SEM. Each symbol represents data from a replicate. Each panel is a representative experiment of at least three independent biological replicates. *, **, *** represent p  < 0.05, p  < 0.01 and p  < 0.001, respectively. Abbreviation: MFI, Mean fluorescence intensity

Both CCL28 and retinoic acid could promote vascular normalization in vivo

Further, we investigated the role of CCL28 in tumor growth and vascular normalization in immunocompetent mice. We overexpressed CCL28 in LLC cells, which was validated by ELISA. Compared to the control group, CCL28 overexpression showed a significant increase in CCL28 concentration in both the cell supernatants and serum in mice implanted with tumors (Supplementary Fig. 4A, 4B). As expected, in vivo studies indicated that CCL28 could promote tumor growth in Lewis lung adenocarcinoma (LLC) (Fig. 5 A). Compared to the control group, NG2 + pericytes accumulation and angiogenesis enhanced in the LLC-CCL28 group (Fig. 5 B).

To more clearly explore the function of CCL28 on stromal cells and immune cells in the tumor microenvironment, we performed single-cell sequencing analysis on tumor tissues of LLC-NC and LLC-CCL28. Single-cell sequencing analysis provided a panoramic study of the tumor microenvironment, and the cell population in the tumor microenvironment underwent significant alterations. In total, 6380 cells were obtained after quality filtering in the 2 conditions. Specifically, there were 3829 cells in the CCL28 overexpression group, and 2551 cells in the control group. The t-SNE diagrams showed that these cells were divided into 19 cell clusters with a total of 10 cell types (Fig. 5 C and Supplementary Fig. 4D, 4E). Of all the cell types, three types showed the most significant changes in cell proportions: fibroblasts, pericytes, and macrophages, with fold changes of 2.39, 1.95, and 1.45, respectively. The percentage of pericytes was 1.09% in LLC-NC and rose to the rate of 2.14% in LLC-CCL28 (Fig. 5 D). In the CCL28 overexpressing group, we identified 82 pericytes, whereas the control group had 28 pericytes. This significant difference suggested that CCL28 may play a pivotal role in influencing pericyte populations within the tumor microenvironment. Moreover, a previous study has reported that CCL28 could recruit Tregs and promote angiogenesis in ovarian carcinoma [ 17 ]. However, the percentage of T cells was reduced in the CCL28 up-regulated LLC, indicating that CCL28 might play different roles in different cancer types. Moreover, the number of DC cells remains unchanged, but their proportion decreased (Fig. 5 D).

Subsequently, we focused on the analysis of pericytes. Cluster 14, which was CSPG4 + and ACTA2 + , was recognized as pericytes. The marker genes t-SNE plot for Cluster 14 was presented in Supplementary Fig. 5. Interestingly, metabolic pathway enrichment analysis revealed that the retinol metabolism was activated in 13 clusters, including DC, macrophages, fibroblasts, and pericytes, suggesting that the synthesis and metabolism of retinoic acid play essential physiological roles in tumor microenvironment (Fig. 5 E). In the cellular communication network, pericytes interact closely with various cell types. The heat map indicated that pericytes exhibited the strongest interaction with themselves, followed by cluster 3 and cluster 6, representing tumor and endothelial cells. Cluster 3 was characterized as Ki-67-positive and Top2a-positive, and cluster 6 was positive for Pecam (Fig. 5 F). Our results in Fig. 3 M also suggested CCL28 can promote retinoic acid accumulation in pericytes in vitro. We further investigated the effects of retinoic acid by daily gavage on tumor growth and vascular normalization in vivo. Knocking out CCL28 (LLC-CCL28-KO) or supplementing retinoic acid (LLC-NC + RA) could suppress tumor growth in LLC models (Fig. 5 G and Supplementary Fig. 4C). However, the suppression effect is more pronounced after knocking out CCL28 than the control group (LLC-NC), and CCL28 knockout with supplementing RA could further synergistically inhibit tumor growth (Fig. 5 G). Multiplex immunofluorescence was used to detect the normalization of tumor blood vessels. We found that compared to the control group, knocking out CCL28 resulted in a significant reduction in the proportion of NG2 + cells and decreased coverage of pericytes. As expected, supplementation of retinoic acid was able to promote a certain degree of restoration of pericytes coverage both in wild-type and CCL28 knockout tumors (Fig. 5 H). Then, we quantified the vascular density in different groups and found that, compared to the control group, CCL28 knockout resulted in significantly decreased vascular density, while retinoic acid had only a slight effect (Fig. 5 H). Additionally, we evaluated the effect of retinoic acid on tumor cell viability using a CCK8 assay (Supplementary Fig. 4F). The results indicated that retinoic acid did not significantly affect tumor cell viability at pharmacological concentrations (1–2 μg/ml, equivalent to 3.33–6.66 μM). However, cytotoxic effects were observed at higher concentrations, which exceed pharmacologically relevant levels in the body necessary to support essential cellular functions. To confirm the influence on tumor hypoxia, we immunofluorescently labeled mouse tumor tissues using CA9 (Carbonic Anhydrase 9), which is considered an indicator of hypoxia. Our findings indicated that compared to the control group, tumor hypoxia significantly increased in tumors with CCL28 knockout. However, retinoic acid does not appear to have a significant effect on hypoxia (Supplementary Fig. 4G). These results indicated that RA inhibited tumor growth and enhanced vascular normalization, rather than angiogenesis.

The above data indicate that CCL28-regulated retinoic acid plays a crucial role in vascular normalization, suggesting its potential as a therapeutic agent in anti-angiogenic tumor treatment. To investigate whether the combination of retinoic acid and bevacizumab has a synergistic effect, we established an A549 mouse model and administered intravenous injections of bevacizumab, coupled with oral gavage of retinoic acid. We observed that retinoic acid and bevacizumab could attenuate tumor growth in mice, respectively. Moreover, simultaneous administration of retinoic acid and bevacizumab had a synergistic effect on tumor growth (Fig. 5 I). These data indicate that retinoic acid can be used in combination with bevacizumab for tumor treatment, providing a new direction for clinical combination therapy.

CCL28 is involved in bevacizumab-mediated vascular normalization

Moreover, we investigated the synergistic effects of CCL28 knock-out and VEGF blocking on the tumor growth and vascular normalization of lung adenocarcinoma. Subcutaneously implanted lung adenocarcinoma cells with CCL28 knock-out grew much slower than wild-type tumor cells, while combination of VEGF blocker could stop the growth of the tumors (Fig. 6 A). In addition, significant promotion of vascular normalization is observed after bevacizumab treatment. However, this effect disappeared after knocking out CCL28, regardless of whether bevacizumab was added (Fig. 6 B). Knocking out CCL28 could inhibit tumor angiogenesis, and the effect was more pronounced when bevacizumab was combined (Fig. 6 B). These results indicated that CCL28 could participate in bevacizumab-mediated vascular normalization, and CCL28 might be a potential target for anti-angiogenesis therapy in lung adenocarcinoma.

Further, we evaluated the expression levels of CCL28 within the tumor tissue (Fig. 6 C, upper panel). The mean fluorescence intensity of CCL28 was significantly upregulated when bevacizumab was used compared to the control group (Fig. 6 C, lower panel), consistent with the results we found in the clinical samples. Also, the expression level of CCL28 in lung adenocarcinoma correlates with therapeutic efficacy. In lung adenocarcinoma patients, based on their treatment outcomes with bevacizumab, they were categorized into two groups: poor responders and good responders. PAN-CK, NG2, CD31, and CCL28 were stained in tumor tissues to analyze their expression levels with the therapeutic efficacy of bevacizumab treatment. Patients who responded well to bevacizumab showed significantly increased CCL28 expression, and high expression of CCL28 in tumor cells,, was associated with enhanced vascular maturity and favorable treatment outcomes (Fig. 6 D, 6E). The conclusion was evidenced by a notable decrease in malignant pericardial and pleural effusion and a significant reduction in metastatic lymph nodes after bevacizumab-based treatment (Fig. 6 F). Then, we analyzed the expression levels of DHRS11 and RDH13 in two groups of patients. Consistently, the therapeutic efficacy of bevacizumab appeared to be positively correlated with RDH13 expression and negatively correlated with DHRS11 expression (Fig. 6 G, H). These results suggest that the favorable response to anti-angiogenic therapy in patients may be attributed to the activation of CCL28, thereby deepening our understanding of treatment response variations.

