Source Of Carcinoma-associated Fibroblasts Research Articles

Mesothelial-to-Mesenchymal Transition Contributes to the Generation of Carcinoma-Associated Fibroblasts in Locally Advanced Primary Colorectal Carcinomas.

During peritoneal metastasis, cancer cells spread from abdominal solid tumors, disseminate through the peritoneal fluid and attach to and invade through mesothelial cells (MCs) that line the peritoneum. Intestinal adenocarcinomas originating in the mucosa infiltrate the submucosa, muscle layer, and serosa in order to finally colonize the peritoneal cavity. However, the mechanism by which metastatic cells leave the primary tumor and reach the peritoneal cavity has not been previously described. Hence, we investigate whether MCs lining visceral peritoneum, through a mesothelial-to-mesenchymal transition (MMT), are a source of carcinoma-associated fibroblasts (CAFs), which could contribute to cancer progression toward the peritoneal cavity. CAFs detected in biopsies from patients with superficially invasive colorectal cancer differed from locally advanced tumors. An aberrant accumulation of myofibroblasts expressing mesothelial markers was found in the stroma of deeply infiltrative tumors located in the neighborhood of a frequently activated mesothelium. We suggest that MMT is a key event in the early stages of peritoneal dissemination.

Abstract 4171: Novel regulatory role of Neuropilin-1 in endothelial to mesenchymal transition as a potential source of carcinoma associated fibroblasts

Abstract BACKGROUND: Endothelial-mesenchymal transition (EndMT) is an extreme form of endothelial cell plasticity whereby endothelial cells acquire mesenchymal phenotype. While EndMT is a significant component during embryogenesis, maladaptive EndMT is a key contributor to fibrotic pathologies including cancer. With regard to this, EndMT accounts for up to 40% of cancer-associated fibroblasts (CAFs) that play an important role in cancer progression through potentially oncogenic signals like transforming growth factor- β (TGF-β). Recent investigations have demonstrated that the angiogenic co-receptor Neuropilin-1 (NRP1) is aberrantly expressed in cancer and modulates cancer progression through interaction with various ligands, including TGF-β. However, the relationship and driving mechanisms linking NRP1 and EndMT remain unexplored. METHODS: SiRNA-mediated NRP1 gene knockdown studies using Dharmafect-4 transfection reagent and siNRP1 or scramble control (Ambion) were performed on human umbilical vein endothelial cells (HUVECs), at baseline and after TGF-β stimulation. Total RNA was extracted using Trizol® reagent (Invitrogen), and precipitated with isopropanol. Complementary DNA was synthesized using iScript cDNA synthesis kit (Bio-Rad) and real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green (Bio-Rad) following manufacturer's protocols. Markers of EndMT and mediators of TGF-β1 signalling were evaluated by RT-PCR, western blotting and immunocytochemistry. RESULTS: RT-PCR and western blots measuring NRP1 mRNA and protein levels in HUVEC lysates confirmed successful silencing. Light and fluorescence microscopy revealed marked morphological changes in TGF-β1 stimulated scramble siRNA-transfected HUVECs. TGF-β1 stimulated HUVECs demonstrated a distinct change from “cobblestone-like endothelial cell morphology” to an enlarged spindle shaped appearance consistent with a “fibroblast like morphology”, accompanied by re-arrangement of the cytoskeleton. We further observed that these characteristic EndMT features were revoked upon NRP1 silencing where cells maintained a conventional cobblestone phenotype. Expression of endothelial cell markers, CD-31 and VE-Cadherin, were significantly higher in NRP1-silenced vs. scramble siRNA-treated HUVECs; additionally, NRP1 silencing was associated with significant reduction in expression of the mesenchymal markers α-smooth muscle actin, Slug and N-Cadherin. Loss of NRP1 was accompanied by decreased expression of TGF-β1 and its receptors as well as significant changes in the key downstream effectors of TGF-β1 signalling. CONCLUSION: Our novel findings demonstrating that loss of NRP1 revokes TGF-β-dependent EndMT-like phenotype switching suggesting that NRP1 may represent a novel therapeutic target to limit the source of EndMT derived CAFs and associated cancer aggression. Citation Format: Pratiek N. Matkar, Krishna Kumar Singh, Gerald Prud'homme, Howard Leong-Poi. Novel regulatory role of Neuropilin-1 in endothelial to mesenchymal transition as a potential source of carcinoma associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4171. doi:10.1158/1538-7445.AM2015-4171

Carcinoma‐associated fibroblasts derive from mesothelial cells via mesothelial‐to‐mesenchymal transition in peritoneal metastasis

