Matrix remodeling-associated protein 8 is a marker of a subset of cancer-associated fibroblasts in pancreatic cancer.

Cancer-associated fibroblasts (CAFs), a compartment of the tumor microenvironment, were previously thought to be a uniform cell population that promotes cancer progression. However, recent studies have shown that CAFs are heterogeneous and that there are at least two types of CAFs, that is, cancer-promoting and -restraining CAFs. We previously identified Meflin as a candidate marker of cancer-restraining CAFs (rCAFs) in pancreatic ductal adenocarcinoma (PDAC). The precise nature of rCAFs, however, has remained elusive owing to a lack of understanding of their comprehensive gene signatures. Here, we screened genes whose expression correlated with Meflin in single-cell transcriptomic analyses of human cancers. Among the identified genes, we identified matrix remodeling-associated protein 8 (MXRA8), which encodes a type I transmembrane protein with unknown molecular function. Analysis of MXRA8 expression in human PDAC samples showed that MXRA8 was differentially co-expressed with other CAF markers. Moreover, in patients with PDAC or syngeneic tumors developed in MXRA8-knockout mice, MXRA8 expression did not affect the roles of CAFs in cancer progression, and the biological importance of MXRA8+ CAFs is still unclear. Overall, we identified MXRA8 as a new CAF marker; further studies are needed to determine the relevance of this marker.

Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer.

The cancer­stromal interaction has been demonstrated to promote tumor progression, and cancer-associated fibroblasts (CAFs), which are the main components of stromal cells, have attracted attention as novel treatment targets. Chitinase 3-like 1 (CHI3L1) is a chitinase-like protein, which affects cell proliferation and angiogenesis. However, the mechanisms through which cells secrete CHI3L1 and through which CHI3L1 mediates tumor progression in the cancer microenvironment are still unclear. Accordingly, the present study assessed the secretion of CHI3L1 in the microenvironment of colorectal cancer and evaluated how CHI3L1 affects tumor angiogenesis. CAFs and normal fibroblasts (NFs) established from colorectal cancer tissue, and human colon cancer cell lines were evaluated using immunostaining, cytokine antibody array, RNA interference, reverse transcription-quantitative PCR (RT-qPCR), ELISA, western blotting and angiogenesis assays. The expression and secretion of CHI3L1 in CAFs were stronger than those in NFs and colorectal cancer cell lines. In addition, interleukin-13 receptor α2 (IL-13Rα2), a receptor for CHI3L1, was not expressed in colorectal cancer cell lines, but was expressed in fibroblasts, particularly CAFs. Furthermore, the expression and secretion of IL-8 in CAFs was stronger than that in NFs and cancer cell lines, and recombinant CHI3L1 addition increased IL-8 expression in CAFs, whereas knockdown of CHI3L1 suppressed IL-8 expression. Furthermore, IL-13Rα2 knockdown suppressed the enhancement of IL-8 expression induced by CHI3L1 treatment in CAFs. For vascular endothelial growth factor-A (VEGFA), similar results to IL-8 were observed in an ELISA for comparison of secretion between CAFs and NFs and for changes in secretion after CHI3L1 treatment in CAFs; however, no significant differences were observed for changes in expression after CHI3L1 treatment or IL-13Rα2 knockdown in CAFs assessed using RT-qPCR assays. Angiogenesis assays revealed that tube formation in vascular endothelial cells was suppressed by conditioned medium from CAFs with the addition of human CHI3L1 neutralizing antibodies compared with control IgG, and also suppressed by conditioned medium from CAFs transfected with CHI3L1, IL-8 or VEGFA small interfering RNA compared with negative control small interfering RNA. Overall, the present findings indicated that CHI3L1 secreted from CAFs acted on CAFs to increase the secretion of IL-8, thereby affecting tumor angiogenesis in colorectal cancer.

Organ Specificity and Heterogeneity of Cancer-Associated Fibroblasts in Colorectal Cancer.

Fibroblasts constitute a ubiquitous mesenchymal cell type and produce the extracellular matrix (ECM) of connective tissue, thereby providing the structural basis of various organs. Fibroblasts display differential transcriptional patterns unique to the organ of their origin and they can be activated by common stimuli such as transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) signaling. Cancer-associated fibroblasts (CAFs) reside in the cancer tissue and contribute to cancer progression by influencing cancer cell growth, invasion, angiogenesis and tumor immunity. CAFs impact on the tumor microenvironment by remodeling the ECM and secreting soluble factors such as chemokines and growth factors. Differential expression patterns of molecular markers suggest heterogeneous features of CAFs in terms of their function, pathogenic role and cellular origin. Recent studies elucidated the bimodal action of CAFs on cancer progression and suggest a subgroup of CAFs with tumor-suppressive effects. This review attempts to describe cellular features of colorectal CAFs with an emphasis on their heterogeneity and functional diversity.