Anti-angiogenesis therapy stands as a pivotal approach for metastatic lung adenocarcinoma [ 3 , 27 ], targeting the well-established VEGF/VEGFR pathway with numerous drugs developed over the last four decades [ 28 , 29 ]. However, its clinical impact has proven more complicated than initially anticipated. Recognizing the vasculature normalization effects of anti-angiogenic drugs has led to their integration into combination regimens with chemotherapy, radiotherapy, immunotherapy, and targeted therapy [ 4 ]. For example, bevacizumab, through the modulation of angiogenesis and improvement of the tumor microenvironment, lowers tumor vascular density and edema, thereby enhancing the efficiency of oxygen supply and making other treatment modalities more effective. Consequently, anti-angiogenesis drugs are employed as modulators for tumor vasculature and the microenvironment within combination regimens.

Anti-angiogenic therapy inhibits abnormal blood vessel formation, fostering vascular normalization through mechanisms like reducing vessel density, remodeling the extracellular matrix, regulating inflammation, balancing growth factors, and modulating the immune system. The tipped balance between pro-angiogenesis and anti-angiogenesis factors contributes to maintaining tumor vascular growth [ 30 ]. Anti-angiogenic therapy disrupts this tipped balance by inhibiting VEGF, enhancing the relative action of angiopoietin-1(ANGPT1), which aids in regulating and inducing vascular normalization. This rebalancing contributes to vascular normalization, ensuring newly formed blood vessels exhibit a more organized structure and function more normally. The action of angiopoietin-1 helps consolidate and stabilize the newly formed vessels, making them a more effective transportation system.

However, it is also widely accepted that agents targeting VEGF/VEGFR may ultimately elevate hypoxia levels within tumors [ 4 ]. This hypoxic microenvironment triggers alternative pro-angiogenesis molecular pathways in tumor or stromal cells within the tumor microenvironment, including FGF-2, HGF, DLL4/Notch, CCL28, and others [ 19 , 21 , 22 , 31 ]. Furthermore, hypoxia prompts a cascade of biological responses, leading tumor cells to adapt their metabolic pathways to low-oxygen conditions. The dysregulated metabolism observed in rapidly proliferating tumor cells is a hallmark of malignancy [ 32 ], contributing to the activation of various metabolism-related genes, including several hypoxia-related transcription factors like hypoxia-inducible factor (HIF), nuclear factor kappa-B, CREB, AP-1, p53, Sp1/3, Egr-1, and CEBPB [ 33 ].

In this study, we identified CCL28 as another crucial molecular target for vascular normalization in lung adenocarcinoma. It was observed that CCL28 could be up-regulated under hypoxic conditions. However, the molecular mechanism underlying hypoxia-induced transcriptional activation of the CCL28 gene remains unclear. Our investigation revealed that the transcription factors CEBEPB could regulate the expression of CCL28. CCL28, belonging to the subfamily of small cytokine CC chemokines, binds to chemokine receptors CCR3 and CCR10 [ 19 ]. It has been reported that CCL28 exhibits chemotactic activity for various immune cells and plays a role in the physiology of extracutaneous epithelial tissues [ 34 ]. Several studies have delved into the functions of CCL28 in the tumor microenvironment [ 17 , 18 , 20 ]. Therefore, we hypothesized that CCL28 might play a pivotal role in modulating the tumor microenvironment in lung adenocarcinoma. Here, we noted an augmentation in pericyte accumulation within the tumor microenvironment associated with the overexpression of CCL28. However, the mechanisms of pericyte recruitment in lung adenocarcinoma remain unknown. Thus, we examined the expression of CCR3/CCR10 on various tumor stromal cells, revealing widespread expression of CCR3 on endothelial cells, cancer-associated fibroblasts, and pericytes in lung adenocarcinoma. Two chemotactic experiments substantiated the effects of CCL28 on recruiting pericytes through the receptor CCR3.

Abnormal differentiation of stromal cells is an essential characteristic of malignant tumors and is correlated with abnormal metabolism. Many tumor stromal cells were differentiated from anti-tumor type to pro-tumor type, such as tumor-associated macrophage, cancer-associated fibroblasts, tumor-associated neutrophils, etc. [ 35 , 36 , 37 ]. Reprogramming the metabolism and modulating tumor stromal cell differentiation is a promising cancer treatment strategy. Interestingly, the present study found that retinoic acid metabolism was activated in a series of stromal cells in lung cancer.

Importantly, chemotactic factors play a crucial role in cell migration, influencing not only the cell's movement [ 38 ] but also closely interacting with cellular metabolism. They regulate energy production, metabolic pathways, and antioxidant responses, ensuring that cells have sufficient energy and resources during migration. Chemotactic factors influence cellular metabolism by regulating intracellular signaling pathways, such as PI3K/AKT, MAPK, AMPK, and mTOR [ 39 , 40 ]. The activation or inhibition of these signaling pathways can modulate the activity of intracellular metabolic enzymes, affecting processes such as glucose metabolism, lipid synthesis, and amino acid utilization. In the present study, we found chemokine CCL28 could influence the cellular levels of retinoic acid by modulating its synthesis.

Retinoic acid (RA), also known as vitamin A acid, and its related analogs participate in regulating the gene networks involved in cell growth, differentiation, homeostasis, and apoptosis. RA is an active metabolite of retinol. Retinol (Vitamin A) is a fat-soluble essential micronutrient that plays a crucial role in embryonic development, organ formation, immune system function, and vision [ 26 ]. Retinol can be metabolized into retinal by the retinol dehydrogenases (RDHs), and retinal can also be metabolized into retinol by the dehydrogenase/reductase SDR family (DHRS) [ 41 ]. Retinal can be further irreversibly metabolized to RA by the retinaldehyde dehydrogenases (ALDHs). The synthesis of RA depends on the balance between RDHs and DHRS. The present study found that CCL28 could disturb the balance between RDH13 and DHRS11. After being treated with CCL28, RDH13 in pericytes was significantly upregulated in a dose-dependent manner. However, the effect curve of CCL28 on DHRS11 expression in pericytes is not linear and might be bell-shaped, like the effects of VEGFA on endothelial cells. In addition, there might be an alternative pathway to maintain the balance of DHRS11 and RDH13 in pericytes. RA binds to RARα, promoting the formation of a heterodimer with retinoid X receptor alpha (RARα/RXRα) in the cell nucleus. This heterodimer binds to retinoic acid response elements (RAREs) in the promoter region of target genes [ 42 ], including ANGPT1 .

The angiopoietin family, including ANGPT1, ANGPT2, ANGPT3, and ANGPT4, is crucial in vascular development and normalization. They regulate endothelial cells' survival, proliferation, and migration by interacting with Tie receptors, thus vital to vascular normalization. ANGPT1 can activate the TIE2 receptor on endothelial cells, maintaining endothelial cell stability, enhancing tight connections between endothelial cells, and reducing microvascular permeability through a series of signaling pathways [ 26 ]. These signaling pathways include tyrosine kinase-related protein DOKR (also known as DOK2), endothelial nitric oxide synthase (eNOS), SH2 domain-containing phosphatase (SHP2), growth factor receptor-binding protein 2 (GRB2), and PI3K-Akt [ 24 ]. Various regulatory mechanisms influence the expression of the ANGPT1 . These include the hypoxia-inducible factor-1α (HIF-1α) signaling pathway under low oxygen conditions, interaction with vascular endothelial growth factor (VEGF), involvement of anti-inflammatory factors and growth factors, cell–cell interactions, as well as the nuclear factor-κB (NF-κB) signaling pathway and hormonal regulation. In the present study, we identified another regulatory mechanism controlling the expression of ANGPT1.

