Expression Of Cancer-associated Fibroblasts Markers Research Articles

Influential upregulation of KCNE4: Propelling cancer associated fibroblasts-driven colorectal cancer progression

BackgroundColorectal cancer (CRC) is a malignancy of remarkable heterogeneity and heightened morbidity. Cancer associated fibroblasts (CAFs) are abundant in CRC tissues and are essential for CRC growth. Here, we aimed to develop a CAF-related classifier for predicting the prognosis of CRC and identify critical pro-tumorigenic genes in CAFs.MethodThe mRNA expression and clinical information of CRC samples were sourced from two comprehensive databases, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Using a weighted gene co-expression network analysis (WGCNA) approach, CAF-related genes were identified and a CAF risk signature was developed through the application of univariate analysis and the least absolute shrinkage and selection operator (LASSO) Cox regression model. EdU cell proliferation assay, and transwell assay were performed to detect the oncogenic role of KCNE4 in CAFs.ResultsWe constructed a prognostic CAF model consisting of two genes (SFRP2 and KCNE4). CRC patients were classified into low- and high-CAF-risk groups using the median CAF risk score, and patients in the high-CAF-risk group had worse prognosis. Meanwhile, a higher risk score for CAFs was associated with greater stromal and CAF infiltrations, as well as higher expression of CAF markers. Furthermore, TIDE analysis indicated that patients with a high CAF risk score are less responsive to immunotherapy. Our further experiments had confirmed the strong correlation between KCNE4 and the malignant phenotypes of CAFs. Moreover, we had shown that KCNE4 could actively promote tumor-promoting phenotypes in CAFs, indicating its critical role in cancer progression.ConclusionThe two-gene prognostic CAF signature was constructed and could be reliable for predicting prognosis for CRC patients. Moreover, KCNE4 may be a promising strategy for the development of novel anti-cancer therapeutics specifically directed against CAFs.

Lactate Transporters Modulate Stromal Cell Remodeling in Myeloproliferative Neoplasms (MPN)

Introduction. Philadelphia-negative myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The diagnostic approach proposed by the World Health Organization (WHO) uses clinical features, bone marrow (BM) morphology, karyotype and molecular genetic tests to classify MPN subtypes. The BM morphology includes cellularity, erythropoiesis, and neutrophil granulopoiesis in context with specific features of megakaryocytes as well as the BM fiber content, especially in early-stage myelofibrosis that present with thrombocytosis and clinically mimic ET. Since alteration of hematopoiesis is constantly associated with profound modifications of the bone marrow (BM), we hypothesize that CD34+ cells from PMF can actively produce lactate and thus rewiring metabolism of immune cells and mesenchymal stromal cells (MSCs). In turn, this could favor immune evasion and remodeling of BM niche. Methods.Cell proliferation was assessed in vitro on primary MSCs, HS-5 cell line, and PBMC cells. Protein expression was performed by Western blot and immunofluorescence assay. The NCBI GEO database was used to select transcriptome datasets and to analyze gene expression in PMF patients. Cytokine detections were performed by Multiplex immunobead assay technology. An adult TPO-mimetic-induced PMF Zebrafish model was used for in vivo evaluation. An immunohistology assay was performed in MPN BM biopsies. Results.CD34+ cells from PMF patients significantly upregulated the lactate monocarboxylate transporters (MCT-1 and -4) compared to healthy CD34+ cells. Consistently, lactate concentration was higher in PMF sera compared to healthy controls. Since PMF patients showed an increased percentage of circulating immunosuppressive cells such as G- and M-MDSC and Treg, we incubated healthy peripheral blood mononucleated cells (PBMNCs) with PMF sera in the presence or absence of MCT1inhibitor (AZD3965) to evaluate the role of lactate in the expansion of these immunological subsets. We found an expansion of both Treg and M-MDSCs, which was reverted after blocking of circulating lactate by AZD3965. In order to study the effect of lactate in BM remodeling, we treated in vitro healthy MSCs with lactate (20 mM) for 24h. Interestingly, lactate induced a modification of extracellular matrix organization, as demonstrated by increased reticulin and collagen deposition and metalloproteases (MMP9, MMP2). Moreover, lactate increased calcium deposit and soluble osteogenic molecular signals (i.e. osteoprotegerin, osteonectin). These results also were obtained after incubation with PMF sera and were reverted after AZD3965 treatment. Since it has been demonstrated the role of cancer-associated fibroblast (CAF) in cancer development through ECM remodeling, we next evaluated the expression of CAF markers in MSCs after lactate exposure. α-SMA, FAP1, and TGFβ were found upregulated and this effect was reverted by AZD3965. Finally, we further confirmed these results in a Zebrafish animal model of PMF. Interestingly, in Zebrafish model we observed a reduction of MCT4 expression that was confirmed in the PBMNCs of the PMF patients. Conclusions. Inhibition of lactate metabolism may represent a strategy to inhibit cancer cells and contribute to restoring the anti-cancer immune response. Therefore, lactate metabolism may represent a promising target to counteract inflammation, osteosclerosis, and fibrosis in PMF patients.