Mesenchymal Stem Cells and Extracellular Vesicles in Osteosarcoma Pathogenesis and Therapy.

Osteosarcoma (OS) is an aggressive bone tumor that mainly affects children and adolescents. OS has a strong tendency to relapse and metastasize, resulting in poor prognosis and survival. The high heterogeneity and genetic complexity of OS make it challenging to identify new therapeutic targets. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into adipocytes, osteoblasts, or chondroblasts. OS is thought to originate at some stage in the differentiation process of MSC to pre-osteoblast or from osteoblast precursors. MSCs contribute to OS progression by interacting with tumor cells via paracrine signaling and affect tumor cell proliferation, invasion, angiogenesis, immune response, and metastasis. Extracellular vesicles (EVs), secreted by OS cells and MSCs in the tumor microenvironment, are crucial mediators of intercellular communication, driving OS progression by transferring miRNAs/RNA and proteins to other cells. MSC-derived EVs have both pro-tumor and anti-tumor effects on OS progression. MSC-EVs can be also engineered to deliver anti-tumor cargo to the tumor site, which offers potential applications in MSC-EV-based OS treatment. In this review, we highlight the role of MSCs in OS, with a focus on EV-mediated communication between OS cells and MSCs and their role in OS pathogenesis and therapy.

Cancer Associated Fibroblasts Derived from Pancreatic Adenocarcinoma and Their Role in Cell Migration.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) shows poor survival and early systemic dissemination. Cancer associated fibroblasts (CAFs) enhance migration and invasion of cancer cells. We aimed to investigate the role of CAFs in cell migration and their underlying paracrine effects. MATERIALS AND METHODS: Using Transwell® migration assays, PDAC cells (PANC-1) and three distinct types of fibroblasts were analyzed: CAFs, genetically transformed human foreskin-fibroblasts (BJeLR), and non-transformed human foreskin-fibroblasts (VH7). IL6 in the culture supernatant was measured to investigate paracrine communication in monocultures and direct/indirect cocultures. RESULTS: CAFs showed a significantly higher capacity to migrate in vitro when compared to benign fibroblasts (p=0.009). They also facilitated the migration of PDAC cells in coculture (p=0.001). Neither BJeLR, nor VH7 displayed such features. This was accompanied by a significant increase in IL-6 when CAFs were cocultured with PANC-1 (p=0.009). CONCLUSION: CAFs are a key element of intra-tumoral migration and should be further investigated as a potential therapeutic target.

Cancer­associated fibroblast­induced M2­polarized macrophages promote hepatocellular carcinoma progression via the plasminogen activator inhibitor­1 pathway.

Targeting the tumor stroma is an important strategy in cancer treatment. Cancer­associated fibroblasts (CAFs) and tumor­associated macrophages (TAMs) are two main components in the tumor microenvironment (TME) in hepatocellular carcinoma (HCC), which can promote tumor progression. Plasminogen activator inhibitor­1 (PAI­1) upregulation in HCC is predictive of unfavorable tumor behavior and prognosis. However, the crosstalk between cancer cells, TAMs and CAFs, and the functions of PAI­1 in HCC remain to be fully investigated. In the present study, macrophage polarization and key paracrine factors were assessed during their interactions with CAFs and cancer cells. Cell proliferation, wound healing and Transwell and Matrigel assays were used to investigate the malignant behavior of HCC cells in vitro. It was found that cancer cells and CAFs induced the M2 polarization of TAMs by upregulating the mRNA expression levels of CD163 and CD206, and downregulating IL­6 mRNA expression and secretion in the macrophages. Both TAMs derived from cancer cells and CAFs promoted HCC cell proliferation and invasion. Furthermore, PAI­1 expression was upregulated in TAMs after being stimulated with CAF­conditioned medium and promoted the malignant behavior of the HCC cells by mediating epithelial­mesenchymal transition. CAFs were the main producer of C­X­C motif chemokine ligand 12 (CXCL12) in the TME and CXCL12 contributed to the induction of PAI­1 secretion in TAMs. In conclusion, the results of the present study suggested that CAFs promoted the M2 polarization of macrophages and induced PAI­1 secretion via CXCL12. Furthermore, it was found that PAI­1 produced by the TAMs enhanced the malignant behavior of the HCC cells. Therefore, these factors may be targets for inhibiting the crosstalk between tumor cells, CAFs and TAMs.

