Roles Of Carcinoma-associated Fibroblasts Research Articles

Abstract A108: Identification of distinct fibroblast populations with unique roles in PDAC progression and tumor immunity

Abstract The desmoplastic reaction in pancreatic ductal adenocarcinomas (PDAC) involves a significant accumulation of immune cells and fibroblasts. Although carcinoma-associated fibroblasts (CAFs) have been associated with immune suppression and immunotherapy resistance in PDAC mouse models, some studies have shown immune-promoting and tumor-suppressing roles of CAFs. The functional diversity of carcinoma-associated fibroblasts (CAFs) remains largely unknown, and identification of immune-regulating subsets would have a substantial impact in augmentation of immunotherapy efficacy. Employing histology, FACs, multiplex immunohistochemistry, single-cell RNA sequencing (sc-RNA-seq), and genetically engineered mouse models, we demonstrate that SMA+ cells are a dominant CAF population in PDAC with tumor-restraining properties (TS-CAFs), as opposed those of the FAP+ CAFs, which demonstrate tumor-promoting activity (TP-CAFs). Analysis of bulk tumor depleted of either TS-CAFs or TP-CAFs showed that TS-CAFs predominantly modulate extracellular matrix (ECM) production, facilitate cell-ECM adhesion, and regulate adaptive immunity, while TP-CAFs exhibit a lineage that is skewed towards a proinflammatory, chemokine-secreting phenotype. Further, scRNA-Seq analyses demonstrate that CAFs share distinct gene expression profiles characteristic of lymphocytic and myeloid lineages. Together our data distinguish two populations of CAFs, one that is tumor suppressing with roles in ECM remodeling and another that is tumor promoting with roles in cytokine production, both with immune-modulating capabilities. Collectively, our study identifies a complex network of functionally heterogeneous fibroblasts during PDAC progression with significant immunotherapeutic implication. Citation Format: J. Kebbeh Darpolor, Xiaofeng Zheng, Sujuan Yang, Kathleen M. McAndrews, Julienne L. Carstens, Patricia E. Phillips, Hikaru Sugimoto, Pedro Correa De Sampaio, Chia-Chin Wu, Valerie S. LeBleu, Raghu Kalluri. Identification of distinct fibroblast populations with unique roles in PDAC progression and tumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A108.

Role of carcinoma-associated fibroblasts in “anoikis resistance” for oral squamous cell carcinoma: Need of the hour

Role of carcinoma-associated fibroblasts in “anoikis resistance” for oral squamous cell carcinoma: Need of the hour

Carcinoma associated fibroblasts derived from oral squamous cell carcinoma promote lymphangiogenesis via c-Met/PI3K/AKT in vitro.

Carcinoma-associated fibroblasts (CAFs) are dominant components of the tumor microenvironment (TME) that promote the development, progression and metastasis of cancer. c-Met is a receptor of the hepatocyte growth factor (HGF), which is involved in lymphangiogenesis. Currently, the roles of CAFs during lymphangiogenesis are unknown. It has been hypothesized that CAFs contribute to lymphangiogenesis of oral squamous cell carcinoma (OSCC) via a HGF/c-Met complex. The expression of HGF in OSCC was determined using CAFs derived from OSCC tissue and it was demonstrated that HGF is overexpressed in OSCC-derived CAFs. It was also revealed that c-Met was highly expressed in human lymphatic endothelial cells (HLECs) when co-cultured with CAFs. Furthermore, it was demonstrated that recombinant human HGF significantly enhanced the proliferation, migration, invasion and tube formation of HLECs. By contrast, the inhibition of c-Met expression suppressed the aforementioned biological activities and also downregulated the expression of c-Met, phosphoinositide 3-kinase and phosphorylated protein kinase B. Taken together, these data demonstrate that c-Met is associated with the regulation of lymphangiogenesis. Thus, the results of the present study indicate that c-Met may be a promising novel therapeutic target to treat patients with OSCC.

Role of Carcinoma Associated Fibroblasts in Anoikis Resistance in Oral Squamous Cell Carcinoma –need of the hour

Role of Carcinoma Associated Fibroblasts in Anoikis Resistance in Oral Squamous Cell Carcinoma –need of the hour

Abstract 4087: Mammary fibroblasts exert divergent effects on the survival of invasive breast cancer cells