Transcutaneous carbon dioxide application suppresses the expression of cancer-associated fibroblasts markers in oral squamous cell carcinoma xenograft mouse model.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Cancer-associated fibroblasts (CAFs) are the main stromal cells in the tumor microenvironment (TME). As CAFs promote tumor progression and hypoxia in the TME, regulating the conversion of normal fibroblasts (NFs) into CAFs is essential for improving the prognosis of patients with OSCC. We have previously reported the antitumor effects of transcutaneous carbon dioxide (CO2) application in OSCC. However, the effects of reducing hypoxia in the TME remain unclear. In this study, we investigated whether CO2 administration improves the TME by evaluating CAFs marker expression. Human OSCC cells (HSC-3) and normal human dermal fibroblasts (NHDF) were coinjected subcutaneously into the dorsal region of mice. CO2 gas was applied twice a week for 3 weeks. The tumors were harvested six times after transcutaneous CO2 application. The expression of CAFs markers (α-SMA, FAP, PDPN, and TGF-β) were evaluated by using real-time polymerase chain reaction and immunohistochemical staining. The expression of α-SMA, FAP, PDPN, and TGF-β was significantly increased over time after co-injection. In the CO2-treated group, tumor growth was significantly suppressed after treatment initiation. In addition, the mRNA expression of these markers was significantly inhibited. Furthermore, immunohistochemical staining revealed a significant decrease in the protein expression of all CAFs markers in the CO2-treated group. We confirmed that transcutaneous CO2 application suppressed CAFs marker expression and tumor growth in OSCC xenograft mouse model.

FAP, CD10, and GPR77-labeled CAFs cause neoadjuvant chemotherapy resistance by inducing EMT and CSC in gastric cancer

ObjectiveA significant proportion of patients can not benefit from neoadjuvant chemotherapy (NCT) due to drug resistance. Cancer-associated fibroblasts (CAFs) influence many biological behaviours of tumors, including chemo-resistance. This study aims to explore whether CAFs expressing FAP, CD10, and GPR77 affect the efficacy of NCT and the prognosis of patients with gastric cancer, and its mechanism.MethodsOne hundred seventy-one patients with locally progressive gastric adenocarcinoma who had undergone NCT and radical surgery were collected. Immunohistochemistry was used to detect the expression of FAP, CD10, and GPR77 in CAFs; the EMT markers (N-cadherin, Snail1, and Twist1) and the CSC markers (ALDH1, CD44, and LGR5) in gastric cancer cells. The χ2 test was used to analyze the relationship between the expression of CAF, EMT, and CSC markers and the clinicopathological factors, as well as the relationship between CAF markers and EMT, and CSC markers. Logistic regression and Cox risk regression were used to analyze the relationship between the expression of CAF, EMT, and CSC markers and TRG grading and OS; Kaplan-Meier analysis was used for survival analysis and plotting the curves.ResultsThe expression of CAF markers FAP, CD10, and GPR77 was closely associated with that of EMT markers; FAP and CD10 were closely related to CSC markers. In the univariate analysis of pathological response, CAF markers (FAP, CD10, GPR77), EMT markers (N-cadherin, Snail1, Twist1), and CSC markers (ALDH1, LGR5, CD44), were all closely associated with pathological response (all p < 0.05). Only Twist1 was an independent factor affecting pathological response in multifactorial analysis (p = 0.001). In a univariate analysis of OS, expression of FAP and CD10 in CAF, as well as expression of EMT biomarkers (N-cadherin, Snail1), were significant factors influencing patient prognosis (all p < 0.05). Multifactorial analysis revealed N-cadherin (p = 0.032) and Snail1 (p = 0.028), as independent prognostic factors affecting OS.ConclusionFAP, CD10, and GPR77 labeled CAF subgroup may lead to NCT resistance and poor prognosis by inducing EMT and CSC of gastric cancer cells in locally advanced gastric cancer patients.