Vitamin A and its metabolites, particularly retinoic acid, exert regulatory effects on the vascular system through various pathways [ 43 ]. These include maintaining endothelial cell function, regulating angiogenesis, inhibiting vascular smooth muscle cell proliferation, suppressing inflammatory responses, and promoting cell differentiation. This comprehensive regulatory process contributes to maintaining blood vessels' stable structure and function, playing a crucial role in embryonic development, tissue repair, angiogenesis, and vascular health during inflammation and diseases. Interestingly, vitamin A and its metabolites have been used as a differentiation modulator of malignant cells for cancer treatment. The present study proposed a new strategy to modulate the differentiation of cancer stromal by vitamin A and its metabolites.

Conclusions

In conclusion, we elucidated that a specific chemokine CCL28 induced after anti-angiogenesis therapy can alter tumor stromal cell metabolism and reshape the tumor microenvironment. In summary, we identified a mechanism through which CCL28 promotes vascular normalization via a CCR3-pericytes-RA-RXRα-ANGPT1-dependent pathway (Fig. 7 ). With an in-depth exploration of the interplay between chemokine and ANGPT1, our work may provide valuable insights into the regulatory network of vascular normalization, offering new avenues for modulating tumor microenvironment and overcoming resistance to anti-angiogenesis therapy in lung adenocarcinoma treatment.

A schematic diagram of tumor microenvironment modulation effects of CCL28

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.Competing interests.

The authors declare that they have no competing interests.

Abbreviations

Platelet-derived growth factor receptor β

Chondroitin sulfate proteoglycan 4

Human CC motif chemokine ligands 28

Human umbilical vein endothelial cell

Dehydrogenase/reductase 11

Retinol dehydrogenase 13

Bevacizumab

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Acknowledgements

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This work was supported by the National Natural Science Foundation of China (Grant No. 82273326), Jiangsu Provincial Youth Medical Key Talents Project (Grant No. QNRC2016887), the Bethune Charitable Foundation (Grant No. KY202301-17) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (Grant No. KYCX22_0176).

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Ying Chen, Zhiyong Zhang, Pengfei Li, Yuxi Chen & Tingting Wang

Jiangsu Key Laboratory of Molecular Medicine, Division of Immunology, Medical School, Nanjing University, Nanjing, 210093, China

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Ying Chen, Zhiyong Zhang, Fan Pan, Pengfei Li, Weiping Yao, Yuxi Chen & Tingting Wang

Department of Respiratory Critical Care Medicine, Affiliated Hospital of Medical School, Nanjing Drum Tower Hospital, Nanjing University, Nanjing, Jiangsu, 210008, China

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TTW, YL and GCH: Provided funding support, conceptualization, methodological guidance, and manuscript revision for this study.. YC: conceived of the study, performed the experiments and wrote the paper. WPY: participated in animal experiments. ZYZ and FP: performed the statistical analysis. PFL, YXC and LX: Participated in experiment design and sample collection. All authors read and approved the final manuscript.

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Correspondence to Tingting Wang , Yan Li or Guichun Huang .

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Chen, Y., Zhang, Z., Pan, F. et al. Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma. J Exp Clin Cancer Res 43 , 210 (2024). https://doi.org/10.1186/s13046-024-03135-3

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DOI : https://doi.org/10.1186/s13046-024-03135-3

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Iron accumulation in tumors contributes to disease progression and chemoresistance. While targeting this process can influence various hallmarks of cancer, the immunomodulatory effects of iron chelation in the tumor microenvironment are unknown. Here, we report that treatment with deferiprone, an FDA-approved iron chelator, unleashes innate immune responses that restrain ovarian cancer. Deferiprone reprogrammed ovarian cancer cells towards an immunostimulatory state characterized by production of type I interferon (IFN) and overexpression of molecules that activate natural killer (NK) cells. Mechanistically, these effects were driven by innate sensing of mitochondrial DNA in the cytosol and concomitant activation of nuclear DNA damage responses triggered upon iron chelation. Deferiprone synergized with chemotherapy and prolonged the survival of mice with ovarian cancer by bolstering type I IFN responses that drove NK cell-dependent control of metastatic disease. Hence, iron chelation may represent an alternative immunotherapeutic strategy for malignancies that are refractory to current T cell-centric modalities.

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The impact of impaired tissue fixation in resected non-small-cell lung cancer on protein deterioration and DNA degradation.

Author(s): Butter R, Halfwerk H, Radonic T, Lissenberg-Witte B, Thunnissen E

Publication: Lung Cancer , 2023, Vol. 178 , Page 108-115

PubMed ID: 36812759 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to assess the effects of inadequate fixation on tissue quality (determined macroscopically and microscopically), immunohistochemical staining of 10 antigens, and DNA degradation using formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue.

Conclusion of Paper

Based on either macroscopic or microscopic review a similar number of fixed tissue specimens were classified as adequately fixed (8 versus 6 specimens), inadequately fixed (2 specimens for each, and having both adequately and inadequately fixed regions (15 versus 17 specimens). 

Mean IHC scores of eight antigens (programmed death-ligand 1, PD-L1; cytokeratin CAM5.2, CK7; mesenchymal-epithelial transition factor, c-MET; cytokeratin KER-MNF116; Napsin A; p40; thyroid transcription factor 1, TTF) were compared between adequately and inadequately fixed areas of the 25 tumors collected for the study; because specimens were excluded from analysis if immunopositive staining was absent in both adequately and inadequately fixed tissue, a different number of specimens were analyzed for each antigen. Mean H-scores for both KER-MNF116 (256 versus 151, respectively; p<0.001) and p40 (293 versus 248, respectively; p=0.028) were significantly higher in regions that were adequately fixed than those that were inadequately fixed. When mean H-scores were only examined in the subset of tumors that displayed both adequately and inadequately fixed regions, KER-MNF116 alone differed significantly, with a higher mean H-score in the adequately fixed region compared to the inadequately fixed region (249 versus 131; p=0.003). Necrotic zones within a tumor had significantly lower mean H-scores than adequately fixed regions for CK7 (80 versus 186; p=0.044), KER-MNF116 (63 versus 256; p<0.001), and p40 (0 versus 293; p=0.002). 

Evidence of DNA fragmentation (the absence of 300 and 400 bp amplicons) was prominent in DNA isolated from both adequately and inadequately fixed regions, and the concentration of PCR amplicons did not differ between the two region types for any of the amplicon lengths investigated.  Interactions between DNA degradation and cold ischemia time and fixation time were observed, and tumor specimens that had a cold ischemia time >16 h had a lower concentration of 300 (0 versus 0.13 ng/µl; p=0.004) and 400 bp (0 versus 0.0059 ng/µl; p=0.046) PCR amplicons than those with a delay of <6 h, and tumor specimens that were fixed in formalin for >24 h had a lower concentration of 300 (0.02 versus 0.14 ng/µl; p=0.024) and 400 bp (0 versus 0.0067 ng/µl; p=0.045) PCR amplicons than those that were fixed for <24 h. A potential influence of cold ischemia time and fixation time on immunohistochemical staining was not investigated.  

