Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis

The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.     

Fibroblast nemosis induces angiogenic responses of endothelial cells

Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-κB. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.     

Gastric Cancer Exosomes Trigger Differentiation of Umbilical Cord Derived Mesenchymal Stem Cells to Carcinoma-Associated Fibroblasts through TGF-β/Smad Pathway

Background

Mesenchymal stem cells (MSCs) promote tumor growth by differentiating into carcinoma-associated fibroblasts (CAFs) and composing the tumor microenvironment. However, the mechanisms responsible for the transition of MSCs to CAFs are not well understood. Exosomes regulate cellular activities by mediating cell-cell communication. In this study, we aimed to investigate whether cancer cell-derived exosomes were involved in regulating the differentiation of human umbilical cord-derived MSCs (hucMSCs) to CAFs.

Methodology/Principal Findings

We first showed that gastric cancer cell-derived exosomes induced the expression of CAF markers in hucMSCs. We then demonstrated that gastric cancer cell-derived exosomes stimulated the phosphorylation of Smad-2 in hucMSCs. We further confirmed that TGF-β receptor 1 kinase inhibitor attenuated Smad-2 phosphorylation and CAF marker expression in hucMSCs after exposure to gastric cancer cell-derived exosomes.

Conclusion/Significance

Our results suggest that gastric cancer cells triggered the differentiation of hucMSCs to CAFs by exosomes-mediated TGF-β transfer and TGF-β/Smad pathway activation, which may represent a novel mechanism for MSCs to CAFs transition in cancer.     

Tenascin-C,a Prognostic Determinant of Esophageal Squamous Cell Carcinoma

Background

Tenascin-C, an adhesion modulatory extracellular matrix molecule, is highly expressed in numerous human malignancies; thus, it may contribute to carcinogenesis and tumor progression. We explored the clinicopathological significance of Tenascin-C as a prognostic determinant of esophageal squamous cell carcinoma (ESCC).

Methods

In ESCC patient tissues and cell lines, the presence of isoforms were examined using western blotting. We then investigated Tenascin-C immunohistochemical expression in 136 ESCC tissue samples. The clinical relevance of Tenascin-C expression and the correlation between Tenascin-C expression and expression of other factors related to cancer-associated fibroblasts (CAFs) were also determined.

Results

Both 250 and 350 kDa sized isoforms of Tenascin-C were expressed only in esophageal cancer tissue not in normal tissue. Furthermore, both isoforms were also identified in all of four CAFs derived from esophageal cancer tissues. Tenascin-C expression was remarkably higher in ESCC than in adjacent non-tumor esophageal epithelium (p < 0.001). Tenascin-C expression in ESCC stromal fibroblasts was associated with patient’s age, tumor (pT) stage, lymph node metastasis, clinical stage, and cancer recurrence. Tenascin-C expression in cancer cells was correlated with an increase in tumor-associated macrophage (TAM) population, cancer recurrence, and hypoxia inducible factor1α (HIF1α) expression. Moreover, Tenascin-C overexpression in cancer cells and stromal fibroblasts was an independent poor prognostic factor for overall survival (OS) and disease-free survival (DFS). In the Cox proportional hazard regression model, Tenascin-C overexpression in cancer cells and stromal fibroblasts was a significant independent hazard factor for OS and DFS in ESCC patients in both univariate and multivariate analyses. Furthermore, Tenascin-C expression in stromal fibroblasts of the ESCC patients was positively correlated with platelet-derived growth factor α (PDGFRα), PDGFRβ, and smooth muscle actin (SMA) expression. The 5-year OS and DFS rates were remarkably lower in patients with positive expressions of both Tenascin-C and PDGFRα (p < 0.001), Tenascin-C and PDGFRβ (p < 0.001), Tenascin-C and SMA (p < 0.001), Tenascin-C and fibroblast activation protein (FAP) (p < 0.001), and Tenascin-C and fibroblast-stimulating protein-1 (FSP1) (p < 0.001) in ESCC stromal fibroblasts than in patients with negative expressions of both Tenascin-C and one of the abovementioned CAF markers.

Conclusion

Our results show that Tenascin-C is a reliable and significant prognostic factor in ESCC. Tenascin-C may thus be a potent ESCC therapeutic target.     

Weighted gene co‐expression network analysis can sort cancer‐associated fibroblast‐specific markers promoting bladder cancer progression

The role of cancer‐associated fibroblasts (CAFs) has been thoroughly investigated in tumour microenvironments but not in bladder urothelial carcinoma (BLCA). The cell fraction of CAFs gradually increased with BLCA progression. Weighted gene co‐expression network analysis (WGCNA) revealed a specific gene expression module of CAFs that are relevant to cancer progression and survival status. Fifteen key genes of the module were consistent with a fibroblast signature in single‐cell RNA sequencing, functionally related to the extracellular matrix, and significant in survival analysis and tumour staging. A comparison of the luminal‐infiltrated versus luminal‐papillary subtypes and fibroblast versus urothelial carcinoma cell lines and immunohistochemical data analysis demonstrated that the key genes were specifically expressed in CAFs. Moreover, these genes are highly correlated with previously reported CAF markers. In summary, CAFs play a major role in the progression of BLCA, and the 15 key genes act as BLCA‐specific CAF markers and can predict CAF changes. WGCNA can, therefore, be used to sort CAF‐specific gene set in cancer tissues.     