Analysis of stromal PDGFR-β and α-SMA expression and their clinical relevance in brain metastases of breast cancer patients

BackgroundBreast cancer brain metastasis (BCBM) is a growing therapeutic challenge and clinical concern. Stromal cancer-associated fibroblasts (CAFs) are crucial factors in the modulation of tumorigeneses and metastases. Herein, we investigated the relationship between the expression of stromal CAF markers in metastatic sites, platelet-derived growth factor receptor-beta (PDGFR-β), and alpha-smooth muscle actin (α-SMA) and the clinical and prognostic variables in BCBM patients.MethodsImmunohistochemistry (IHC) of the stromal expression of PDGFR-β and α-SMA was performed on 50 cases of surgically resected BCBM. The expression of the CAF markers was analyzed in the context of clinico-pathological characteristics.ResultsExpression of PDGFR-β and α-SMA was lower in the triple-negative (TN) subtype than in other molecular subtypes (p = 0.073 and p = 0.016, respectively). And their expressions were related to a specific pattern of CAF distribution (PDGFR-β, p = 0.009; α-SMA, p = 0.043) and BM solidity (p = 0.009 and p = 0.002, respectively). High PDGFR-β expression was significantly related to longer recurrence-free survival (RFS) (p = 0.011). TN molecular subtype and PDGFR-β expression were independent prognostic factors of recurrence-free survival (p = 0.029 and p = 0.030, respectively) and TN molecular subtype was an independent prognostic factor of overall survival (p < 0.001).ConclusionsExpression of PDGFR-β in the stroma of BM was associated with RFS in BCBM patients, and the clinical implication was uniquely linked to the low expression of PDGFR-β and α-SMA in the aggressive form of the TN subtype.

YAP, a novel target regulates F-actin rearrangement-associated CAFs transformation and promotes colorectal cancer cell progression

Colorectal cancer (CRC) progression is strongly influenced by the tumor microenvironment (TME) in which cancer-associated fibroblasts (CAFs) are the major components influencing CRC growth and progression. The present study aimed to investigate the effect of YAP on F-actin arrangement in CAF transformation and the possibility of using YAP as a target for inhibiting CRC growth and progression. Conditioned media were collected from direct interaction between CRC cells and fibroblasts. CAF markers were investigated by flow cytometry, western blot analysis, and immunofluorescence assay in CM-treated fibroblasts. Promoting the CRC progression of conditioned media was determined in CRC cells by using MTT assay, fluorescence assay, wound healing assay, transwell migration assay, and tubulogenesis. The results showed that the conditioned media induced the expression of CAF markers associated with the central rearrangement of F-actin in colon fibroblasts, upregulating and promoting the nuclear translocation of YAP. The conditioned media also significantly promoted the proliferation, migration, invasion, and angiogenesis of CRC cells. Interestingly, Verteporfin, a YAP inhibitor during cocultivation, abolished the conversion of CAFs and inhibited proliferation, migration, invasion, and angiogenesis in CRC cells. Moreover, bioinformatics analysis was employed to determine the potential role of YAP as a prognostic marker in CRC patients from databases. The results suggested that YAP has higher expression in CRC patients and is associated with a poor prognosis. In conclusion, these findings demonstrate that YAP-related F-actin rearrangement may be a potential new target of combination therapy with a focus on targeting TME.

Proteomic characterisation of prostate cancer intercellular communication reveals cell type-selective signalling and TMSB4X-dependent fibroblast reprogramming

BackgroundIn prostate cancer, the tumour microenvironment (TME) represents an important regulator of disease progression and response to treatment. In the TME, cancer-associated fibroblasts (CAFs) play a key role in tumour progression, however the mechanisms underpinning fibroblast-cancer cell interactions are incompletely resolved. Here, we address this by applying cell type-specific labelling with amino acid precursors (CTAP) and mass spectrometry (MS)-based (phospho)proteomics to prostate cancer for the first time.MethodsReciprocal interactions between PC3 prostate cancer cells co-cultured with WPMY-1 prostatic fibroblasts were characterised using CTAP-MS. Signalling network changes were determined using Metascape and Enrichr and visualised using Cytoscape. Thymosin β4 (TMSB4X) overexpression was achieved via retroviral transduction and assayed by ELISA. Cell motility was determined using Transwell and random cell migration assays and expression of CAF markers by indirect immunofluorescence.ResultsWPMY-1 cells co-cultured with PC3s demonstrated a CAF-like phenotype, characterised by enhanced PDGFRB expression and alterations in signalling pathways regulating epithelial-mesenchymal transition, cytoskeletal organisation and cell polarisation. In contrast, co-cultured PC3 cells exhibited more modest network changes, with alterations in mTORC1 signalling and regulation of the actin cytoskeleton. The expression of the actin binding protein TMSB4X was significantly decreased in co-cultured WPMY-1 fibroblasts, and overexpression of TMSB4X in fibroblasts decreased migration of co-cultured PC3 cells, reduced fibroblast motility, and protected the fibroblasts from being educated to a CAF-like phenotype by prostate cancer cells.ConclusionsThis study highlights the potential of CTAP-MS to characterise intercellular communication within the prostate TME and identify regulators of cellular crosstalk such as TMSB4X.