Study Purpose

The purpose of this study was to assess the effects of under- and over-fixation of formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue on tissue quality (determined macroscopically and microscopically), immunohistochemical staining of 10 antigens, and DNA degradation.  Surgically resected lung tissue from 25 patients diagnosed with NSLC(9 with non-mucinous adenocarcinoma, 9 with squamous cell carcinoma, 2 with pleomorphic adenoma, 2 with carcinoid tumors, 1 with mucinous adenocarcinoma, and two with large cell carcinoma) were partially sliced along the axial plane every 1.5 cm, bandage mesh was placed between slices, and the tissue specimen was fixed in 10% neutral buffered formalin for 12-24 h. Cold ischemia time (time between resection and fixation) ranged between 0.5 to 21.5 h (mean 3.4 h).  Fixed specimens were sectioned again (0.5 cm thick sections) and examined macroscopically for adequate (specimen was a white/gray color throughout) or inadequate (specimen contained pink/red areas) fixation.  Specimens then underwent additional tissue processing and paraffin embedding (no additional details provided).  FFPE tissue sections were stained with hematoxylin and eosin for microscopic evaluation and by immunohistochemistry (IHC) for the following antigens: ALK, PD-L1, GAM2.5, CK7, c-Met, KER-MNF116, NapsinA, p40, ROS1, TTF1. For tissue slides stained by immunohistochemistry, areas containing the maximum and minimum staining intensity, necrotic zones, and evidence of adequate and inadequate fixation (detachment of the basement membrane) fixation were estimated.  To evaluate DNA degradation, adequately fixed and inadequately fixed regions were macroscopically dissected from 10 µm thick FFPE sections and DNA was isolated with the QIAamp DNA FFPE Tissue Kit, then assessed by multiplex PCR (100, 200, 300, 400 bp amplicons; gene not specified). The concentration of PCR amplicons was determined by electrophoresis with an Agilent Bioanalyzer. 

Summary of Findings:

Based on macroscopic evaluation of the fixed tissue segment before paraffin-embedding, 8 of the 25 specimens were adequately fixed (tissue was white or grey), 2 of the specimens were inadequately fixed (tissue was pink or red), and 15 specimens contained regions of both. Microscopic review of H&E-stained slides generated similar results, as 6 specimens were adequately fixed, 2 were inadequately fixed (detachment of the basement membrane), and 17 specimens contained regions of both.  Mean IHC scores of eight antigens were compared between adequately and inadequately fixed areas of the 25 tumors collected for the study; because specimens were excluded from analysis if immunopositive staining absent in both adequately and inadequately fixed tissue, a different number of specimens were analyzed for each antigen. Mean H-scores for both KER-MNF116 (256 versus 151, respectively; p<0.001) and p40 (293 versus 248, respectively; p=0.028) were significantly higher in regions that were adequately fixed and those that were inadequately fixed. Necrotic zones within a tumor had significantly lower mean H-scores than adequately fixed regions for CK7 (80 versus 186; p=0.044), KER-MNF116 (63 versus 256; p<0.001), and p40 (0 versus 293; p=0.002).  When mean H-scores were only examined in the subset of tumors that displayed both adequately and inadequately fixed regions, KER-MNF116 alone differed significantly, with a higher mean H-score in the adequately fixed region compared to the inadequately fixed region (249 versus 131; p=0.003). The minimum and maximum H-scores within a tumor differed significantly for all eight antigens evaluated (PD-L1, CAM5.2, CK7, c-MET, KER-MNF116, Napsin A, p40, TTF1; p≤0.008 for all).  Evidence of DNA fragmentation (the absence of 300 and 400 bp amplicons) was prominent in both adequately and inadequately fixed regions, and the concentration of PCR amplicons did not differ between the two region types for any of the amplicon lengths investigated.  Interactions between DNA degradation and cold ischemia time and fixation time were observed. Tumor specimens that had a cold ischemia time >16 h had a lower concentration of 300 (0 versus 0.13 ng/µl; p=0.004) and 400 bp (0 versus 0.0059 ng/µl; p=0.046) PCR amplicons than those with a delay of <6 h. Tumor specimens that were fixed in formalin for >24 h had a lower concentration of 300 (0.02 versus 0.14 ng/µl; p=0.024) and 400 bp (0 versus 0.0067 ng/µl; p=0.045) PCR amplicons than those that were fixed for <24 h. A potential influence of cold ischemia time and fixation time on immunohistochemical staining was not investigated.  

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• Neoplastic - Benign
• Neoplastic - Carcinoma

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IMPDH2 suppression impedes cell proliferation by instigating cell cycle arrest and stimulates apoptosis in pediatric hepatoblastoma

• Open access
• Published: 01 August 2024
• Volume 150 , article number  377 , ( 2024 )

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• Linman Li 1 , 3   na1 ,
• Yichi Wu 1 , 3   na1 ,
• Hong-ting Huang 1   na1 ,
• June-kong Yong 1 ,
• Zicheng Lv 1 , 2 ,
• Yi Zhou 3   na1 ,
• Xuelin Xiang 3 ,
• Jie Zhao 1 ,
• Zhifeng Xi 1 , 3 ,
• Hao Feng 1 , 2 , 3 &
• Qiang Xia 1 , 3  

Hepatoblastoma (HB) is the most common pediatric liver tumor, presenting significant therapeutic challenges due to its high rates of recurrence and metastasis. While Inosine Monophosphate Dehydrogenase 2(IMPDH2) has been associated with cancer progression, its specific role and clinical implications in HB have not been fully elucidated.

This study utilized Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Tissue Microarray (TMA) for validation. Following this, IMPDH2 was suppressed, and a series of in vitro assays were conducted. Flow cytometry was employed to assess apoptosis and cell cycle arrest. Additionally, the study explored the synergistic therapeutic effects of mycophenolate mofetil (MMF) and doxorubicin (DOX) on HB cell lines.

The study identified a marked overexpression of IMPDH2 in HB tissues, which was strongly correlated with reduced Overall Survival (OS) and Event-Free Survival (EFS). IMPDH2 upregulation was also found to be associated with key clinical-pathological features, including pre-chemotherapy alpha-fetoprotein (AFP) levels, presence of preoperative metastasis, and the pre-treatment extent of tumor (PRETEXT) staging system. Knockdown of IMPDH2 significantly inhibited HB cell proliferation and tumorigenicity, inducing cell cycle arrest at the G0/G1 phase. Notably, the combination of MMF, identified as a specific IMPDH2 inhibitor, with DOX, substantially enhanced the therapeutic response.

The overexpression of IMPDH2 was closely linked to adverse outcomes in HB patients and appeared to accelerate cell cycle progression. These findings suggest that IMPDH2 may serve as a valuable prognostic indicator and a potential therapeutic target for HB.

The present study unveiled a significant overexpression of inosine monophosphate dehydrogenase 2 (IMPDH2) in hepatoblastoma (HB) tissues, particularly in association with metastasis and recurrence of the disease. The pronounced upregulation of IMPDH2 was found to be intimately correlated with adverse outcomes in HB patients. This overexpression appears to accelerate the progression of the cell cycle, suggesting that IMPDH2 may serve as a promising candidate for both a prognostic marker and a therapeutic target in the context of HB.

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Introduction

Hepatoblastoma (HB) is the predominant pediatric liver cancer, typically affecting children aged below three years (Czauderna et al. 2014 ). Despite its rarity, the prevalence of HB has climbed annually in recent decades, constituting approximately 80% of pediatric liver malignancies (Bell et al. 2017 ; Linabery and Ross 2008 ). Clinical presentations often include abdominal distension and the detection of an abdominal mass through imaging. Elevated levels of AFP, a fetal hepatocyte-derived protein, are commonly observed and considered as a crucial tumor indicator for monitoring disease progression and response to comprehensive treatment. The rising incidence is speculated to be linked to improved survival rates among prematurely born or with extreme low weight, a population susceptible to HB (Heck et al. 2013 ; Fine Licht et al. 2012 ).