Abstract Cancer development and progression are inherently inefficient processes riddled with anti-tumorigenic obstacles. Anoikis, or apoptosis induced by detachment from the extracellular matrix (ECM), represents one prominent hindrance to cancer progression that transformed cells must overcome in order to survive, particularly during the ECM-bereft metastatic cascade. Far from acting in isolation, we report that one mechanisms of anoikis evasion by invasive breast cancer cells involves the exploitation of carcinoma-associated fibroblasts (CAFs) from the surrounding tumor microenvironment. CAFs secrete insulin-like growth factor-binding proteins (IGFBPs) that facilitate non-canonical activation of integrin-linked kinase (ILK), activation of ERK/MAPK, and ultimately stabilization of the anti-apoptotic protein Mcl-1 to promote cancer cell survival during ECM detachment. Further investigation into the influence of the tumor microenvironment on cancer cell survival revealed that, contrary to the role of CAFs, normal mammary fibroblasts (NMFs) appear to exert anti-tumorigenic effects on ECM attached cancer cells. Utilizing conditioned media from NMFs, we have found that NMFs secrete factors that increase caspase activation in multiple cancer cell lines, suggestive of NMF-mediated cancer cell death. Because cancer progression necessitates the intermingling of cancer cells with normal stromal cells, particularly at early time points in primary tumor and metastases formation, we hypothesize that this NMFs pose yet another obstacle to disease progression that may compliment the process of anoikis by capping the metastatic cascade with environments that are inhospitable to cancer cell survival. Similar to anoikis evasion, our data suggests that cancer cells must evade NMF-induced death stimuli in order to survive, possibly through the accrual of CAFs in the tumor microenvironment to combat or replace the pro-apoptotic NMFs. Using traditional and 3D mammalian cell culture in combination with in vivo studies, we aim to delineate the roles of both CAFs and NMFs on cancer cell survival under conditions of differential ECM availability. A more complete understanding of the nuances dictating cancer cell-fibroblast interactions could yield informative data relevant to chemotherapeutic research and development. Citation Format: Kimberly Hagel, Kelsey Weigel, Meaghan Boyd, Luke McCormack, Matthew Champion, Zachary Schafer. Mammary fibroblasts exert divergent effects on the survival of invasive breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4087.

Carcinoma-Associated Fibroblasts Lead the Invasion of Salivary Gland Adenoid Cystic Carcinoma Cells by Creating an Invasive Track.

Carcinoma-associated fibroblasts (CAFs) are critical in determining tumor invasion and metastasis. However the role of CAFs in the invasion of salivary gland adenoid cystic carcinoma (ACC) is poorly understood. In this study, we isolated primary CAFs from two ACC patients. ACC-derived CAFs expressed typical CAF biomarkers and showed increased migration and invasion activity. Conditioned medium collected from CAFs significantly promoted ACC cell migration and invasion. Co-culture of CAFs with ACC cells in a microfluidic device further revealed that CAFs localized at the invasion front and ACC cells followed the track behind the CAFs. Interfering of both matrix metalloproteinase and CXCL12/CXCR4 pathway inhibited ACC invasion promoted by CAFs. Overall, our study demonstrates that ACC-derived CAFs exhibit the most important defining feature of CAFs by promoting cancer invasion. In addition to secretion of soluble factors, CAFs also lead ACC invasion by creating an invasive track in the ECM.

Abstract 175: Carcinoma-associated fibroblasts promote breast cancer cell invasion via IGF-1 and RhoA

Abstract In triple-negative breast cancer (TNBC) and other breast cancer subtypes, the presence of a fibrotic focus - composed of carcinoma-associated fibroblasts (CAFs) and collagen fibers correlates with high-grade malignancies and reduced survival. CAFs secrete specific soluble pro-survival factors and extracellular matrix (ECM) molecules that modulate the adhesion of cancer cells, leading to the remodeling of a permissive and supportive environment for tumor progression and dissemination. Hence, the main goal of our study was to determine how CAFs affect breast cancer cell invasion. To study in vitro invasion, we established a 3D co-culture model by seeding human TNBC spheroids (MDA-MB-231 and HCC-1937), alone or in presence of human mammary CAFs, in a collagen I gel. Our findings show that CAFs increase active RhoA-GTP expression in MDA-MB-231 cells and significantly promote their invasion, by inducing cellular scattering and a switch from an elongated to an amœboid mode of migration. In presence of CAFs, the ROCK inhibitor Y27632, as well as the genetic silencing of RhoA, inhibited the rounding of MDA-MB-231 cells and significantly reduced their invasion. We then determined the expression of pro-invasive cytokines in CAFs and we found that IGF-1 expression was increased in CAFs co-cultured with MDA-MB-231 cells. To date, the IGF-1R pathway has been shown to stimulate proliferation and cell survival in TNBC cells in vitro, and several studies have suggested that the presence of stromal IGF-1 could modulate TNBC cell plasticity and drive tumors to metastasize to bone. To assess the role of IGF-1 in CAF-promoted invasion of TNBC cells, we first assayed the effect of recombinant IGF-1 on RhoA activation and the invasion of MDA-MB-231 cells. IGF-1 increased RhoA-GTP expression and promoted invasion in a RhoA-dependent manner. Furthermore, the addition of an IGF-1 blocking antibody to the co-culture of MDA-MB-231 cells and CAFs reduced the expression of RhoA-GTP, and the scattering and invasion of MDA-MB-231. Together, our results suggest that CAFs increase the invasion of MDA-MB-231 by promoting cell scattering and amœboid migration, which is dependent on the IGF-1/RhoA/ROCK pathway. Interestingly, we also found that CAFs as well as recombinant IGF-1, promote multicellular invasion of HCC-1937, in a ROCK-dependent manner, indicating that CAFs, via the IGF-1/RhoA/ROCK pathway, can support different modes of invasion. In conclusion, our results highlight the different roles of CAFs in invasion of TNBC cells. In breast cancer patients, the expression of RhoA and its downstream effector ROCK1 has been associated with cancer outgrowth and dissemination. Our findings may lead to the use of ROCK inhibitors (such as Fasudil), potentially in combination with IGF-1R targeting therapy, in the treatment of invasive breast cancer. Citation Format: Julien Daubriac, Yves Boucher. Carcinoma-associated fibroblasts promote breast cancer cell invasion via IGF-1 and RhoA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 175. doi:10.1158/1538-7445.AM2014-175

Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line

Conclusions: Carcinoma-associated fibroblasts (CAFs) can influence the biological characteristics of a laryngeal carcinoma cell line. These results could lay the foundation for further studies on the role of CAFs in the laryngeal tumor–host microenvironment. Objective: CAFs are important contributors to the microenvironment in determining the fate of tumors. The aim of this study was to separate, culture, and identify laryngeal CAFs and investigate their biological influence on the laryngeal carcinoma cell line. Methods: The primary CAFs and normal fibroblasts (NFs) of the larynx were obtained by tissue culture. The cells were verified according to immunohistochemical and immunofluorescence staining of certain proteins. Conditioned medium (CM) from CAFs and NFs was obtained. Functional assays were performed to test the influence of each CM on laryngeal carcinoma cell lines. Results: Third-passage purified laryngeal CAFs and NFs were successfully attained. The CAFs showed positive staining for vimentin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The migration ability of the CAFs increased significantly compared with that of NFs (p < 0.05). CM from CAFs (compared with CM from NFs) stimulated proliferation, migration, and invasion to a greater extent.

Abstract 48: Activation of c-Met receptor signaling by carcinoma-associated fibroblasts in the tumor microenvironment

Abstract Objective: Carcinoma-associated fibroblasts (CAFs) play a key role in tumorigenesis by promoting tumor growth, invasion, and metastasis. At sites of intra-abdominal ovarian cancer metastases, more than half of the tumor microenvironment is composed of CAFs, however, the mechanism for how CAFs promote metastatic disease in ovarian cancer is not known. Because of the prominent role that the receptor tyrosine kinase c-Met plays in metastasis, we hypothesized that CAFs within the metastatic tumor microenvironment may activate c-Met signaling in ovarian cancer cells leading to tumor progression at a metastatic site. Methods: Human cancer cell lines and primary human carcinoma-associated fibroblasts isolated from patients’ ovarian cancer metastases were used in multiple biochemical assays to analyze the role of CAFs in c-Met receptor signaling. Results: Using a receptor tyrosine kinase array to analyze phosphorylation of 42 different receptor tyrosine kinases (RTKs), we found that c-Met was the only RTK that was phosphorylated in cancer cells upon co-culture with CAFs. Further analysis of the co-culture revealed that direct contact between cancer cells and CAFs was required for activation of c-Met. Interestingly, the unique ligand for c-Met, hepatocyte growth factor (HGF), could not be detected in conditioned media from CAFs by ELISA and addition of HGF neutralizing antibodies to the co-culture did not prevent activation of the receptor. Furthermore, CAFs transiently transfected with HGF siRNA were still able to activate c-Met in the human ovarian cancer cell line SKOV3ip1 suggesting that c-Met activation by CAFs is HGF-independent. This effect was not limited to ovarian cancer cell lines as co-culture of CAFs with the cervical cancer cell line, HeLa, which expresses wild-type c-Met, and the breast cancer cell line, T47D, showed the same results as the two ovarian cancer cell lines, SKOV3ip1 and OVCAR-5. Remarkably, membrane fragments isolated from CAFs were sufficient to stimulate phosphorylation of c-Met in cancer cells in an HGF-independent manner. Several candidate factors were identified in the CAF membrane proteome which can activate c-Met signaling. Conclusions: Collectively, these results suggest a potentially novel mechanism whereby CAFs activate c-Met in cancer cells through direct contact which provides an additional rationale for the therapeutic targeting of c-Met in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 48. doi:1538-7445.AM2012-48

Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer

The tumor microenvironment strongly influences cancer development, progression, and metastasis. The role of carcinoma-associated fibroblasts (CAFs) in these processes and their clinical impact has not been studied systematically in non-small cell lung carcinoma (NSCLC). We established primary cultures of CAFs and matched normal fibroblasts (NFs) from 15 resected NSCLC. We demonstrate that CAFs have greater ability than NFs to enhance the tumorigenicity of lung cancer cell lines. Microarray gene-expression analysis of the 15 matched CAF and NF cell lines identified 46 differentially expressed genes, encoding for proteins that are significantly enriched for extracellular proteins regulated by the TGF-β signaling pathway. We have identified a subset of 11 genes (13 probe sets) that formed a prognostic gene-expression signature, which was validated in multiple independent NSCLC microarray datasets. Functional annotation using protein-protein interaction analyses of these and published cancer stroma-associated gene-expression changes revealed prominent involvement of the focal adhesion and MAPK signaling pathways. Fourteen (30%) of the 46 genes also were differentially expressed in laser-capture-microdissected corresponding primary tumor stroma compared with the matched normal lung. Six of these 14 genes could be induced by TGF-β1 in NF. The results establish the prognostic impact of CAF-associated gene-expression changes in NSCLC patients.

Inoculated mammary carcinoma-associated fibroblasts: contribution to hormone independent tumor growth

BackgroundIncreasing evidence has underscored the role of carcinoma associated fibroblasts (CAF) in tumor growth. However, there are controversial data regarding the persistence of inoculated CAF within the tumors. We have developed a model in which murine metastatic ductal mammary carcinomas expressing estrogen and progesterone receptors transit through different stages of hormone dependency. Hormone dependent (HD) tumors grow only in the presence of progestins, whereas hormone independent (HI) variants grow without hormone supply. We demonstrated previously that CAF from HI tumors (CAF-HI) express high levels of FGF-2 and that FGF-2 induced HD tumor growth in vivo. Our main goal was to investigate whether inoculated CAF-HI combined with purified epithelial (EPI) HD cells can induce HD tumor growth.MethodsPurified EPI cells of HD and HI tumors were inoculated alone, or together with CAF-HI, into female BALB/c mice and tumor growth was evaluated. In another set of experiments, purified EPI-HI alone or combined with CAF-HI or CAF-HI-GFP were inoculated into BALB/c or BALB/c-GFP mice. We assessed whether inoculated CAF-HI persisted within the tumors by analyzing inoculated or host CAF in frozen sections of tumors growing in BALB/c or BALB/c-GFP mice. The same model was used to evaluate early stages of tumor development and animals were euthanized at 2, 7, 12 and 17 days after EPI-HI or EPI-HI+CAF-HI inoculation. In angiogenesis studies, tumor vessels were quantified 5 days after intradermal inoculation.ResultsWe found that admixed CAF-HI failed to induce epithelial HD tumor growth, but instead, enhanced HI tumor growth (p < 0.001). Moreover, inoculated CAF-HI did not persist within the tumors. Immunofluorescence studies showed that inoculated CAF-HI disappeared after 13 days. We studied the mechanisms by which CAF-HI increased HI tumor growth, and found a significant increase in angiogenesis (p < 0.05) in the co-injected mice at early time points.ConclusionsInoculated CAF-HI do not persist within the tumor mass although they play a role during the first stages of tumor formation promoting angiogenesis. This angiogenic environment is unable to replace the hormone requirement of HD tumors that still need the hormone to recruit the stroma from the host.

Separation, cultivation and biological characteristics of oral carcinoma‐associated fibroblasts

Carcinoma-associated fibroblasts (CAFs) have been suggested to regulate the initiation and progression of many types of solid tumors. The aim of the study was to separate, cultivate, identify oral CAFs, and to investigate their biological characteristics. The primary CAFs and normal fibroblasts (NFs) of the tongue were obtained by tissue culture. Then cells were dissociated by 0.25% trypsin and purified by curettage method combining with trypsinization. The cells were verified according to morphological observation and immunohistochemical staining of certain proteins. Multiple proliferation indexes and karyotype of the cells were assayed. Third passage purified oral CAFs and NFs were attained successfully. The morphological characteristics of the CAFs changed significantly comparing to the NFs. The CAFs showed positive staining for vimentin, alpha-smooth muscle actin and matrix metalloproteinases-2. The proliferation and mitosis ability of the CAFs were significantly increased compared with the NFs (P < 0.05). No karyotypic abnormalities were found in the CAFs. There were obvious differences in the biological characteristics between oral CAFs and NFs. The results may provide us an experimental foundation for further studies on the roles of CAFs in the initiation and progression of oral cancer.