BioIns-PO-01 - Distinction between CTCL and inflammatory dermatoses using mRNA expression of cancer-associated fibroblast markers from skin biopsies

BioIns-PO-01 - Distinction between CTCL and inflammatory dermatoses using mRNA expression of cancer-associated fibroblast markers from skin biopsies

Abstract 6137: Tumor-derived endothelin-1 recruits and activates fibroblasts to support tumor aggressiveness

Abstract The recruitment of fibroblasts to tumor and their activation to cancer-associated fibroblast (CAF) is an exploited strategy used by tumor to guide matrix remodeling, supporting cancer invasion and metastatic disease. CAFs are the major effectors of extracellular matrix (ECM) remodelling through secretion of collagens, cross-linking enzymes and proteases, and in turn engagement of integrins. Although several tumor cell-secreted factors have been identified in generating tumor-promoting stroma, there has been a great interest to define factors and associated molecular mechanisms involved in promoting CAF premetastatic function. Tumor-related endothelin-1 (ET-1) has an important role in the crosstalk between cancer and stromal cells supporting serous ovarian cancer (SOC) progression. By binding its receptors (ETAR and ETBR), ET-1 favours the recruitment of the protein β-arrestin-1 and the generation of signaling complexes regulating also cytoskeleton reorganization, thereby fine-tuning SOC cell invasion and metastasis. However, how the integration of ET-1 receptors might “educate” stroma to become permissive for invasion, supplying an altered ECM governing metastasis needs to be investigated. In this study, we identified endothelin-1 as a key factor in priming CAF conversion in SOC. We demonstrate that primary ovarian fibroblasts express ETA and ETB, along with β-arrestin1, and secrete ET-1; b) the autocrine and cancer-secreted ET-1 promotes fibroblast proliferation, and an increase in the expression of CAF markers (αSMA, vimentin and FAP), secretion of proinflammatory cytokines, collagen contraction ability and cell motility. Moreover, ET-1 induces activation of b1 integrin and downstream signaling. Mechanistically, ET1R/intb1 signaling allows ECM remodeling, either by promoting and invasive protrusion formation, invadosome, and collagen secretion. Importantly, therapeutical inhibition of ET-1 receptors with Ambrisentan and intβ1 with ATN-116 markedly reduced the ability of SOC cells to adhere in the intraperitoneal organs and to metastasize. Our findings support a model in which ET-1 educates the stromal fibroblasts to become permissive for tumor invasion and demonstrate for the time the significance of an ET-1-dependent integrin signaling in this process. Citation Format: Danila Del Rio, Valentina Caprara, Ilenia Masi, Francesca Spadaro, Sara Giannitelli, Alberto Rainer, Anna Bagnato, Laura Rosanò. Tumor-derived endothelin-1 recruits and activates fibroblasts to support tumor aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6137.

TGF-β1-activated cancer-associated fibroblasts promote breast cancer invasion, metastasis and epithelial-mesenchymal transition by autophagy or overexpression of FAP-α

Cancer-associated fibroblasts (CAFs) play an important role in the initiation, metastasis, and invasion of breast cancer. However, whether autophagy acts as a tumor promotion mechanism by inducing epithelial-mesenchymal transition (EMT) is still controversial and remains undefined at the mechanistic levels. In this study, we investigated whether autophagy or FAP-α is required for the invasion, pulmonary metastasis and EMT of breast cancer cells and underlying mechanism. We employed an in vitro model of NIH3T3 fibroblasts treated with H2O2 and confirmed that TGF-β1 could convert fibroblasts into CAFs through autophagy under oxidative stress in the tumor microenvironment. Modulation of autophagy by rapamycin, 3-methyladenine or ATG-5 knockdown regulated the expression of CAFs markers, suggesting a role of autophagy in the tumor promotion mechanism of TGF-β1-induced CAFs activation. Furthermore, we established an indirect co-culture model and a mixed xenograft as a corresponding in vivo model. We demonstrated that TGF-β1-activated CAFs promote tumor invasion, pulmonary metastasis and EMT, which act through autophagy and overexpression of FAP-α in both models, while autophagy inhibitor 3-methyladenine blocked these effects induced by TGF-β1-activated CAFs. Moreover, the co-localization of LC3β and EMT marker vimentin in mixed xenograft also revealed that TGF-β1-activated CAFs promote tumor growth, pulmonary metastasis, and EMT program partly through autophagy. In addition, knockdown of FAP-α resulted in reversed EMT and abolished tumor invasion and pulmonary metastasis induced by TGF-β1-activated CAFs. Taken together, we conclude that both autophagy and FAP-α are required for breast cancer cell invasion and metastasis. Targeting autophagy or FAP-α rather than both can serve as a potential approach to improve the prognosis for human breast cancer.