Primary therapeutic approaches for HB involve complete surgical resection and chemotherapy, with cisplatin-based regimens proving effective for unresectable tumors and contributing to increased survival rates (Davies et al. 2004 ). Advances in surgical techniques and postoperative chemotherapy have enhanced overall outcomes, yielding 5-year survival rates averaging 82% (Tulla et al. 2015 ). However, approximately 30% of patients, even with surgery and neoadjuvant chemotherapy, exhibit suboptimal outcomes (Aronson et al. 2019 ; Meyers et al. 2017 ). In cases of advanced HB, liver transplantation stands as the sole effective treatment option (Trobaugh-Lotrario et al. 2016 ).

Mechanistically, ever-increasing evidence implicates that HB originates from less differentiated cells, with disorders of developmental and self-regeneration pathways playing a pivotal role in hepato-carcinogenesis. The Wnt/beta-catenin pathway, in relation to stem cells, is critically important, influencing the activation and expansion of these cells during embryonic development and liver regeneration, thus fostering liver homeostasis (Monga 2015 ; Russell and Monga 2018 ). HB often exhibits resistance to chemotherapy at high levels (Marin et al. 2018 ). Unfortunately, the mechanisms underlying this resistance remain poorly understood.

IMPDH is recognized as a pivotal rate-controlling enzyme within the purine de novo synthesis pathway (Shu and Nair 2008 ). This enzyme comprises two isotypes: IMPDH1 and IMPDH2 (Natsumeda et al. 1990 ). with IMPDH1 exhibiting relatively stable expression in both normal and tumor cells. In contrast, IMPDH2 manifests significantly heightened expression levels across various malignant tumors (Collart et al. 1992 ). Accumulating evidence from previous investigations has underscored the substantial elevation of IMPDH2 expression, which correlates closely with tumor progression and a poor prognosis in a diverse array of malignancies (Zhou et al. 2014 ). Notably, overexpression of IMPDH2 has been identified in specific patient subgroups that demonstrate a diminished response to chemotherapy, thereby establishing it as an independent prognostic indicator for chemotherapeutic response and event-free survival (EFS) (Fellenberg et al. 2007 ). Recent research has unveiled markedly upregulated IMPDH2 expression in prostate, kidney, bladder, and liver cancers, suggesting its potential utility as a biomarker for these diseases(Yang et al. 2014 ; Zou et al. 2015 ; Jia et al. 2022 ). However, the precise role of IMPDH2 in the molecular mechanisms underlying hepatoblastoma (HB) remains insufficiently characterized within the current literature.

Cyclin-Dependent Kinase Inhibitor 1 A (CDKN1A), commonly referred to as p21, is a critical regulator of cell cycle progression, particularly during the G1 phase (Xiong et al. 1993 ). Functionally, p21 plays a multifaceted role in orchestrating cell cycle arrest, facilitating DNA repair, and regulating apoptosis (Mauro et al. 2012 ; Gartel and Tyner 2002 ). By inhibiting the activity of cyclin-dependent kinases (CDKs), p21 effectively prevents the transition of cells from the G1 to the S phase, thereby significantly contributing to the regulation of cell division and preventing aberrant cell proliferation. Furthermore, in response to DNA damage, p21 can induce cell cycle arrest, providing sufficient time for DNA repair to occur. Under certain conditions, p21 also participates in the modulation of programmed cell death, or apoptosis.

Mycophenolate mofetil (MMF), approved by the FDA in 1995 for the prevention of transplant graft failure (Lipsky 1996 ), is a widely utilized immunosuppressive agent in the post-organ transplantation setting. Acting as a prodrug, MMF is rapidly metabolized into mycophenolic acid (MPA), which targets IMPDH, thereby reducing the synthesis of guanine nucleotides (Ransom 1995 ; Suthanthiran and Strom 1997 ). In the therapeutic management of hepatoblastoma (HB), particularly in high-risk patients, a combination regimen of cisplatin and doxorubicin (DOX) is frequently employed (Trobaugh-Lotrario and Katzenstein 2012 ). However, both of these chemotherapy agents are associated with specific side effects. Cisplatin, for example, is known to induce severe ototoxicity. Dox, an anthracycline, exerts its therapeutic effects through a variety of mechanisms, including DNA intercalation, interference with type II topoisomerases, generation of free radicals, and inhibition of DNA and RNA synthesis (Zsíros et al. 2010 ). Cardiomyopathy stands as a recognized side effect of Dox therapy (Zsiros et al. 2013 ). Unfortunately, specific medications targeting DOX-induced cardiotoxicity are currently unavailable.

In the present study, our analysis of public databases revealed a significant overexpression of IMPDH2 in hepatoblastoma (HB) tissues, particularly in cases associated with metastasis and recurrence of the disease. Subsequently, we conducted an immunohistochemical assessment of IMPDH2 protein expression using tissue microarray (TMA) analysis. This was followed by an investigation into the correlations between IMPDH2 protein expression levels and various clinicopathological variables, as well as the overall survival (OS) and event-free survival (EFS) of the patients. Furthermore, we experimentally knocked down IMPDH2 in HB cell lines to determine its effects on cellular functions and its relationship with the cell cycle. Ultimately, our research led to the discovery of a synergistic interaction between mycophenolate mofetil (MMF), an inhibitor of the IMPDH2 target, and the chemotherapeutic agent doxorubicin (DOX).

Materials and methods

Patients and specimens.

Tissue samples were obtained from a cohort of 129 patients diagnosed with hepatoblastoma (HB) at Renji Hospital, Shanghai, over a decade-long period from May 2013 to August 2023. For the study, we constructed tissue microarrays (TMAs) that included a total of 129 paraffin-embedded HB tumor specimens, 21 paraffin-embedded specimens of adjacent normal liver tissue from HB patients, and 10 paraffin-embedded specimens of normal liver tissue from controls. Furthermore, we randomly selected eight pairs of tumor and corresponding normal tissues for analysis via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Surgical interventions for all patients included in the study were performed at Renji Hospital. The study protocol was approved by the Ethics Committee of Renji Hospital, and we obtained written informed consent from the guardians of all participating patients.

Cell culture

The HB cell lines (Huh6, HepG2) and embryonic kidney cell lines (HEK293) were nurtured in Dulbecco’s modified Eagle’s medium, complemented with 10% fetal bovine serum. Incubation was sustained in a humidified environment at 37 °C, with 5% carbon dioxide. Cell passages were restricted to < 6 months before experimentation.

Dataset acquisition and process

Three microarray datasets (GSE151347, GSE131329, GSE133039) sourced from the GEO database were utilized for investigating IMPDH2 expression in HB cancer tissues and normal tissues. Differential expression profiles of genes were generated through GEO2R analysis. Subsequent analysis and visualization of Gene Ontology (GO) results were conducted employing R Studio software (version 4.2.3). The HSA Synergy Score was assessed utilizing the online tool SynergyFinder. ( https://tangsoftwarelab.shinyapps.io/synergyfinder/_w_f98c38d7/#!/dashboard ).

TMA staining and immunohistochemistry (IHC) assay

TMA sections, each 4 μm thick and prepared using a Leica RM2245 microtome, underwent heat treatment at 60 °C for 30 min, followed by three cycles of immersion in xylene for 10 min per cycle and subsequent immersion in absolute ethanol for 10 min per cycle. After rinsing with running water, experiments were conducted. IHC was performed to investigate IMPDH2 expression in the TMA, utilizing anti-IMPDH2 as the primary antibody. A semi-quantitative scoring system was devised, considering both the proportion of cells displaying positive staining and the intensity of staining. The positive staining proportions were graded as follows: 0 for absence, 1 for 0–25%, 2 for 25–50%, 3 for 50–75%, and 4 for 75–100%. Staining intensity was rated on a scale of 0 for absence, 1 for weak, 2 for moderate, and 3 for strong. The combined score was calculated by multiplying these two sub-scores, allowing for the classification of samples into categories reflecting negative expression (0), low expression (1–3), moderate expression (4–6), and high expression (9–12). Two blinded pathologists independently assessed the IHC staining results without access to clinicopathologic data.