Improving function of cytotoxic T-lymphocytes by transforming growth factor-ß inhibitor in oral squamous cell carcinoma.

Immunotherapy with immune-checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor-ß (TGF-ß) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T-cells (Tregs) and cancer-associated fibroblasts and inhibiting the function of cytotoxic T-lymphocytes (CTLs) and natural killer cells. TGF-ß may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF-ß on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T-cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF-ß suppressed the function of antigen-specific CTLs in the priming and effector phases in vitro. Additionally, TGF-ß inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T-cell/Treg ratio and between TGFB1 mRNA expression and the Ki-67 expression in CD8+ T-cells, indicating that TGF-ß also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF-ß function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune-checkpoint inhibitors and TGF-ß inhibitors, for OSCCs.

Protocol to establish cancer-associated fibroblasts from surgically resected tissues and generate senescent fibroblasts.

Accumulating evidence suggests that the malignant behavior of cancer is influenced by stromal activity in the tumor microenvironment. Cancer-associated fibroblasts (CAFs), which are the main component of the cancerous stroma, play an important role in cancer development. Here, we describe a protocol to establish CAFs from surgically resected tissues. CAFs could be a vital tool for understanding the microenvironment and its impact on tumor progression and metastasis. Moreover, we generated inflammation-induced senescent fibroblasts that more closely mimic the tumor microenvironment. For complete details on the use and execution of this protocol, please refer to Yasuda et al. (2021).

Impairment of a distinct cancer-associated fibroblast population limits tumour growth and metastasis.

Profiling studies have revealed considerable phenotypic heterogeneity in cancer-associated fibroblasts (CAFs) present within the tumour microenvironment, however, functional characterisation of different CAF subsets is hampered by the lack of specific markers defining these populations. Here we show that genetic deletion of the Endo180 (MRC2) receptor, predominantly expressed by a population of matrix-remodelling CAFs, profoundly limits tumour growth and metastasis; effects that can be recapitulated in 3D co-culture assays. This impairment results from a CAF-intrinsic contractility defect and reduced CAF viability, which coupled with the lack of phenotype in the normal mouse, demonstrates that upregulated Endo180 expression by a specific, potentially targetable CAF subset is required to generate a supportive tumour microenvironment. Further, characterisation of a tumour subline selected via serial in vivo passage for its ability to overcome these stromal defects provides important insight into, how tumour cells adapt to a non-activated stroma in the early stages of metastatic colonisation.

Perivenous Stellate Cells Are the Main Source of Myofibroblasts and Cancer-Associated Fibroblasts Formed After Chronic Liver Injuries.

BACKGROUND AND AIMS: Studies of the identity and pathophysiology of fibrogenic HSCs have been hampered by a lack of genetic tools that permit specific and inducible fate-mapping of these cells in vivo. Here, by single-cell RNA sequencing of nonparenchymal cells from mouse liver, we identified transcription factor 21 (Tcf21) as a unique marker that restricted its expression to quiescent HSCs. APPROACH AND RESULTS: Tracing Tcf21+ cells by Tcf21-CreER (Cre-Estrogen Receptor fusion protein under the control of Tcf21 gene promoter) targeted ~10% of all HSCs, most of which were located at periportal and pericentral zones. These HSCs were quiescent under steady state but became activated on injuries, generating 62%-67% of all myofibroblasts in fibrotic livers and ~85% of all cancer-associated fibroblasts (CAFs) in liver tumors. Conditional deletion of Transforming Growth Factor Beta Receptor 2 (Tgfbr2) by Tcf21-CreER blocked HSC activation, compromised liver fibrosis, and inhibited liver tumor progression. CONCLUSIONS: In conclusion, Tcf21-CreER-targeted perivenous stellate cells are the main source of myofibroblasts and CAFs in chronically injured livers. TGF-ß signaling links HSC activation to liver fibrosis and tumorigenesis.

Small extracellular vesicles containing miR-192/215 mediate hypoxia-induced cancer-associated fibroblast development in head and neck squamous cell carcinoma.