The interaction between cancer associated fibroblasts and tumor associated macrophages via the osteopontin pathway in the tumor microenvironment of hepatocellular carcinoma.

Background: Cancer-tumor associated macrophage (TAM)-cancer associated fibroblast (CAF) interactions are an important factor in the tumor microenvironment of hepatocellular carcinoma.Materials and Methods: Hepatic stellate cells (HSCs) were cultured with cancer cell-conditioned medium (Ca.-CM), TAM-CM and CAF-CM, and the expression of CAF markers were evaluated by RT-PCR. Whether HSCs cultured with Ca.-CM, TAM-CM and CAF-CM contributed to the enhanced malignancy of cancer cells was examined using proliferation, invasion and migration assays. Furthermore, the differences between these three types of CM were evaluated using cytokine arrays.Results: HSCs cultured with Ca.-CM, TAM-CM and CAF-CM showed significantly increased mRNA expression of αSMA, FAP and IL-6. All HSCs cultured with each CM exhibited significantly increased proliferation, invasion and migration of cancer cells. The osteopontin concentration was significantly higher in HSCs cultured with TAM-CM than the other CAF-CMs. Osteopontin inhibition significantly reduced osteopontin secretion from HSCs cultured with TAM-CM and suppressed the proliferation and invasion of cancer cells enhanced by HSCs cultured with TAM-CM.Conclusions: We observed enhanced osteopontin secretion from TAMs, and this increased osteopontin further promoted osteopontin secretion from HSCs cultured with TAM-CM, leading to increased malignancy. For the first time, we demonstrated the importance of cancer-TAM-CAF interactions via osteopontin in hepatocellular carcinoma.

Exosomes derived from chronic lymphocytic leukaemia cells transfer miR-146a to induce the transition of mesenchymal stromal cells into cancer-associated fibroblasts.

Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia and remains incurable. Mesenchymal stem cells (MSCs) can promote tumour progression by differentiating into cancer-associated fibroblasts (CAFs). However, the mechanisms by which tumour cells induce the transition of MSCs to CAFs are still largely undefined. Exosomes can regulate recipient cellular function by mediating intracellular communication. This study aimed to investigate whether CLL cells regulate the transition of bone marrow-derived MSCs (BM-MSCs) to CAFs via exosomal miR-146a delivery. The exosomes were isolated from CLL cell line MEC-1 (CLL-Exo) and then co-cultured with BM-MSCs. The expression of α-smooth muscle actin (α-SMA) and fibroblast-activated protein (FAP) were determined by immunofluorescence, quantitative real-time polymerase chain reaction and western blot. A luciferase reporter assay was performed to verify whether ubiquitin-specific peptidase 16 (USP16) was a target of miR-146a. CLL-Exo treatment up-regulated miR-146a and down-regulated expression of CAF markers (α-SMA and FAP) and USP16. The inducing effect of CLL-Exo on CAF marker expression was compromised when miR-146a expression was inhibited in CLL-Exo. USP16 was confirmed as a direct target of miR-146a and USP16 overexpression in BM-MSCs abrogated the CLL-Exo-mediated up-regulation of CAF markers. Collectively, CLL-Exo delivered miR-146a into BM-MSCs where miR-146a mediated transition of BM-MSCs into CAFs by targeting USP16.

Immunohistochemical analysis of the expression of cancer-associated fibroblast markers in esophageal cancer with and without neoadjuvant therapy.

Esophageal carcinoma (EC) is one of the most aggressive human malignancies with high rates of resistance to conventional anticancer treatment. Cancer-associated fibroblasts (CAFs) are an important part of the tumor microenvironment and associated with tumor progression. COL11A1, SPARC, and CD90 have been identified as rather specific CAF markers, with COL11A1 expression particularly shown to influence response to chemotherapy. We investigated the impact of CAFs in esophageal cancer with a special focus on response to neoadjuvant treatment (nTX). Two collections of esophageal carcinomas were investigated: 164 cases treated with primary resection and 256 cases receiving nTX before resection. The expression of CAF markers was determined using next-generation tissue microarray (ngTMA®) technology and immunohistochemistry. The presence of COL11A1 and SPARC in fibroblasts within both primary resected cases and nTX-treated cases was associated with unfavorable clinicopathological variables such as higher (y)pT category and lymphatic invasion (p<0.001 each). The presence of COL11A1-positive CAFs was associated with worse overall survival in primary resected cases (HR: 2.162, p = 0.004, CI 95% 1.275-3.686). While in tumors showing regression after nTX, COL11A1-positive CAFs were detected less frequently, SPARC-positive CAFs were enriched after nTX, in both responding and non-responding patients (p < 0.001). Our results support the concept of CAFs as an important factor of tumor promotion and maintenance in EC. The population of CAFs increases with tumor progression and decreases, partly depending on the subtype, after regression following nTX. CAFs may serve as potential target for future therapeutic approaches for these highly aggressive tumors.