Fucosyltransferase 1 mediates angiogenesis,cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

Introduction

We previously reported that sialyl Lewis^y, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewis^y antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.

Methods

Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.

Results

Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.

Conclusions

These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.     

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

Background

Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells.

Methods

In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC–MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells.

Results

We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication.

Conclusions

Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

     

The role of CAF derived exosomal microRNAs in the tumour microenvironment of melanoma

Exosomes play a crucial role in the crosstalk between cancer associated fibroblasts (CAFs) and cancer cells, contributing to carcinogenesis and the tumour microenvironment. Recent studies have revealed that CAFs, normal fibroblasts and cancer cells all secrete exosomes that contain miRNA, establishing a cell-cell communication network within the tumour microenvironment. For example, miRNA dysregulation in melanoma has been shown to promote CAF activation via induction of epithelial-mesenchymal transition (EMT), which in turn alters the secretory phenotype of CAFs in the stroma. This review assesses the roles of melanoma exosomal miRNAs in CAF formation and how CAF exosome-mediated feedback signalling to melanoma lead to tumour progression and metastasis. Moreover, efforts to exploit exosomal miRNA-mediated network communication between tumour cells and their microenvironment, and their potential as prognostic biomarkers or novel therapeutic targets in melanoma will also be considered.     

Tumor-produced, active interleukin-1β regulates gene expression in carcinoma-associated fibroblasts

Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial–mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1β (IL1-β) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-β expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-β processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-β. IL1-β signaling was investigated by western blot and immunocytochemistry. IL1-β-regulated genes were analyzed by real-time qPCR.SCC-25 cells produced 16 kD active IL1-β, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NFκBα. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-β reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-β-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-β in the tumor cells leads to IL1-β-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression.     

The gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro

Background

It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro.

Results

A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p < 0.05). Bulk of the up-regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal transition (EMT) and generally take part in the developmental processes. These results were further confirmed using real-time qPCR. Moreover, a wound-healing assay and growth characteristics on Matrigel matrix showed that CAFs increase cancer cell migration and matrix invasion.

Conclusion

The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.     

Cancer-Associated Fibroblasts Promote Proliferation of Endometrial Cancer Cells

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.     

Caveolin-1 Expression Level in Cancer Associated Fibroblasts Predicts Outcome in Gastric Cancer

Aims

Altered expression of epithelial or stromal caveolin-1 (Cav-1) is observed in various types of human cancers. However, the clinical significance of Cav-1 expression in gastric cancer (GC) remains largely unknown. The present study aims to explore the clinicopathological significance and prognostic value of both tumor cells and cancer associated fibroblasts (CAFs) Cav-1 in GC.

Methods and Results

Quantum dots immunofluorescence histochemistry was performed to examine the expression of Cav-1 in 20 cases of gastritis without intestinal metaplasia (IM), 20 cases of gastritis with IM and 286 cases of GC. Positive rates of epithelial Cav-1 in gastritis without IM, gastritis with IM and GC showed a decreasing trend (P = 0.012). Low expression of Cav-1 in CAFs but not in tumor cells was an independent predictor of poor prognosis in GC patients (P = 0.034 and 0.005 respectively in disease free survival and overall survival). Cav-1 level in tumor cells and CAFs showed no significant correlation with classic clinicopathological features.

Conclusions

Loss of epithelial Cav-1 may promote malignant progression and low CAFs Cav-1 level herald worse outcome of GC patient, suggesting CAFs Cav-1 may be a candidate therapeutic target and a useful prognostic marker of GC.     

Nemosis, a novel way of fibroblast activation, in inflammation and cancer

Malignant cells when grown in suspension, as a rule, proliferate and can form spheroids that have been used as a model of tumor nodules, micrometastases and avascular tumors. In contrast, normal adherent cells cannot be stimulated to grow as multicellular aggregates. Now, recent results show that normal fibroblasts if forced to cluster (spheroid formation) do not grow but undergo a new pathway of cell activation (nemosis) leading to a massive proinflammatory, proteolytic and growth factor response. The clustering and activation are initiated by fibronectin-integrin interaction. The activated fibroblasts are able to modulate the behavior of cancer cells and, furthermore malignant cells boost this activation even further. In this model, the activation of fibroblasts terminates in programmed necrosis-like cell death. Activation of the tumor stroma, especially of fibroblasts, is of critical importance for tumor progression, although mechanisms leading to their activation are still largely uncharacterized. In summary, our results suggest that this kind of fibroblast activation (nemosis) may be involved in pathological conditions such as inflammation and cancer.     

