The Promoting Effect Of Cancer Associated Fibroblasts On Metastasis And Its Possible Regulatory Mechanism In Colorectal Cancer

BackgroundColorectal cancer(CRC)is a common malignancy in the world.Metastasis is the major cause of death in colorectal cancer.Inhibition of metastasis and reducing the tumor burden will prolong the survival of patients with colorectal cancer.The metastasis is not entirely determined by the tumor cells.Cells in the tumor microenvironment also play an important role during the metastasis.Cancer associated fibroblasts(CAFs)is an important part of the tumor microenvironment.Cancer cells bring CAFs to metastasize together.Circulating CAFs could be distinguished in the peripheral blood of patients with breast cancer,prostate cancer,colorectal cancer and lung cancer.The level of circulating CAFs was associated with clinical metastasis and worse prognosis.Inhibition of CAFs metastasis is expected to reduce the incidence of colorectal cancer metastasis.Therefore,we planned to study the possible mechanism by which CAFs metastasize in CRC.The results will provide new insights for tumor treatment.Methods1 CAFs were derived from adipose mesenchymal stem cells(MSCs)after co-culture with CRC cell lines.Western Blot was used to detect the expression of the CAFs characteristic proteins.Flow cytometry assay was used to analyze the cell mesenchymal markers of CAFs and MSCs.The multiline differentiation ability of MSCs and CAFs was detected by the adipogenic and osteogenic differentiation assay.Adhesion assay was used to detect the adhesion ability of CAFs and MSCs.The migration and invasion of CAFs were evaluated by transwell in vitro.The metastasis of CAFs and its effect on colorectal cancer metastasis were evaluated in nude mice subcutaneous xenograft model.2 We then explored the mechanism of up-regulation of the migration and invasion ability in CAFs.Western Blot and RT-PCR were used to analyse the differences in gene expression.Gene interference and overexpression experiment was used to analyze the regulation function of these genes.CHIP was used to verify that miR210 was regulated by HIF-1α,Dual luciferase reporter assay was used to identify the target gene of miR210.The effect of miR210 interference on both CAFs and CRC metastasis was verified in nude mice subcutaneous transplantation model.3 The subcutaneous transplantation model of colorectal cancer was constructed in mice.The effect of CAFs on the colonization of metastatic colorectal cancer cells was detected.Results1 The expressions of q-SMA and FAPA were significantly upregulated after MSCs were co-cultured with CRC cells.In addition,CAFs had the same mesenchymal markers as MSCs.But the multiline differentiation ability of MSCs disappeared after co-culture.The expression of IL-8,IL-10,MCP-1,HGF in CAFs was higher than that in MSCs,while the adhesion ability of CAFs was lower than that of MSCs.Further analysis indicated that CAFs had stronger migration and invasion ability than MSCs.In xenograft assay,CAFs could metastasize from primary tumor to lung and promote the formation of more colorectal cancer metastasis.2 The expression of HIF-la was up-regulated when MSCs differentiated into CAFs.Inhibition of HIF-1α expression could inhibit the migration and invasion of CAFs.ChIP assay showed that HIF-1α could bind to the miR210 promoter region in CAFs.In addition,HIF-la regulated the migration and invasion of CAFs by up-regulating miR210 transcription.Bioinformatics and luciferase reporter assay revealed that miR210 specifically targeted the 3’-UTR of VMP1 and regulated its expression.Down-regulation of VMP1 or overexpression of miR210 enhanced the migration and invasion of CAFs.A Rescue experiment revealed that the ability of miR210 to upregulate the migration and invasion of CAFs could be impaired by overexpressing VMP1.Therefore,the HIF-1α/miR210/VMP1 pathway could regulate the migration and invasion of CAFs.3 Mice with subcutaneous tumor were divided into four groups.Mice of two groups were injected Matrigel or CAFs mixed with Matrigel into the contralateral limbs.After five weeks,metastases were observed at the injection site in the CAFs mixed with Matrigel group.The other two groups were injected PBS or CAFs into caudal vein.After five weeks,the number of lung metastases in the CAFs group was more than that in the PBS group,indicating that CAFs could promote the colonization of cancer cells in CRC.Conclusions:1 HIF-1α/miR210/VMP1 pathway maybe regulate the migration and invasion of CAFs in CRC.2 Reducing CAFs metastasis by regulating HIF-1α/miR210/VMP1 pathway might decrease colorectal metastasis.3 CAFs might have the ability to promote colonization of cancer cells in CRC.