The mechanisms underlying the hypoxic cancer cell-mediated differentiation of cancer-associated fibroblasts (CAFs) have not been elucidated yet. The present study showed that the hypoxic head and neck squamous cell carcinoma (HNSCC) cells promoted CAF-like differentiation through secreting TGF-ß and small extracellular vesicles (sEVs) that contain enhanced levels of miR-192/215 family miRNAs. Caveolin-1 (CAV1), which is a target gene of miR-192/215, inhibited the TGF-ß/SMAD signaling and promoted CAF-like differentiation of the fibroblasts. Restoring the levels of CAV1 inhibited the hypoxic sEV- and TGF-ß-induced CAF-like differentiation. The enhanced levels of miR-192/215 encapsulated in the HNSCC tissue-derived sEVs (but not serum-derived sEVs) indicated hypoxic and aggressive cancer stroma. miR-215 in the tumor tissue-derived sEVs (but not circulating sEVs) was correlated with poor overall survival of patients with HNSCC. This study demonstrated that sEVs function as a "courier" to deliver miRNAs from the cancer cells to the fibroblasts, which promotes the remodeling of the hypoxic tumor microenvironment, and that cancer tissue-derived sEV could potentially serve as a source of biomarker.

Re-expression of REG family and DUOXs genes in CRC organoids by co-culturing with CAFs.

Organoids derived from epithelial tumors have recently been utilized as a preclinical model in basic and translational studies. This model is considered to represent the original tumor in terms of 3D structure, genetic and cellular heterogeneity, but not tumor microenvironment. In this study, we established organoids and paired cancer-associated fibroblasts (CAFs) from surgical specimens of colorectal carcinomas (CRCs), and evaluated gene expression profiles in organoids with and without co-culture with CAFs to assess interactions between tumor cells and CAFs in tumor tissues. We found that the expression levels of several genes, which are highly expressed in original CRC tissues, were downregulated in organoids but re-expressed in organoids by co-culturing with CAFs. They comprised immune response- and external stimulus-related genes, e.g., REG family and dual oxidases (DUOXs), which are known to have malignant functions, leading tumor cells to proliferative and/or anti-apoptotic states and drug resistant phenotypes. In addition, the degree of differential induction of REG1 and DUOX2 in the co-culture system varied depending on CAFs from each CRC case. In conclusion, the co-culture system of CRC organoids with paired CAFs was able to partially reproduce the tumor microenvironment.

Mechanism of tumour microenvironment in the progression and development of oral cancer.

Oral cancer has been a major problem all across the globe, majorly in the developing countries. With a growing emphasis in the field of cancer research, the contribution of the tumour microenvironment has been gaining a lot of importance in identifying the role of components other than the tumour cells that cause the development of cancer, thus changing the outlook. The review will shed light on the studies that describe the role of microenvironment, its components as well as summarize the studies related to their mechanism in the progression of oral cancer. The literature for the review was derived mainly from Google Scholar and PubMed, in particular concentrating on the most recent papers published in 2019 and 2020, by using the keywords "Cancer, Oral Cancer, Metastasis, OSCC, Tumour microenvironment, CAFs, ECM, Cytokines, Hypoxia, Therapeutics targeting the microenvironment". The study provides insight into the world of micro-environmental regulation of oral cancer, the mechanism by which they interact and how to exploit it as a potential therapeutic haven for treating the disease. The components Cancer-Associated Fibroblasts (CAFs), Tumour-associated Macrophages (TAMs), Tumour-associated neutrophils (TANs), Hypoxic environment, myeloid-derived stem cells (MDSCs) and T regulatory (Tregs) cells and underlying mechanisms that control them will be the targets of study to understand the microenvironment.

Expression of asporin reprograms cancer cells to acquire resistance to oxidative stress.

Asporin (ASPN), a small leucine-rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC-43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC-43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1α and upregulated lactate dehydrogenase A (LDHA) and PDH-E1α, suggesting that ASPN reprograms HSC-43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC-44PE. By contrast, 44As3 cells expressed high levels of HIF1α in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN-/- mice revealed that growth of HSC-43 tumors with increased micro blood vessel density was significantly accelerated by ASPN; however, ASPN increased the invasion depth of both HSC-43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1α-mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44-Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas.

CAFs and Cancer Cells Co-Migration in 3D Spheroid Invasion Assay.