Melanoma-derived exosomes induce reprogramming fibroblasts into cancer-associated fibroblasts via Gm26809 delivery

ABSTRACT Cancer-associated fibroblasts (CAFs) are activated fibroblasts and can interact with cancer cells to promote tumor progression. The process of how tumor cells reprogram normal fibroblasts (NFs) to tumor-promoting CAFs regulated by long non-coding RNA (lncRNA) remains incompletely understood. The tumor cells-released exosomes can induce reprogramming of NFs into CAFs. This study aimed to explore the role of melanoma-derived exosomes in regulating NF-CAF transition and to clarify whether lncRNA Gm26809 was involved in this process. The results showed that the exosomes secreted by melanoma cell B16F0 induced reprogramming of fibroblast NIH/3T3 cells into CAFs, as evidenced by increased expression of CAFs markers (α-SMA and FAP) and facilitated cell migration. Mechanistically, B16F0-secreted exosome delivered Gm26809 into NIH/3T3 cells where Gm26809 mediated reprogramming of fibroblast NIH/3T3 cells into CAFs. Furthermore, the conditioned medium from the co-culture of NIH/3T3 cells and B16F0-exosomes facilitated cell proliferation and migration in a melanoma cell line (Cloundman S91), and the effect was abrogated by Gm26809 knockdown in B16F0 cells. In summary, melanoma-derived exosomes facilitate melanoma cell proliferation and migration through reprogramming fibroblasts into tumor-promoting CAFs via transferring Gm26809.

Abstract 1907: Tenascin-C promotes the activation of mammary fibroblasts to calponin-expressing myofibroblasts

Abstract [Introduction] Tenascin-C (TN-C), an extracellular matrix molecules, is highly expressed in cancer stroma. Recent studies have demonstrated that cancer associated fibroblasts (CAFs) expressed TN-C in many tumors, including breast cancer. Here, we clarified the effects of TN-C on phenotype changes of the fibroblasts. [Methods] We examined the cell morphology, expression of CAFs markers, and contractile ability in the fibroblasts after TNC treatment in vitro, using human mammary gland fibroblasts (HMFs) and dermal ones (HDFs). We also investigated the correlation between the expression of the CAF markers and TNC deposition in human breast cancer tissues, using immunohistochemical staining. [Results] TN-C (10μg/mL) induced morphological changes of HMFs from spindle-shaped cells to stellate-shaped cells on plastic as well as on glass coverslips. After the treatment of TN-C, the protein expressions of α-SMA and calponin were significantly increased. Immunofluorescence analysis revealed TN-C increased the actin-stress fiber formation and the expression of α-SMA and calponin. Furthermore, TN-C promoted contraction of collagen gel by HMFs. These results indicated TN-C induces transformation of HMFs into high-contractile myofibroblasts. In human breast cancer tissues, α-SMA and calponin-positive fibroblasts were localized in TN-C-positive cancer stroma. Interestingly, calponin-expressing fibroblasts were not observed in the skin metastatic lesions of breast cancer. In addition, the increased expression of α-SMA and calponin after TN-C treatment in HDFs were significantly lower compared to HMFs in vitro. These results suggested the responses of TN-C on fibroblasts differs according to the residential tissues. Next, we examined the receptor to TNC and signaling pathway for TNC-induced change of HMFs. TN-C increased the colocalization of integrin αv and β1 in focal adhesion. Furthermore, phosphorylation of SMAD2 and SMAD3 were significantly increased and SMAD3 translocated from cytoplasm to nucleus after TN-C addition. Inhibition of TGF-β type I receptor (SB-505124) or SMAD3 (SIS3) suppressed the expression of α-SMA and calponin after TN-C treatment. [Conclusion] TNC could contribute cancer stroma formation characteristics of breast cancer tissues, following induction of phenotypical change to myofibroblasts and enhanced stromal contraction via integrin αvβ1/TGF-β/SMAD signaling. Phenotypic differences between HMFs and HDFs after TN-C administration may be present. Citation Format: Daisuke Katoh, Aya Noro, Kyoko Imanaka-Yoshida, Tomoko Ogawa, Toshimichi Yoshida. Tenascin-C promotes the activation of mammary fibroblasts to calponin-expressing myofibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1907.