Western blot

Twenty micrograms of protein samples were applied onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After separation, these samples were transferred from the gel to nitrocellulose membranes (Millipore, MA, USA), which underwent blocking using 5% bovine serum albumin for 1 h. Subsequently, the membranes were subjected to an overnight incubation at 4 °C with anti-β-actin, IMPDH2, or specific antibodies as indicated. Following washing with PBST, secondary antibody incubation was conducted for 1 h, and subsequent visualization was achieved by exposure of the membranes to photographic film. Table S2 contains detailed information about the utilized antibodies.

Quantitative real-time PCR (RT-qPCR)

Real-time PCR analyses were conducted in triplicate, utilizing 25 ng of cDNA per reaction and 10 µM forward and reverse primers, employing 2× ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech) on a Bio-Rad CFX 96 system (Bio-Rad, California, USA). Details of primer sequences are available in Tables S3. The PCR cycling protocol initiated with a 30-second denaturation at 95 °C, succeeded by 40 cycles consisting of 10 s at 95 °C for denaturation and 30 s for annealing/elongation. Normalization of target gene expression to GAPDH was performed, and the relative expression of target genes was determined employing the 2 −ΔΔCT method.

Cell proliferation and colony formation assays

Cell proliferation was evaluated utilizing the CCK-8 Kit (Beyotime, China). Assessment of cell colony-forming ability post-IMPDH2 silencing was conducted via colony formation assay. Cells, transfected with shIMPDH2 or shNC controls, were seeded into 12-well plates and incubated for a duration of 2 weeks. Subsequently, they were rinsed twice with PBS, followed by fixation using 4% formaldehyde for 15 min, staining with crystal violet for an additional 15 min, and subsequent photographic documentation.

HepG2 and Huh6 cell lines were maintained in a serum-depleted medium within the upper transwell chamber (Corning, USA) containing an 8.0 μm pore polycarbonate membrane. The lower chamber was supplemented with medium containing 20% FBS. After a 24-hour incubation period, cells that traversed the membrane and adhered to its undersurface were fixed and stained using crystal violet.

Cell transfection

Lentiviral vectors containing IMPDH2 shRNA plasmids (shIM#1, shIM#2, Supplement) and an empty vector (shNC) were procured from Shanghai Jiaotong University School of Medicine. To develop stable IMPDH2 knockdown HB cell lines, lentiviral particles were produced through co-transfection of the shIMPDH2 plasmid with a mix of packaging plasmids (pMDL, VSVG, pRSV-Rev at a ratio of 5:3:2) using PEI MAX transfection reagent (Polysciences, 24765-100) in HEK-293T cells. The viral particles were collected 48–72 h post-transfection. Subsequently, HepG2 and Huh6 cells were exposed to the lentivirus containing IMPDH2 shRNA or control lentivirus for 48 h and subsequently subjected to puromycin screening to select for cells exhibiting stable knockdown.

Flow Cytometry

Cell cycle analysis was performed using the Cell Cycle Staining Kit, while apoptosis assessment utilized the Annexin V-FITC/PI apoptosis Kit (MULTI SCIENCES, Shanghai, China) as per the manufacturer’s protocols. BD FACSCanto II (USA) was employed for cell collection and analysis. ModFit LT 3.2 software was utilized to compute cell cycle distribution and apoptosis ratio, and Flowjo 10.8.1 was used for the visualization of apoptosis-related images.

Tumorigenesis assays in nude mice

All animal experiments adhered to the Institutional Animal Care and Use Committee guidelines at Shanghai Jiaotong University. Four-week-old male BALB/c nude mice were randomly allocated to specified groups. Subcutaneous tumor models were established using HepG2 and Huh6 cells. The study comprised three groups: two experimental groups with IMPDH2 knockdown (HepG2 shIM#1, HepG2 shIM#2) and one control group (HepG2 shNC), each consisting of three nude mice. Each mouse received subcutaneous injections of HepG2 cells (5 × 10 6 in 50% Matrigel™, BD Biosciences). The huh6 groups were similar to HepG2 groups, differing only in the number of injected cells (1.0 × 10 7 in 25% Matrigel™). Tumor volume was assessed every 2 days using calipers and calculated using the formula (width 2 x length)/2. Finally, humane euthanasia using carbon dioxide was performed, and tumors were harvested and prepared for further analysis.

Statistical analysis

All experiments were conducted in triplicate, and GraphPad Prism software was utilized for data analysis. Results are expressed as mean ± standard error of mean (SEM). Unpaired and paired two-tailed student’s t-tests were employed as the analytical method to assess differences between the two groups. Kaplan–Meier methodology was applied for OS and EFS calculations, and between-group disparities were evaluated using the log-rank test. Clinicopathological characteristics in HB were subjected to analysis using χ2 tests conducted via SPSS software. A P value less than 0.05 was considered statistically significant (* P  < 0.05, ** P  < 0.01, *** P  < 0.001).

IMPDH2 is highly expressed in HB tissues and is associated with metastasis and recurrence

To explore the differential expression of IMPDH2 in hepatoblastoma (HB) relative to healthy tissue, an extensive analysis was undertaken, leveraging datasets from the Gene Expression Omnibus (GEO). The datasets included GSE151347, GSE131329 (comparing tumor to non-tumor tissue), GSE133039 (analyzing recurrence versus non-recurrence), and again GSE151347 (examining metastasis versus non-metastasis). Our results consistently revealed a significant overexpression of IMPDH2 in HB tissue, which was particularly pronounced in cases associated with metastasis and recurrence in HB patients ( Fig.  1 A ) . To substantiate the validity of these public data, we conducted a preliminary assessment of IMPDH2 expression levels using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a series of HB tissues and their matched healthy liver tissue counterparts at the transcriptome level. These analyses confirmed a marked upregulation of IMPDH2 in HB patients ( Fig.  1 B ) . With the goal of achieving a comprehensive understanding of IMPDH2 expression at the protein level, we constructed a tissue microarray (TMA) encompassing a considerable number of HB cases. Examination of the TMA with an IMPDH2 antibody led to the clear stratification of cases into negative, low, moderate, and high expression groups based on IMPDH2 levels ( Fig.  1 C-D ) . It is noteworthy that the vast majority, 90.2%, showed positive IMPDH2 expression ( Fig.  1 E ) . Furthermore, stark differences were observed between the high/medium and negative/low IMPDH2 expression groups with respect to pre-chemotherapy alpha-fetoprotein (AFP) levels, pre-operative metastasis, and the PRETEXT staging system ( Table  1 ), along with a notable correlation with Ki67 expression. In addition, HB patients with negative/low IMPDH2 expression exhibited significantly better overall survival (OS) and event-free survival (EFS) compared to those with high/medium IMPDH2 expression ( Fig.  1 F-G ) . In aggregate, these observations strongly suggest that the pronounced overexpression of IMPDH2 adversely affects the prognosis of HB patients. Moreover, IMPDH2 holds promise as a potential prognostic and risk prediction biomarker.