In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell-cell interactions and cell-matrix adhesions. Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

Characterization and oncolytic virus targeting of FAP-expressing tumor-associated pericytes in glioblastoma.

Cancer-associated fibroblasts (CAFs) are activated fibroblasts constituting the major stromal components in many types of cancer. CAFs contribute to hallmarks of cancer such as proliferation, invasion and immunosuppressive tumor microenvironment, and are associated with poor prognosis of patients with cancer. However, in glioblastoma (GBM), the most common and aggressive primary malignant brain tumor, our knowledge about CAFs or CAF-like stromal cells is limited. Here, using commonly accepted CAF markers, we characterized CAF-like cell populations in clinical glioma specimens and datasets along with mouse models of GBM. We found that tumor-associated pericytes marked by co-expression of fibroblast activation protein α (FAP) and PDGFRß represent major stromal cell subsets in both human GBM and mouse GBM models, while a fraction of mesenchymal neoplastic cells also express FAP in patient tumors. Since oncolytic viruses can kill cancer cells and simultaneously modulate the tumor microenvironment by impacting non-neoplastic populations such as immune cells and tumor vasculature, we further investigated the ability of oncolytic viruses to target GBM-associated stromal cells. An oncolytic adenovirus, ICOVIR15, carrying ∆24-E1A and an RGD-fiber, infects and depletes FAP+ pericytes as well as GBM cells in murine GBM. Our study thus identifies FAP+/PDGFRß+ pericytes as a major CAF-like stromal cell population in GBM, and highlights the unique property of this oncolytic adenovirus to target both GBM cells and GBM-associated stromal FAP+ cells.

The promising role of noncoding RNAs in cancer-associated fibroblasts: an overview of current status and future perspectives.

As the most important component of the stromal cell population in the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) are crucial players in tumor initiation and progression. The interaction between CAFs and tumor cells, as well as the resulting effect, is much greater than initially expected. Numerous studies have shown that noncoding RNAs (ncRNAs) play an irreplaceable role in this interplay, and related evidence continues to emerge and advance. Under the action of ncRNAs, normal fibroblasts are directly or indirectly activated into CAFs, and their metabolic characteristics are changed; thus, CAFs can more effectively promote tumor progression. Moreover, via ncRNAs, activated CAFs can affect the gene expression and secretory characteristics of cells, alter the TME and enhance malignant biological processes in tumor cells to contribute to tumor promotion. Previously, ncRNA dysregulation was considered the main mechanism by which ncRNAs participate in the crosstalk between CAFs and tumor cells. Recently, however, exosomes containing ncRNAs have been identified as another vital mode of interaction between these two types of cells, with a more direct and clear function. Gaining an in-depth understanding of ncRNAs in CAFs and the complex regulatory network connecting CAFs with tumor cells might help us to establish more effective and safer approaches for cancer therapies targeting ncRNAs and CAFs and offer new hope for cancer patients.

Neoplastic-Stromal Cell Cross-talk Regulates Matrisome Expression in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic reaction, warranting intense cancer-stroma communication. In this study, we interrogated the contribution of the BET family of chromatin adaptors to the cross-talk between PDAC cells and the tumor stroma. Short-term treatment of orthotopic xenograft tumors with CPI203, a small-molecule inhibitor of BET proteins, resulted in broad changes in the expression of genes encoding components of the extracellular matrix (matrisome) in both cancer and stromal cells. Remarkably, more than half of matrisome genes were expressed by cancer cells. In vitro cocultures of PDAC cells and cancer-associated fibroblasts (CAF) demonstrated that matrisome expression was regulated by BET-dependent cancer-CAF cross-talk. Disrupting this cross-talk in vivo resulted in diminished growth of orthotopic patient-derived xenograft tumors, reduced proliferation of cancer cells, and changes in collagen structure consistent with that of patients who experienced better survival. Examination of matrisome gene expression in publicly available data sets of 573 PDAC tumors identified a 65-gene signature that was able to distinguish long- and short-term PDAC survivors. Importantly, the expression of genes predictive of short-term survival was diminished in the cancer cells of orthotopic xenograft tumors of mice treated with CPI203. Taken together, these results demonstrate that inhibiting the activity BET proteins results in transcriptional and structural differences in the matrisome are associated with better patient survival. IMPLICATIONS: These studies highlight the biological relevance of the matrisome program in PDAC and suggest targeting of epigenetically driven tumor-stroma cross-talk as a potential therapeutic avenue.