Peritoneal dissemination is a frequent metastatic route for cancers of the ovary and gastrointestinal tract. Tumour cells metastasize by attaching to and invading through the mesothelial cell (MC) monolayer that lines the peritoneal cavity. Metastases are influenced by carcinoma-associated fibroblasts (CAFs), a cell population that derives from different sources. Hence, we investigated whether MCs, through mesothelial-mesenchymal transition (MMT), were a source of CAFs during peritoneal carcinomatosis and whether MMT affected the adhesion and invasion of tumour cells. Biopsies from patients with peritoneal dissemination revealed the presence of myofibroblasts expressing mesothelial markers in the proximity of carcinoma implants. Prominent new vessel formation was observed in the peritoneal areas harbouring tumour cells when compared with tumour-free regions. The use of a mouse model of peritoneal dissemination confirmed the myofibroblast conversion of MCs and the increase in angiogenesis at places of tumour implants. Treatment of omentum MCs with conditioned media from carcinoma cell cultures resulted in phenotype changes reminiscent of MMT. Adhesion experiments demonstrated that MMT enhanced the binding of cancer cells to MCs in a β1-integrin-dependent manner. Scanning electron microscopy imaging showed that the enhanced adhesion was mostly due to increased cell-cell interaction and not to a mere matrix exposure. Invasion assays suggested a reciprocal stimulation of the invasive capacity of tumour cells and MCs. Our results demonstrate that CAFs can derive from mesothelial cells during peritoneal metastasis. We suggest that MMT renders the peritoneum more receptive for tumour cell attachment/invasion and contributes to secondary tumour growth by promoting its vascularization.

Inhibition of TGF‐β/Smad Signaling by BAMBI Blocks Differentiation of Human Mesenchymal Stem Cells to Carcinoma‐Associated Fibroblasts and Abolishes their Protumor Effects

Bone marrow mesenchymal stem cells (BM-MSCs) have multiple therapeutic potentials for regenerative, anti-inflammatory, and immunomodulatory purposes and also show promise as vehicles for gene therapy of various metastatic cancers based on their tumor-tropic capacity. However, BM-MSCs are also a source of carcinoma-associated fibroblasts (CAFs) and may promote growth and metastasis of cancer. Transforming growth factor β (TGF-β) signaling is required to induce CAF differentiation of mouse BM-MSCs in vivo and can induce expression of some CAF markers in human BM-MSCs in vitro. To determine whether inhibiting TGF-β signaling in human BM-MSCs can block their differentiation to CAFs induced by tumor microenvironments and the consequent protumor effects, we transduced human BM-MSCs with a lentiviral vector encoding bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a decoy TGF-β receptor. BAMBI transduction significantly inhibited TGF-β/Smad signaling and expression of CAF markers in human BM-MSCs treated with TGF-β1 or tumor-conditioned medium or cocultured with cancer cells, but did not alter the stem cell properties and the tumor-tropic property of MSCs. In addition, BAMBI transduction disrupted the cytokine network mediating the interaction between MSCs and breast cancer cells. Consequently, BAMBI transduction abolished protumor effects of BM-MSCs in vitro and in an orthotopic breast cancer xenograft model, and instead significantly inhibited growth and metastasis of coinoculated cancer. These results indicated that TGF-β signaling is essential for differentiation of human BM-MSCs to CAFs in tumor microenvironments and the consequent protumor effects, and inhibiting TGF-β/Smad pathway may improve the safety of MSC-based therapies in cancer patients.

Adipose tissue derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor derived factors

Carcinoma-associated fibroblasts (CAF) are considered to contribute to tumor growth, invasion and metastasis. However, the cell type of origin remains unknown. Since human adipose tissue derived stem cells (hASCs) are locally adjacent to breast cancer cells and might directly interact with tumor cells, we investigated whether CAFs may originate from hASCs. hASCs cultured under different conditions were quantified for the expression of alpha smooth muscle actin. ELISA was performed using the human TGFβ1, SDF-1α and CCL5 Quantikine Kit. The invasion potential of MDAMB231 cancer cells was evaluated using a Boyden chamber with filter inserts coated with Matrigel in 24-well dishes. We demonstrated that a significant percentage of hASCs differentiated into a CAF-like myofibroblastic phenotype (e.g. expression of alpha smooth muscle actin and tenascin-C) when exposed to conditioned medium from the human breast cancer lines MDAMB231 and MCF7. The conditioned medium from MDAMB231 and MCF7 contains significant amounts of transforming growth factor-beta 1 (TGFβ1) and the differentiation of hASCs towards CAFs is dependent on TGFβ1 signaling via Smad3 in hASCs. The induction of CAFs can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of the hASCs with SB431542, a TGFβ1 receptor kinase inhibitor. Additionally, we found that these hASC-derived CAF-like cells exhibit functional properties of CAFs, including the ability to promote tumor cell invasion in an in vitro invasion assay, as well as increased expression of stromal-cell derived factor 1 (SDF-1) and CCL5. Our data suggest that hASCs are a source of CAFs which play an important role in the tumor invasion.