IMPDH2 is highly expressed in HB tissues and is associated with metastasis and recurrence. A. Left, Venn diagrams displayed DEGs retrieved from the GEO database (GSE151347 T vs. N, GSE131329 T vs. N); the Right, illustrated Venn diagrams of DEGs from the GEO database (GSE133039 R vs. T, GSE151347 M + vs. M-); The middle section depicted the collective genes overlapping across all datasets. P  < 0.001, log2FC > 1. B. The RT-qPCR analysis unveiled the mRNA expression levels of IMPDH2 in HB tissues, encompassing eight pairs of tumors and their corresponding paired normal tissues. C: The schematic diagram illustrating the collection, classification, and analysis of tumor samples. D: Representative images and proportion of HB TMA with different staining intensities. E: The proportion of HB TMA with different staining intensities. F. Patient EFS between IMPDH2 −/low and IMPDH2 medium/high group. G. Patient OS between IMPDH2 −/low and IMPDH2 medium/high group

Knockdown of IMPDH2 suppresses HB cell malignancy in vitro

To elucidate the biological role of inosine monophosphate dehydrogenase 2 (IMPDH2) in hepatoblastoma (HB) cells, we experimentally downregulated IMPDH2 expression in HB cell lines ( Fig.  2 A ) . Subsequently, the proliferation rate of these IMPDH2-knockdown cell lines was evaluated using the CCK-8 assay. Our results demonstrated a significant reduction in the proliferative capacity of the HB cell lines upon IMPDH2 suppression ( Fig.  2 B-C ) . In parallel, the clonogenic potential of the HB cell lines was markedly diminished following IMPDH2 knockdown ( Fig.  2 D ) . Furthermore, transwell migration assays indicated a pronounced inhibition of the migratory capacity of the HB cell lines post-IMPDH2 knockdown ( Fig.  2 E ) . Importantly, previous studies have reported a significant increase in apoptosis following IMPDH2 inhibition in cell lines derived from triple-negative breast cancer and diffuse large B-cell lymphoma(Wang et al. 2021 ; Gao et al. 2023 ). Prompted by these findings, we investigated the impact of IMPDH2 knockdown on apoptosis using flow cytometry. Our analysis revealed a substantial increase in apoptotic cell death following IMPDH2 knockdown ( Fig.  2 F ) . Collectively, these observations suggest that IMPDH2 plays a critical role in promoting the malignant progression of HB cells while concurrently suppressing apoptotic processes within these cell lines.

Knockdown of IMPDH2 suppresses HB cell malignancy in vitro and in vivo. A. Western blot assays validating the efficiencies of IMPDH2 knockdown in HB cell lines. B-C. The CCK-8 assay was employed to evaluate proliferative potential in HB cell lines post transfection with IMPDH2 shRNA (shIM#1 and shIM#2) and control shRNA. ( n  = 3, t-test). D. Colony formation assays were utilized to assess the proliferative capacity of HB cell lines following transfection with IMPDH2 shRNA and control shRNA. ( n  = 3, t-test). E. Transwell assays were employed to evaluate the metastatic potential of HB cell lines subsequent to transfection with IMPDH2 shRNA and control shRNA. ( n  = 3, t-test). F. A Flow cytometry analysis was used to assess the apoptosis rate of HB cell lines post transfection with IMPDH2 shRNA and control shRNA. ( n  = 3, t-test). G. The in vivo tumorigenic potential of HepG2, Huh6 cells transfected with IMPDH2 shRNA and control shRNA, respectively. B-C. Assessment of tumor volumes and weights of transfected HepG2 and Huh6 cells was conducted every 2 days, respectively

Knockdown of IMPDH2 inhibits tumor proliferation in vivo

To further investigate the impact of IMPDH2 on hepatoblastoma (HB) proliferation in vivo, HepG2 and Huh6 cells were transfected with stable short hairpin negative control (shNC) and short hairpin IMPDH2 (shIMPDH2) constructs, specifically shIM#1 and shIM#2. These cells were then inoculated into nude mice. After a period of 19 days following the injection of the HepG2 cell line, xenograft tumors formed at the injection site in all mice. Notably, the group treated with shIMPDH2 demonstrated significantly reduced tumor growth rates when compared to the shNC group. A comparable trend was observed in the results from the Huh6 cell line ( Fig.  2 G ) . These findings corroborate that the suppression of IMPDH2 markedly hinders tumor formation and growth.

IMPDH2 regulates the progression of HB through the cell cycle signaling pathway

To delineate the molecular mechanisms underlying the inhibitory effects observed upon IMPDH2 knockdown, we performed RNA sequencing on hepatoblastoma (HB) cell lines with IMPDH2 knockdown. The analysis of differentially expressed genes (DEGs) revealed a significant upregulation of genes linked to cell cycle regulation. Gene Ontology (GO) term enrichment analysis underscored the role of these genes in processes such as the regulation of the mitotic cell cycle, cell cycle phase control, and the transition through the mitotic cell cycle phase ( Fig.  3 A ). This prompted us to focus on the influence of IMPDH2 on the cell cycle in HB cells. The impact of IMPDH2 knockdown on the cell cycle was evaluated using flow cytometry. We observed a pronounced accumulation of cells in the G0/G1 phase in the IMPDH2 knockdown group compared to the IMPDH2 short hairpin negative control (shNC) group, accompanied by a significant decrease in the G2/M phase cell population ( Fig.  3 B ) . The increase in apoptosis observed following IMPDH2 knockdown ( Fig.  2 F ) supports our inference that IMPDH2 knockdown results in G0/G1 cell cycle arrest, which subsequently initiates apoptosis. It is noteworthy that the cell cycle inhibitory protein p21 was significantly upregulated in the IMPDH2 knockdown group relative to the IMPDH2 shNC group. In contrast, genes associated with the G1/S transition, such as CDK6 and cyclin D1, showed marked downregulation. These results were further validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis ( Fig.  3 C-D ) and confirmed through Western blot analysis ( Fig.  3 E). In summary, our results elucidate that IMPDH2 knockdown induces G0/G1 phase cell cycle arrest and triggers apoptosis in HB cell lines. This is achieved through the upregulation of the cell cycle inhibitory protein p21 and the concurrent downregulation of cell cycle transition genes CDK6 and cyclin D1.

IMPDH2 regulates the progression of HB through the Cell Cycle Signaling Pathway. Combination of Doxorubicin with MMF remarkably reduced tumor proliferation and promoted apoptosis in vitro. A. Results of Gene Ontology analysis in HB cell lines. B. Analysis of cell cycle distribution in HB cell lines using flow cytometry following IMPDH2 knockdown: graphical representation of cell cycle distribution and statistical analysis. C-D. Quantification of IMPDH2, P21, CDK6, and cyclin D1 mRNA expression levels in HB cell lines transfected with IMPDH2 shRNA and control shRNA, assessed via real-time PCR. E. Validation of changes in cell cycle-related proteins following knockdown of IMPDH2 using Western blot assays. F. Cellular viability of HB cell lines post-administration of DOX and MMF was evaluated utilizing the CCK-8

Combination of doxorubicin with mmf remarkably reduced tumor proliferation and promoted apoptosis in vitro

Cisplatin, a chemotherapy agent extensively utilized in the treatment of hepatoblastoma (HB), exerts its therapeutic effect by disrupting DNA repair mechanisms, thereby inhibiting cancer cell growth. The efficacy of cisplatin is significantly enhanced when administered in combination with doxorubicin (DOX). Mycophenolate mofetil (MMF) functions as an inhibitor of purine synthesis by targeting inosine monophosphate dehydrogenase (IMPDH) activity, which in turn suppresses the de novo biosynthesis of purine nucleotides. Our hypothesis was centered on the potential synergistic effect of combining DOX’s inhibition of DNA synthesis with MMF’s blockade of de novo DNA synthesis, thereby amplifying their therapeutic impact on tumors. To evaluate this hypothesis, HB cell lines were treated with DOX, MMF, and their combination. The resulting dose-response relationships for each drug were depicted (Fig.  3 F). Subsequently, the computation of the HSA Synergy Score revealed the striking superiority of the DOX-MMF combination treatment (40 nM DOX and 1 µM MMF) over the individual drugs (Fig.  3 G-H). These results indicate that the addition of a specific dosage of MMF can significantly enhance the tumor-suppressive effects of DOX. Furthermore, our examination of the impact of combination therapy on cell apoptosis clearly demonstrated a pronounced pro-apoptotic effect of the combined treatment when compared to monotherapy (Fig. 5I-K). Consequently, we propose that optimizing the concentration of MMF in conjunction with DOX administration could lead to improved treatment outcomes. This strategy could potentially enable the use of reduced DOX dosages, thereby minimizing the side effects associated with MMF.

