Study On Interaction Between Colorectal Cancer Cells And Fibroblasts By Co-culture Model In Vitro

1. Background:According to World Health Organization, cancer is becoming the biggest cause of death in the world, leaving behind heart and vascular disease. Cancer research has focused on elucidating the mechanisms of cancer cell progression for improved diagnosis and treatment. The emergence of the crucial role of the tumor microenvironment has brought to light the need for stroma specific therapeutic strategies The tumor microenvironment (TME) is complex and constantly evolving. TME consists of several cell types, including distinct cell types and subtypes that collectively promote tumor growth and progression, which involves tumor cells, endothelial cells, pericytes, immune inflammatory cells, tumor-associated fibroblasts, stem and progenitor cells of the tumor stroma and extra cellular matrix.Fibroblasts in TME also known as tumor-associated fibroblasts (CAFs),assume an aberrant stimulatory role in modulating TME and influence the behavior of tumor cells in either a tumor-promoting or tumor-inhibiting manner. Expression of a-SMA is the most commonly used marker for CAFs, but because of their heterogeneity, employing solely a-SMA expression will not identify all CAFs . Other widely used CAFs markers are fibroblast specific protein(FSP, aka S100A4), fibroblast activation protein(FAP, aka seprase) and platelet-derived growth factor receptor (PDGFR)a/β. FSP1 regulates cell cycle progression and cytoskeletal integrity Aberrant expression of FSP1 has been shown to promote tumor growth and metastasis.FAP is a gelatinase expressed selectively in pericytes and inactivated fibroblasts during wound healing and tumor-stroma reaction.Its expression is activated in over 90% of human carcinomas . Likewise, expression of PDGFRa/β has been detected in up to 90% of fibroblasts in solid tumors.CAFs found in different cancers are highly heterogeneous, and they are possibly derived from resident fibroblasts, epithelia cells, endothelia cells or mesenchymal cells. The results of several in vitro and in vivo experiments indicate that CAFs promote cancer progression in both proliferation and invasion through multiple growth factors and signaling pathways. As the most abundant cell type in the tumor stroma and their tumor-promoting abilities, there is an increasing interest to study CAFs as drug targets for anticancer therapies. Therefore, we are dedicated to simulating the activation process of normal fibroblast in vitro and exploring the changes of tumor cells and fibroblasts respectively.2. Methods:2.1 Morphological changes of mouse embryonic fibroblasts before and after co-culture were observed under the light microscope. The source of fibroblasts was identified by immunofluorescence.2.2 Mouse embryonic fibroblast cell line NIH/3T3 and mouse colon cancer cell line CT26 were indirectly co-cultured in vitro and a-SMA protein staining was detected in the co-cultured cells and normal fibroblasts. Fibroblasts disposed by lOng/ml TGF-β were known as activated fibroblasts. Compare the changes of the activation degree of fibroblasts after co-culture.2.3 Taking a-SMA、FAP、FSP1、PDGFRβ as specific markers of activated fibroblasts, compare the protein expression level changes between co-cultured and normal fibroblasts.2.4 CT26 after co-cultured and untreated were both digested with trypsin, centrifugalized, and suspended with 2% serum medium. The cell suspension concentration was 5*105/ml and 200ul was added to the upper chamber.,600ul 10% serum medium was added to the lower chamber. In the cell culture box after 12h, remove the chamber, HE staining and observe under microscope.2.5 CT26 after co-cultured 0d,15d,30d were under scratch test. After 0h,6h,12h,24h, tumor cell coverage area was observed under the microscope and take photos. Then upload photos to www.wimasis.com for data analysis.2.6 CT26 after co-cultured Od and 30d were exposed with cisplatin, oxaliplatin, fluorouracil, irinotecan under different density respectively. After 24h,digest cells with trypsin, centrifugate, suspend and calculate the number of living cells after trypan blue stain. Each drug was repeated at least three times.2.7 By Agilent microRNA chip, analyse the differentiation in microRNA expression profile between NFs and CAFs.3.Results:3.1 Fibroblast cell morphology did not change significantly after co-culture. Fibroblast cells expressed Vimentin (+),α-SMA (+),Pan cytokeratin(-), Desmin (-) by immunofluorescence staining, suggesting that the cells were homogeneous and mesenchymal components.3.2 With the prolongation of the co-culture time, the proportion of a-SMA positive cells was significantly increased, the expression of a-SMA was enhanced and the cytoskeleton became wide. This indicated that normal fibroblasts transformed to muscle derived fibroblasts or activated fibroblasts.3.3 With the prolongation of the culture time, the expression level of a-SMA, FAP, FSP, PDGFRβ increased gradually. After co-culture 2W, the expression levels of these proteins were equal to the positive control group.3.4 There was almost no untreated CT26 cells through the 8.0um polycarbonate membrane after 12h.The CT26 cell number of co-cultured 6d,15d,30d through the polycarbonate membrane gradually increased. As the prolongation of the culture time, the ability of metastasis of tumor cells enhanced.3.5 At Oh, the tumor cell coverage areas of the three groups (co-culture 30d, co-culture 15d,co-culture Od)were 62.9%,62.6%,62.2%, there was no significant difference; At 6h, the tumor cell coverage areas were 71.6%,67.3%,65.5%; At 12h, the tumor cell coverage areas were 88%,81.3%,68%;At 24h, the tumor cell coverage areas were 99.3%,91%,76.6%. Thus, tumor cell coverage area were higher in co-cultured group than that of ordinary cultured tumor cells.3.6 Drug resistance of two groups of tumor cells to cisplatin. oxaliplatin. fluorouracil、 irinotecan did not change obviously.3.7 A set of microRNA expression quantity changed significantly in co-cultured fibroblasts.4.Conclusions:4.1 Normal fibroblasts can be activated by CT26 cells even if a direct cancer cell-fibroblasts contact is not present.4.2 The activation level of fibroblasts is critical in enhancing tumor migration and invasion.4.3 As the main ingredient in the tumor microenvironment, exporing microRNA expression of CAFs is essential to throw light upon the possibility and feasibility of CAFs regulation on tumor metastasis.