MCP1 Directs Trafficking of Hematopoietic Stem Cell-Derived Fibroblast Precursors in Solid Tumor

Our previous studies have demonstrated that hematopoietic stem cells (HSCs) are a novel source of carcinoma-associated fibroblasts. However, the mechanisms regulating recruitment and homing of HSC-derived carcinoma-associated fibroblasts or their precursors to the tumor microenvironment are unknown. Herein, we demonstrate using a single cell transplantation model that circulating fibroblast precursors (CFPs) are of HSC origin. This population increased with tumor burden in vivo and functional in vitro studies showed that CFPs preferentially migrated and differentiated into fibroblasts in response to tumor, suggesting that HSC-derived CFPs serve as an intermediate between the bone marrow and tumor. Based on this chemotactic ability and our demonstration of a monocyte lineage origin for CFPs, we investigated the role of monocyte chemoattractant protein (MCP1) in mediating CFP recruitment/homing. Blocking tumor-produced MCP1 inhibited in vitro migration of CFPs in response to multiple tumor types, indicating broad biological significance for this CFP/chemokine interaction. In vivo, CCR2-expressing CFPs increased in circulation during the period of active tumor growth and stromal development. Inhibition of MCP1 during tumor development resulted in decreased tumor volume in tumor-bearing mice. Together these findings confirm an HSC origin for CFPs, demonstrate a role for MCP1 in regulating their contribution to the tumor microenvironment, and suggest a potential therapeutic target for limiting tumor growth.

Adipose tissue-derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor-derived factors.

Carcinoma-associated fibroblasts (CAF) are considered to contribute to tumor growth, invasion and metastasis. However, the cell type of origin remains unknown. Since human adipose tissue-derived stem cells (hASCs) are locally adjacent to breast cancer cells and might directly interact with tumor cells, we investigated whether CAFs may originate from hASCs. We demonstrated that a significant percentage of hASCs differentiated into a CAF-like myofibroblastic phenotype (e.g., expression of alpha smooth muscle actin and tenascin-C) when exposed to conditioned medium from the human breast cancer lines MDAMB231 and MCF7. The conditioned medium from MDAMB231 and MCF7 contains significant amounts of transforming growth factor-beta 1 (TGFβ1) and the differentiation of hASCs towards CAFs is dependent on TGFβ1 signaling via Smad3 in hASCs. The induction of CAFs can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of the hASCs with SB431542, a TGFβ1 receptor kinase inhibitor. Additionally, we found that these hASC-derived CAF-like cells exhibit functional properties of CAFs, including the ability to promote tumor cell invasion in an in vitro invasion assay, as well as increased expression of stromal-cell-derived factor 1 (SDF-1) and CCL5. Taken together, these data suggest that hASCs are a source of CAFs which play an important role in the tumor invasion.

Adipose Tissue-Derived Stem Cells Differentiate into Carcinoma-Associated Fibroblast-Like Cells under the Influence of Tumor-Derived Factors

Carcinoma-associated fibroblasts (CAF) are considered to contribute to tumor growth, invasion and metastasis. However, the cell type of origin remains unknown. Since human adipose tissue-derived stem cells (hASCs) are locally adjacent to breast cancer cells and might directly interact with tumor cells, we investigated whether CAFs may originate from hASCs. We demonstrated that a significant percentage of hASCs differentiated into a CAF-like myofibroblastic phenotype (e.g., expression of alpha smooth muscle actin and tenascin-C) when exposed to conditioned medium from the human breast cancer lines MDAMB231 and MCF7. The conditioned medium from MDAMB231 and MCF7 contains significant amounts of transforming growth factor-beta 1 (TGFβ1) and the differentiation of hASCs towards CAFs is dependent on TGFβ1 signaling via Smad3 in hASCs. The induction of CAFs can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of the hASCs with SB431542, a TGFβ1 receptor kinase inhibitor. Additionally, we found that these hASC-derived CAF-like cells exhibit functional properties of CAFs, including the ability to promote tumor cell invasion in anin vitroinvasion assay, as well as increased expression of stromal-cell-derived factor 1 (SDF-1) and CCL5. Taken together, these data suggest that hASCs are a source of CAFs which play an important role in the tumor invasion.

Mesenchymal Stem Cells: Flip Side of the Coin

Tumor-associated fibroblasts or carcinoma-associated fibroblasts (CAF) play an important role in the growth of epithelial solid tumors. Although the cell type of origin of CAFs has not been conclusively established, it has been shown that they may be bone marrow derived. One side of the mesenchymal stem cell (MSC) coin is the well-accepted therapeutic potential of these cells for regenerative and immunomodulatory purposes. The ominous dark side is revealed by the recent work demonstrating that hMSCs may be a source of CAFs. In this review, we discuss the role of stromal cells in the tumor microenvironment and suggest that by exploring the in vitro/in vivo interplay between different cell types within the tumor milieu, strategies for improved tumor therapy can be developed.