| Objective:This study was designed to investigate the characteristics of MSC in gastritis microenvironment and how MSC reshape gastritis microenvironment. We could understand the effects of MSC on nonresolving inflammation-mediated malignant transformation and the relationship between nonresolving inflammation and gastric carcinogenesis. The study was also designed to reveal the biological function of MSC in gastric cancer microenvironment and the underlying mechanisms. This study will provide a theoretical basis and experimental evidence for the prevention and treatment of gastric cancer.Methods:(1) Male Wistar rats were divided into two groups randomly, the control group were feed with normal food and water while the model group feed with aqueous solution containing N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and high-salt diet. The malignant transformation of gastritis in rats was observed dynamically by using magnetic resonance imaging to show the changes in the structure of rat stomach, Luminex and tissue staining to detect serum and tissue samples at different time points. MSC from normal gastric tissue (RGN-MSC) and from gastric tissue in inflammation microenvironment (RGI-MSC) were isolated and their multiple differentiation potential and self-renewal ability were determined by using cell staining, flow cytometry and Western-blot.(2) The proliferation rate, the migration rate and the secretion of inflammatory factors of RGN-MSC and RGI-MSC were compared by using cell growth assay, cell colony formation assay, cell cycle, transwell migration assay and Luminex analyses. The effects of RGN-MSC and RGI-MSC on normal gastric epithelial cells GES-1 and myeloid monocytes THP-1 were determined by using transwell assays. The expression of EMT-related proteins was detected by using Western-blot.(3) Exosome derived from MSC (MSC-exosome) was isolated and identified; HGC-27 cells were treated with MSC-exosome ex vivo. The migration, invasion and the changes of EMT-related proteins of gastric cancer cells were observed and compared with normal gastric epithelial cells GES-1. The ability of self-renewal in gastric cancer cells were observed by using serum-free culture and soft agar assays and Western-blot. The signaling pathways involved in the effects of MSC-exosome on gastric cancer cells were verified by using specific inhibitor.(4) Immunodeficient mouse bearing subcutaneous gastric cancer were treated with FU (fluorouracil,5-FU) intraperitoneally and MSC-exosome/HFL-exosome intratumorally. The tumor growth curves were plotted and the drug resistance related genes and proteins such as MDR, MRP and LRP in tumor tissues were detected. Gastric cancer cell models were treated with chemotherapeutic agents (5-FU and cisplatin) and MSC-exosome. The sensitivity of gastric cancer cells to chemotherapy was observed by MTT assays. The expression of drug resistance related genes and proteins such as MDR, MRP and LRP in gastric cancer cells was detected by using real-time quantitative PCR and Western-blot. The ability of MDR to efflux drugs was detected by flow cytometry. The anti-apoptosis effects of gastric cancer cells were detected by using TUNEL staining and Annexin V-FITC/PI double staining combined with flow cytometry. The mechanisms for MSC-exosome conferring drug resistance in gastric cancer cells were explored by using Western-blot, real-time quantitative RT-PCR, MTT assays and cell transfection.Results:(1) The gastric mucosa of rats changed from normal to superficial gastritis, atrophic gastritis, intestinal metaplasia, and dysplasia during the treatment of MNNG, the level of MCP-1, VEGF, TNF-α, IL-1β and IL-6 in rat serum were statistically increased when rats treated with MNNG for 48 weeks. The RGN-MSC and RGI-MSC had strong potential of osteogenic and adipogenic differentiation, surface antigens CD29, CD90 and CD44H positive, CD45 negative.(2) Compared with RGN-MSC, RGI-MSC had higher proliferation rate and mobility, secreted more inflammatory factors including MCP-1, IL-6 and CXCL10. RGI-MSC promoted the migration of GES-1 and THP-1, enhanced the expression of N-cadherin and Vimentin, and reduced the expression of E-cadherin in GES-1.(3) MSC-exosome with CD9 and CD63 could enter into the gastric cancer cells after treatment for 4h. MSC-exosome promoted the migration and invasion of gastric cancer cells, increased their formation of spheroids in the liquid and semi-solid medium, enhanced the expression of N-cadherin, Vimentin, Twist, Oct4, Sox2, Lin28B, reduced the expression of E-cadherin and stimulated the phosphorylation of Akt in gastric cancer cells. LY294002, the specific inhibiter of PI3K, could inhibit these changes.(4) In vivo and ex vivo, MSC-exosome could active the calmodulin kinases, trigger Raf/MEK/ERK cascade, increase the expression of MDR, MRP and LRP in gene and protein levels, improve MDR efflux drugs, help gastric cancer cells against apoptosis induced by chemotherapeutics, reverse the effect of inhibiting tumor growth from chemotherapeutics.Conclusions:Inflammatory microenvironment promotes proliferation, migration and secretion of inflammatory cytokines of MSC, while MSC in the inflammatory microenvironment can induce normal gastric epithelial cells to acquire EMT, recruit immune cells, involve in the maintenance and malignant transformation of gastritis microenvironment together. MSC involve in the formation of tumor microenvironment by secreting exosome and target gastric cancer cells directly to promote the development of gastric cancer cells including growth/self-renewal, metastasis/EMT and drug resistance. The proteins in MSC-exosome trigger the activation of Akt and CaM-Ks-MAPK signaling pathways is responsible for MSC-exosome conferring the development of drug resistance in gastric cancer cells. This study describes the roles and mechanisms of MSC in gastritis/gastric cancer microenvironment, and will provide new perspectives and strategies for the prevention and treatment of gastric cancer. |
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