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 fibroblas...
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| Published in: | The Journal of pathology Vol. 231; no. 4; pp. 517 - 531 |
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| Main Authors: | , , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Chichester, UK
John Wiley & Sons, Ltd
01-12-2013
Wiley Subscription Services, Inc |
| Subjects: | |
| Online Access: | Get full text |
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| Summary: | 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. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. |
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| Bibliography: | istex:DBA31232C8F731BFF13A7C63F83392057A448303 Appendix S1. Supplementary materials and methodsFigure S1 Analysis of human lung cancer-derived metastasis in the pleura. Two representative slides show calretinin-positive fibroblasts coexisting with large-cell lung carcinoma implants in pleura. These results support the mesothelial origin of CAFs. T, tumour. Scale bars = 100 µmFigure S2 MC markers are not expressed in CAFs of tumours located outside the coelomic cavities. (A, B) Serial sections of a cutaneous basal cell carcinoma show CAFs negative for mesothelial markers (cytokeratin AE1/AE3 and calretinin) accompanying the tumour. (C) An infiltrating breast carcinoma positive for cytokeratin AE1/AE3 shows proximal CAFs negative for this mesothelial marker. Scale bars = 25 µm. T, tumourFigure S3 Expression pattern of mesothelial markers in primary MCs, either treated or not with TGFβ1 plus IL-1β, and in SKOV-3 cells. Western blot analysis shows that SKOV-3 cells are negative for WT1, α-SMA and calretinin. The expression of pan-cytokeratin in SKOV-3 cells reveals its epithelial origin. On the contrary, omental-derived MCs express WT1, pan-cytokeratin and calretinin. The levels of these markers are dramatically down-regulated in MCs treated with TGFβ1 + IL-1β. The molecule α-SMA is increased in MCs transdifferentiated in vitro. Expression of β-actin is employed as a loading controlFigure S4 Increased angiogenesis is found in sites where MC-derived CAFs expressing high amounts of VEGF accumulate. (A, B) CD31 staining reveals that non-tumour zones do not possess a high number of vessels. At the sites of tumour implants, there is a dramatic increase of vessel density, particularly located in the upper compact zone where MC-derived CAFs tend to accumulate. (C, D) VEGF is expressed in a preserved mesothelium distant from tumour implants. At tumour zones, MCs and MC-derived CAFs, as well as cancer cells proximal to vascularized areas, express high levels of VEGF. Arrows, angiogenesis zones. Scale bars = 25 µm. T, tumourFigure S5 Large tumour implants coexist with fibrotic areas where CAFs express mesothelial markers. H&E analysis of advanced peritoneal disseminations (8 weeks after i.p. SKOV-3 cell injection) shows larger metastatic masses coexisting with widespread areas of fibrosis. IHC studies reveal the existence of CAFs expressing mesothelial markers, not only in the proximity of carcinoma implants but also integrated within the tumour stroma. The expression of cytokeratin is restricted exclusively to carcinoma cells. However, extended WT1 and α-SMA staining can be observed cohabiting with the large peritoneal tumours. The inset shows a higher magnification of the WT1 nuclear staining. Scale bars = 100 µm. T, tumourFigure S6 Analysis of omental metastasis in the mouse model of peritoneal dissemination. (A) Visceral macroscopic metastases are observed in mice i.p. inoculated with SKOV-3 cells. (B) Immunohistochemical analyses of omental tissues reveal that MCs positive for cytokeratin are lining omentum in tumour-free areas. (C) Fibroblastic cells (CAFs) express pan-cytokeratin in the stroma interstices between cancer cells. (D) α-SMA is negative in MCs lining tumour-free omental tissue. (E) A higher magnification of the tumour mass shows spindle-like cells positive for α-SMA embedded in the tumour parenchyma. Scale bars = 100 µm (B, D) and 25 µm (C, E). Arrows, MCs expressing cytokeratin or α-SMA; T, tumourFigure S7 Conditioned medium from HT29 cells induces MMT in vitro. (A) Conditioned medium obtained from the colorectal adenocarcinoma cell line HT29 was applied for 6 days to omentum-derived MCs to induce MMT. The treatment induced the acquisition of a spindle-like morphology in MCs. Scale bars = 100 µm. (B-D) MMT molecular reprogramming, including the repression of E-cadherin and the up-regulation of mesenchymal markers such as fibronectin and collagen-I, reach statistical significance at 6 days of exposure to HT29-conditioned medium. Bar graphics represent mean ± SEM; symbols represent the statistical differences between groups. FI, fold induction; HT29 CM, HT29 conditioned medium; T + I, TGFβ1 plus IL-1βTable S1 Specific primers for real-time PCR ark:/67375/WNG-SGZ2HWMB-R ArticleID:PATH4281 No conflicts of interest were declared. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0022-3417 1096-9896 |
| DOI: | 10.1002/path.4281 |