Mechanisms Of Cancer-associated Fibroblasts In Promoting Gastric Cancer Metastasis In Tumor Microenvironment

Objective:Increased evidence suggested that a unique microenvironment created by the interaction between stromal and tumor cells is crucial for tumor progression.Cancer-associated fibroblasts(CAFs),the major components of tumor stroma,play a key role in the tumor progression.CAFs were known to promote tumor cell invasion and metastasis by overexpression of a variety of factors,such as chemokines,cytokines and growth factors(HGF,VEGF,TGF-P,IL-22,TGF-?,IL-6)that trigger the degradation of ECM,regulate metabolic reprogramming,enhance EMT and angiogenesis,and promote proliferation and chemotherapy resistance.Current research indicates that CAFs in gastric cancer can upregulate MiR-106b and promote cell migration and invasion by targeting PTEN.At the same time,miRNA-200b was down-regulated,and ZEB expression can be up-regulated,thus inducing tumor cell invasion and peritoneal dissemination.In addition,CXCL12 secreted by CAFs in gastric cancer activated CXCL12/CXCR4 signaling pathway,thereby promoting tumor growth and invasion.However,current understanding about the mechanisms of CAFs on the promotion of tumor cell invasion and metastasis is still limited in gastric cancer.We co-cultured fibroblast TIG-3-20 with gastric cancer cells,and found that the ability of migration and invasion was enhanced by fibroblasts in gastric cancer cells.Therefore,this study further explored the mechanism of CAFs-induced gastric cancer metastasis.Methods:1.The concentration of IL-11 in the cell supernatant was detected by ELISA.2.Cell migration and invasion ability was measured using Transwell assay.3.Protein expression were analyzed by Western blot.4.RT-qPCR was used to detected mRNA transcription of MUC1,IL-11 gene.5.Mouse model detects peritoneal metastasis of gastric cancer cells.6.Lipofectamin 2000 and siRNA were used to knockdown gene expression.7.Affymetrix sanner 3000 was used to analyse the microarray genome-wide expression.8.Statistical analysis.All values are expressed as means ± SD.The differences of the results between two groups were evaluated by Student's t-test.P<0.05 was considered to be statistically significant.Results:1.CAFs enhance the migration and invasion of gastric cancer cells.The markers of cancer-associated fibroblasts(a-SMA,Fibronectin and Vimentin)were highly expressed in TIG-3-20 cells both alone and in co-culture with gastric cancer cells,indicating that TIG-3-20 cells have CAF characters.Transwell assay revealed The ability of migration and invasion of gastric cancer cells was significantly enhanced when co-cultured with CAFs.These results indicated that CAFs could promote migration and invasion of gastric cancer.2.CAFs promote the peritoneal metastasis of gastric cancer cells.To investigate whether CAFs could promote peritoneal metastasis of gastric cancer cells,MGC-803 cells alone or mixed with CAFs were intraperitoneally injected into the nude mice.MGC-803 cells mixed with CAFs developed more peritoneal metastases nodes.These results indicated that CAFs enhance the ability of gastric cancer cells to peritoneally diffuse and metastasize in vivo.3.IL-11 is highly expressed in the tumor microenvironment when CAFs were co-cultured with gastric cancer cells.To clarify the mechanisms of enhanced migration and invasion of gastric cancer cells after co-cultivation,gene array analysis was carried out.TheCYTOKINE CYTOKINE RECEPTOR INTERACTION pathway was screened out with KEGG pathway enrichment analysis in CAFs.Of all the significantly up-regulated genes,IL-11 was markedly increased in CAFs co-cultured with gastric cancer cells.Meanwhile,the expression of IL-11 in gastric cancer cells was also significantly increased,indicating that IL-11 may play an important role in the microenvironment.Then,qRT-PCR was performed to further confirm that IL-11 mRNA was up-regulated in CAFs and gastric cancer cells after co-cultured.Moreover,ELISA assays showed that IL-11 protein in the GC cells and CAFs co-culture system was elevated.These results showed that IL-11 was markedly secreted in the tumor microenvironment when CAFs and gastric cancer cells were co-cultured.4.CAFs enhance the migration and invasion of gastric cancer cells via the expression of IL-11,In order to clarify the role of IL-11 in this co-culture system,neutralizing IL-11 antibody was added into the co-culture system.Results revealed that the migration and invasion of both MGC-803 and SGC-7901 cells co-cultured with