Focal adhesion kinase is required for synovial fibroblast invasion,but not murine inflammatory arthritis

Introduction

Synovial fibroblasts invade cartilage and bone, leading to joint destruction in rheumatoid arthritis. However, the mechanisms that regulate synovial fibroblast invasion are not well understood. Focal adhesion kinase (FAK) has been implicated in cellular invasion in several cell types, and FAK inhibitors are in clinical trials for cancer treatment. Little is known about the role of FAK in inflammatory arthritis, but, given its expression in synovial tissue, its known role in invasion in other cells and the potential clinical availability of FAK inhibitors, it is important to determine if FAK contributes to synovial fibroblast invasion and inflammatory arthritis.

Methods

After treatment with FAK inhibitors, invasiveness of human rheumatoid synovial fibroblasts was determined with Matrigel invasion chambers. Migration and focal matrix degradation, two components of cellular invasion, were assessed in FAK-inhibited rheumatoid synovial fibroblasts by transwell assay and microscopic examination of fluorescent gelatin degradation, respectively. Using mice with tumor necrosis factor α (TNFα)–induced arthritis in which fak could be inducibly deleted, invasion and migration by FAK-deficient murine arthritic synovial fibroblasts were determined as described above and arthritis was clinically and pathologically scored in FAK-deficient mice.

Results

Inhibition of FAK in human rheumatoid synovial fibroblasts impaired cellular invasion and migration. Focal matrix degradation occurred both centrally and at focal adhesions, the latter being a novel site for matrix degradation in synovial fibroblasts, but degradation was unaltered with FAK inhibitors. Loss of FAK reduced invasion in murine arthritic synovial fibroblasts, but not migration or TNFα-induced arthritis severity and joint erosions.

Conclusions

FAK inhibitors reduce synovial fibroblast invasion and migration, but synovial fibroblast migration and TNFα-induced arthritis do not rely on FAK itself. Thus, inhibition of FAK alone is unlikely to be sufficient to treat inflammatory arthritis, but current drugs that inhibit FAK may inhibit multiple factors, which could increase their efficacy in rheumatoid arthritis.     

Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

Cancer-associated fibroblasts (CAFs) are commonly acquired activated extracellular matrix (ECM)-producing myofibroblasts, a phenotypes with multiple roles in hepatic fibrogenesis and carcinogenesis via crosstalk with cohabitating stromal/cancer cells. Here, we discovered a mechanism whereby CAF-derived cytokines enhance hepatocellular carcinoma (HCC) progression and metastasis by activating the circRNA-miRNA-mRNA axis in tumor cells. CAFs secreted significantly higher levels of CXCL11 than normal fibroblasts (NFs), and CXCL11 also had comparatively higher expressions in HCC tissues, particularly in metastatic tissues, than para-carcinoma tissues. Both CAF-derived and experimentally introduced CXCL11 promoted HCC cell migration. Likewise, CAFs promoted tumor migration in orthotopic models, as shown by an increased number of tumor nodules, whereas CXCL11 silencing triggered a decrease of it. CXCL11 stimulation upregulated circUBAP2 expression, which was significantly higher in HCC tissues than para-carcinoma tissues. Silencing circUBAP2 reversed the effects of CXCL11 on the expression of IL-1β/IL-17 and HCC cell migration. Further downstream, the IFIT1 and IFIT3 levels were significantly upregulated in HCC cells upon CXCL11 stimulation, but downregulated upon circUBAP2 silencing. IFIT1 or IFIT3 silencing reduced the expression of IL-17 and IL-1β, and attenuated the migration capability of HCC cells. Herein, circUBAP2 counteracted miR-4756-mediated inhibition on IFIT1/3 via sponging miR-4756. miR-4756 inhibition reversed the effects induced by circUBAP2 silencing on the IL-17 and IL-1β levels and HCC cell migration. In orthotopic models, miR-4756 inhibition also reversed the effects on metastatic progression induced by silencing circUBAP2.Subject terms: Tumour biomarkers, Cancer     

Cancer-associated fibroblasts: Secretions,interactions, and therapy

Tumor microenvironment (TME) could impose a great challenge for cancer targeted therapies. Immunosuppression within the TME creates a barrier between cancer cells and therapeutic approaches. A number of cells are hosted within this milieu, among them cancer-associated fibroblasts (CAFs) are the most abundant cell populations playing major roles in mediating an immunosuppressive TME. CAFs have cross-talks with almost all cells within the TME for reprogramming them into being tumorigenic. This reprogramming reduces the pre-existing tumor immunity and dampens the efficacy of chemotherapeutic approaches. CAFs would do this through releasing a myriad of factors to the TME making it an appropriate nest for tumor growth. The cells degrade and deposit extracellular matrix components, both of which are tumorigenic. Therefore, disruption of cross-talks between CAFs with other cells within the TME would be a promising approach in cancer targeted therapies. This approach is applicable through dampening dominant signals mediated by CAFs. Another interesting approach would be reprogramming of CAFs toward their normal counterpart. This would need identification of different subtypes for these cells and their functions. More knowledge is also required about selective markers for each CAF subtype.