Proteomics reveals NNMT as a master metabolic regulator of cancer-associated fibroblasts.

High grade serous carcinoma (HGSC) has a poor prognosis primarily due to its early dissemination throughout the abdominal cavity. Genomic and proteomic approaches have provided snapshots of the proteogenomics of ovarian cancer (OvCa)1,2, but a systematic examination of both the tumor and stromal compartments is critical to understanding OvCa metastasis. We developed a label-free proteomic workflow to analyze as few as 5,000 formalin-fixed, paraffin embedded cells microdissected from each compartment. The tumor proteome was stable during progression from in situ lesions to metastatic disease; however, the metastasis-associated stroma was characterized by a highly conserved proteomic signature, prominently including the methyltransferase nicotinamide N-methyltransferase (NNMT) and several proteins it regulates. Stromal NNMT expression was necessary and sufficient for functional aspects of the cancer associated fibroblast (CAF) phenotype, including the expression of CAF markers and the secretion of cytokines and oncogenic extracellular matrix. Stromal NNMT expression supported OvCa migration, proliferation, and in vivo growth and metastasis. Expression of NNMT in CAFs led to a depletion of S-adenosyl methionine (SAM) and a reduction in histone methylation associated with widespread gene expression changes in the tumor stroma. This work supports the use of ultra-low input proteomics to identify candidate drivers of disease phenotypes. NNMT is a central, metabolic regulator of CAF differentiation and cancer progression in the stroma that may be therapeutically targeted.

Role of Phosphodiesterase 5 (PDE5) in Breast Cancer Stroma: Implications for Targeted Therapy

Breast cancers are heterogeneous tissues comprised of multiple components, including tumor and microenvironment cells. Cancer‐associated fibroblasts (CAFs) are an important cell type in tumor‐associated stroma with multiple roles in tumor initiation and promotion. While the significance of CAFs is well recognized, the treatment of CAFs in clinical practice has not yet been established. Our previous studies have shown that the overexpression of phosphodiesterase (PDE) 5, a metallohydrolase which catalyzes the hydrolytic breakdown of cGMP, enhances the invasive potential of breast cancer cells and reduces survival in breast cancer patients. To extend these observations, we propose to assess the function of PDE5 in tumor microenvironment, to further highlight the potential benefit of targeting PDE5.Our results evidenced, for the first time, PDE5 mRNA and protein expression in CAFs isolated from biopsies of primary human breast tumors. PDE5 expression is increased in human breast cancer stroma compared with normal breast stroma and was correlated to a shorter overall survival in patients. Treatment with PDE5 inhibitors (i.e. sildenafil and tadalafil) significantly inhibited CAF proliferation, motility and invasiveness. To better examine the significance of PDE5 expression in stromal fibroblasts, PDE5 was stably integrated into mouse embryonic fibroblasts (MEFs) and cell proliferation, motility and invasion were investigated. PDE5 transformed MEFs towards a cancer‐associated fibroblast phenotype, as evidenced by increased expression of CAF markers along with enhanced proliferative, migratory and invasive capabilities. Coculture experiments showed that proliferation and motility of human breast cancer cells was increased by PDE5‐overexpressing MEFs compared to vector‐expressing MEFs, suggesting that stromal PDE5 contributes to heterotypic communications within breast cancer. An enriched production of the chemokine CXCL16 was revealed in PDE5‐expressing clones, as evidenced by cytokine array and confirmed by realtime PCR, suggesting a role for this molecule in mediating the tumor‐promoting effects of PDE5 expression in breast stroma. In line with these findings, exposure of CAFs to PDE5 inhibitors was associated with a reduced expression of CXCL16. More importantly, expression of CXCL16 in breast cancer stroma showed a strong correlation with PDE5 as well as with a poor patient survival.In conclusion, PDE5 is overexpressed in breast cancer stroma and enhances the tumor‐stimulatory activities of fibroblasts, highlighting this enzyme as a novel prognostic candidate and an attractive target for future therapy in breast cancers, having already addressed the delivery, stability, and toxicity issues of PDE5 inhibitors in other diseases.Support or Funding InformationMy First AIRC Grant (MFAG) #16899 to I. BaroneThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased expression of cancer-associated fibroblast markers at the invasive front and its association with tumor-stroma ratio in colorectal cancer