As the most prevalent class of primary liver malignancies, hepatoblastoma (HB) exhibits a pronounced propensity for metastasis, particularly to the lungs, and often displays inherent resistance to chemotherapy. The poor prognosis and overall survival rates observed in HB patients are largely attributable to recurrent episodes and the dissemination of metastases. Consequently, our study was designed to explore the fundamental mechanisms and potential biomarkers underlying HB.

Within this investigation, elevated IMPDH2 expression was observed among HB patients, which correlated with unfavorable prognostic indicators. Silencing IMPDH2 expression resulted in a significant deceleration of in vitro cellular proliferation and induced cell cycle arrest at the G0/G1 phase in HB cells. Additionally, IMPDH2 suppression led to an increase in P21 expression, disrupting the functionality and binding capacities of CDK6 and Cyclin D1, thereby promoting cellular apoptosis and inhibiting cellular proliferation. These observations position IMPDH2 as a significant contributor to HB progression and suggest its potential as a promising prognostic marker for this disease.

In our study, we collected postoperative samples from HB patients at Renji Hospital spanning the past decade. These samples encompassed tumor tissues as well as paired adjacent normal tissues, with comprehensive clinical data gathered from the corresponding patients. Utilizing these samples, we established the most extensive hepatoblastoma tissue microarray (TMA) to date. Subsequent analysis of publicly accessible databases concerning HB revealed a notable increase in IMPDH2 expression at the transcriptional level, significantly correlating with instances of metastasis and recurrence.

Further evaluation of IMPDH2 protein expression using the TMA platform confirmed its elevated levels in HB. Integration of clinical parameters revealed a significant association, with patients exhibiting heightened IMPDH2 expression tending to present at older ages (Table  1 ), validating prior research findings (Haeberle et al. 2020 ). Moreover, approximately 20% of pediatric HB cases present with metastatic disease at diagnosis, predominantly affecting the lungs and significantly impacting patient prognosis(Hu et al. 2021 ; Angelico et al. 2019 ). Our investigation identified a strong correlation between HB metastasis and increased IMPDH2 expression. Elevated AFP levels serve as a crucial diagnostic criterion for HB, with the majority of patients demonstrating abnormally high levels. Clinical conditions often parallel AFP levels, and existing literature suggests that both low and extremely high AFP levels in pediatric HB patients correspond to poorer prognoses (Meyers et al. 2017 ; Schweinitz et al. 1997 ). Our findings indicate that among patients with heightened IMPDH2 expression, there is a notably higher prevalence of individuals with AFP levels exceeding 10,000 ng/mL, aligning with previous literature findings in patients exhibiting high IMPDH2 expression. Remarkably, both overall survival (OS) and event-free survival (EFS) demonstrated a significant decline in the cohort characterized by increased IMPDH2 expression, mirroring outcomes observed in various solid malignancies(Duan et al. 2018 ; He et al. 2018 ; Shireman et al. 2021 ). These outcomes underscore the robust reliability of our TMA platform in identifying novel biomarkers. We propose that our platform holds promise for uncovering additional pertinent biomarkers in future research endeavors.

In addition to the cell line sequencing outcomes following IMPDH2 knockdown, which affirmed the correlation between IMPDH2 and the cell cycle, our analysis of GSE131329 yielded substantial findings. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway examination (Supplemental Fig.  1 ) identified the cell cycle and Wnt signaling pathway as the foremost pathways of significance. The Wnt/beta-catenin pathway, a pivotal developmental pathway associated with progenitor/stem cells, plays a critical role in activating and expanding these cells during embryogenesis and liver regeneration, thereby facilitating hepatic homeostasis(Monga 2015 ; Russell and Monga 2018 ). Notably, HB demonstrates the highest incidence of beta-catenin mutations among human cancers, closely associated with aberrant Wnt/beta-catenin signaling, reaching rates of up to 90%. This not only validates the reliability of the dataset but also substantiates our investigation. This is evident in our observation that the inhibition of IMPDH2 expression in HB cells resulted in the arrest of the cell cycle at the G0/G1 phase.

Purine nucleotides hold a pivotal role in governing cell cycle dynamics, where IMPDH2 actively participates in their synthesis. These nucleotides are indispensable for DNA and RNA construction, fundamental processes driving cell growth and division. Consequently, IMPDH2 indirectly modulates cell cycle progression by influencing the synthesis of purine nucleotides. Our findings substantiate an expanding body of literature indicating IMPDH2 involvement across diverse cancer types. Notably, heightened IMPDH2 expression in colon cancer cells has been linked to methotrexate resistance(Peñuelas et al. 2005 ). In glioblastoma, IMPDH2 amplifies rRNA and tRNA synthesis, fostering accelerated cell proliferation(Kofuji et al. 2019 ). Furthermore, elevated IMPDH2 levels have demonstrated suppression of cancer cell apoptosis through the regulation of multifaceted pathways, including the PI3K/AKT/mTOR and PI3K/c-Myc/AFF4 pathways (Gao et al. 2023 ; Ni et al. 2023 ). In this current investigation, we elucidate a novel mechanism whereby IMPDH2 augments the proliferative and tumorigenic potential of HB cells by modulating the cell cycle pathway.

Surprisingly, our investigation unveiled mycophenolate mofetil (MMF), an FDA-approved medication widely used for post-transplant rejection, as a specific inhibitor of IMPDH2(Lipsky 1996 ). Its combination with doxorubicin (DOX) demonstrated a notably synergistic effect. Therefore, we postulate that, for post-transplant HB patients, utilizing MMF, as opposed to other immunosuppressants, may offer a dual benefit of anti-rejection and tumor suppression. This hypothesis warrants further substantiation through clinical investigations, forming the cornerstone of our subsequent research endeavors.

In summary, our identification of IMPDH2 as a novel prognostic marker and therapeutic target in HB, particularly in cases of metastasis or recurrence, suggests that targeting this factor holds potential to enhance patient prognosis and overall quality of life.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

This study was funded by Shanghai Science and Technology Development Foundation (Outstanding academic leader) (HF, 23XD1423100, National Natural Science Foundation, China (XQ, 82241221). G-H. The HSA Synergy Score was computed using the online analytical tool SynergyFinder. I-K. Apoptosis in HB cell lines under single (40 nM DOX; 1µM MMF) and combination drug treatments was analyzed using flow cytometry.

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Linman Li, Yichi Wu, Hong-ting Huang and Yi Zhou contributed equally to this work.

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Department of Liver Surgery, Renji Hospital (Punan Branch), School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China

Linman Li, Yichi Wu, Hong-ting Huang, June-kong Yong, Zicheng Lv, Jie Zhao, Zhifeng Xi, Hao Feng & Qiang Xia

Clinical Research Unit, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China

Zicheng Lv & Hao Feng

Shanghai Engineering Research Centre of Transplantation and Immunology, Shanghai, 200127, China

Linman Li, Yichi Wu, Yi Zhou, Xuelin Xiang, Zhifeng Xi, Hao Feng & Qiang Xia

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LLM, FH, and XQ participated in the conception and design of this study. HHT, LLM, and FH was the project manager and coordinated patient recruitment. LLM, ZZJ, YJ, HHT, TH were involved in the acquisition, analysis, or interpretation of data. LLM, WYC, LZ, HHT and ZZJ drafted the manuscript. All the authors contributed to the critical review and final approval of the manuscript. FH, XQ, and LZC accessed and verified the underlying study data. All authors were responsible for the decision to submit the manuscript.

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Li, L., Wu, Y., Huang, Ht. et al. IMPDH2 suppression impedes cell proliferation by instigating cell cycle arrest and stimulates apoptosis in pediatric hepatoblastoma. J Cancer Res Clin Oncol 150 , 377 (2024). https://doi.org/10.1007/s00432-024-05858-4

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Received : 28 March 2024

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DOI : https://doi.org/10.1007/s00432-024-05858-4

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