CAFs were significantly decreased.Moreover,MGC-803 and SGC-7901 cells were treated with exogenous IL-11.Transwell assay showed that IL-11 enhanced migration and invasion than gastric cancer cells alone.These results suggested that CAFs could facilitate the migration and invasion of GC cells via the expression of IL-11.5.CAFs and gastric cancer cells-derived IL-11 enhances the ability of migration and invasion via the activation of JAK/STAT3 and MAPK/ERK signaling pathways in gastric cancer cells.The results revealed that phosphorylation level of STAT3 and ERK was increased with IL-11 treatment.Similarly,the activation of STAT3 and ERK was also observed in GC cells co-cultured with CAFs.On the contrary,IL-11 neutralizing antibody significantly suppressed IL-11-induced STAT3 and ERK activation.To further confirm whether both STAT3 and ERK pathways are necessary for IL-11-promoting metastasis of gastric cancer,ERK inhibitor(PD98059)or STAT3 inhibitor(Stattic)were used to suppress IL-11-induced activation of STAT3 and ERK in MGC-803 and SGC-7901 cells.Simultaneously,the migration and invasion of GC cells were inhibited,indicating that IL-11-promoting metastasis was dampened for both JAK-STAT3 and MAPK/ERK signaling pathways.6.CAFs promote migration and invasion of GC cells via up-regulation of MUC1.MUC1 was significantly up-regulated in MGC-803 cells after co-cultivation with CAFs by gene array analyses.Similar results were obtained by qRT-PCR and Western blot.When MUC1 was knocked-down with si-RNA,the migration and invasion of GC cells were significantly reduced even after co-cultured with CAFs.Furthermore,a time-dependent increase of MUC1 expression was observed with IL-11-treatment.On the contrary,IL-11-induced MUC1 was significantly suppressed by IL-11 neutralizing antibody,Stattic and PD980589 in GC cells.These data indicated that MUC1 was involved in CAFs in promoting the migration and invasion of GC cells.7.CAFs enhance the migration of gastric cancer cells through the expression of VEGFA.We measured the effect of CAFs on gastric cancer cell migration by Transwell.VEGFA neutralizing antibody bevacizumab was added into the co-culture system,the migration of MGC-803 co-cultured with CAFs was significantly reduced.Therefore,these results indicate that CAFs can promote the migration of gastric cancer cells via the expression of VEGFA.8.VEGFA promotes peritoneal metastasis of gastric cancer cells.To investigate whether VEGFA can promote peritoneal metastasis of gastric cancer cells,MGC-803 cells alone were intraperitoneally injected into nude mice.The experimental group was given 200 ?g of Bevacizumab every other day.The peritoneal metastatic nodules in the Bevacizumab group were significantly inhibited.These results indicated that VEGFA enhances the gastric cancer cells' ability to diffuse and metastasize in the peritoneum.9.VEGFA promotes the migration of gastric cancer cells through VEGFR1.We used Transwell to perform further experiments.The results showed the migration of MGC-803 was increased with VEGFA stimulation.This phenomenon was significantly inhibited after the addition of Bevacizumab,but the small molecule inhibitor of VEGFR2,apatinib,did not inhibit this phenomenon.This result suggested that VEGFA mediates gastric cancer cell migration mainly through VEGFR1 but not VEGFR2.We further collected gastric cancer cell lines.We found that VEGFR1 was highly expressed in gastric cancer cell lines,while VEGFR2 expression was extremely low.We used VEGFA to stimulate gastric cancer cells and found elevated levels of p-VEGFR1.These results indicated that VEGFA promoted migration of gastric cancer cells through VEGFR1.10.VEGFR1/VEGF was a poor prognostic factor for gastric cancer.We used the KM-Plotter database to perform survival analyses on gastric cancer patients with different expression levels of VEGFR1 and VEGF.The overall survival of patients was associated with high expression of VEGFR1/VEGF.These results suggested that VEGFR1/VEGF may be a poor prognostic factor in the progression of gastric cancer.Conclusion:1.CAFs enhanced the ability of migration,invasion and peritoneal metastasis of gastric cancer cells.2.After co-culture of CAFs and gastric cancer cells,IL-11 in the microenvironment increased significantly,which enhanced the ability of migration and invasion via the activation of JAK/STAT3 and MAPK/ERK signaling pathways to up-regulate MUC1 expression in gastric cancer cells.3.CAFs-derived VEGFA promotes the migration capacity of gastric cancer cells by activating VEGFR1.