BackgroundThe tumor microenvironment has a critical role in regulating cancer cell behavior. Tumors with high stromal content are associated with poor patient outcome. The tumor-stroma ratio (TSR) identifies colorectal cancers (CRC) with poor patient prognosis based on hematoxylin & eosin stained sections. The desmoplastic reaction consists to a great extent of cancer-associated fibroblasts (CAFs) of which different subtypes are known. The aim of this study is to investigate and quantify CAFs present in the tumor stroma of CRC stratified by the TSR to possibly add prognostic significance to the TSR.MethodsThe expression of established CAF markers was compared between stroma-low and stroma-high tumors using transcriptomic data of 71 stage I – III CRC. Based on literature, fibroblast and stromal markers were selected to perform multiplex immunofluorescent staining on formalin fixed, paraffin-embedded tumor sections of patients diagnosed with stage III colon cancer. Antibodies against the following markers were used: αSMA, PDGFR -β, FAP, FSP1 and the stromal markers CD45 and CD31 as reference. The markers were subsequently quantified in the stroma using the Vectra imaging microscope.ResultsThe transcriptomic data showed that all CAF markers except one were higher expressed in stroma-high compared to stroma-low tumors. Histologically, stroma-high tumors showed a decreased number of FSP1+/CD45+ cells and a trend of an increased expression of FAP compared to stroma-low tumors. FAP was higher expressed at the invasive part compared to the tumor center in both stroma-high and stroma-low tumors.ConclusionsThe increased expression of FAP at the invasive part and in stroma-high tumors might contribute to the invasive behavior of cancer cells. Future functional experiments should investigate the contribution of FAP to cancer cell invasion. Combining the quantity of the stroma as defined by the TSR with the activity level of CAFs using the expression of FAP may result in an expanded stroma-based tool for patient stratification.

Exosomes Induce Fibroblast Differentiation into Cancer-Associated Fibroblasts through TGFβ Signaling.

A particularly important tumor microenvironment relationship exists between cancer cells and surrounding stromal cells. Fibroblasts, in response to cancer cells, become activated and exhibit myofibroblastic characteristics that favor invasive growth and metastasis. However, the mechanism by which cancer cells promote activation of healthy fibroblasts into cancer-associated fibroblasts (CAF) is still not well understood. Exosomes are nanometer-sized vesicles that shuttle proteins and nucleic acids between cells to establish intercellular communication. Here, bladder cancer-derived exosomes were investigated to determine their role in the activation of healthy primary vesical fibroblasts. Exosomes released by bladder cancer cells are internalized by fibroblasts and promoted the proliferation and expression of CAF markers. In addition, cancer cell-derived exosomes contain TGFβ and in exosome-induced CAFs SMAD-dependent signaling is activated. Furthermore, TGFβ inhibitors attenuated CAF marker expression in healthy fibroblasts. Therefore, these data demonstrate that bladder cancer cells trigger the differentiation of fibroblasts to CAFs by exosomes-mediated TGFβ transfer and SMAD pathway activation. Finally, exosomal TGFβ localized inside the vesicle and contributes 53.4% to 86.3% of the total TGFβ present in the cancer cell supernatant. This study highlights a new function for bladder cancer exosomes as novel modulators of stromal cell differentiation.Implication: This study identifies exosomal TGFβ as new molecular mechanism involved in cancer-associated fibroblast activation. Mol Cancer Res; 16(7); 1196-204. ©2018 AACR.

Expression of cancer-associated fibroblast markers in advanced colorectal cancer.

Colorectal cancer is one of the most common causes of mortality from cancer worldwide. Previous studies have demonstrated that cancer-associated fibroblasts (CAFs) promote neoangiogenesis and tumor growth for various tumors. The present study analyzed CAF markers, including α-smooth muscle actin (α-SMA), collagen I, platelet-derived growth factor receptor-β (PDGFR-β), and D2-40 (antibody recognizing podoplanin), and vessel markers, including cluster of differentiation (CD)31 and CD34, for 121 advanced colorectal cancer cases using a digital image analyzing technique. The association between CAF markers and vessel markers with clinicopathological factors was investigated. Furthermore, the association between CAF markers with each other, and their association with vessel markers was analyzed. Mean/median expression area of stromal and vessel markers in tumors were collagen I, 26.787%; D2-40, 1.372%; PDGFR-β, 11.646%; α-SMA-positive and desmin-negative myofibroblasts (α-SMA subtraction), 15.372%; CD31, 3.635%; and CD34, 2.226%. The expression area of α-SMA subtraction was significantly correlated with collagen I (P<0.001, correlation rho=0.509). High levels of α-SMA subtraction (P=0.002), collagen I (P=0.040), and PDGFR-β (P=0.040) expressions tended to be associated with high venous invasion. D2-40 did not correlate with other CAF and vessel markers. These results indicated that individual CAFs may have different expression patterns, and different strength effects for venous invasion in advanced colorectal cancer stroma.