The Role And Mechanisms Of Exosomes Derived From Lymph Node Metastatic Gastric Cancer In Remodeling BM-MSCs

Objective: Bone marrow mesenchymal stem cells(BM-MSCs)are prone to chemotaxis and recruitment to tumors.Exosomes secreted by tumor cells can remodel BM-MSCs into tumor-associated MSCs involved in tumor microenvironment formation.Gastric cancer is more common in regional lymph node metastasis.This study aimed to analyze and elucidate the role of exosomes derived from lymph node metastatic gastric cancer in remodeling BM-MSCs and its molecular mechanism.Methods: The supernatants of lymph node metastatic gastric cancer cell lines(SGC-7901 and HGC-27)and non-metastatic gastric cancer cell lines(AGS and MGC-803)were collected and treated with BM-MSCs.Cellular immunofluorescence staining and Transwell migration assay were used to detect the changes of a-SMA expression and migration ability of BM-MSCs.Transwell migration assays were used to detect the changes of gastric cancer cell metastasis after BM-MSCs supernatant treatment in vitro.To observe and compare the effects of two group cells supernatants on the phenotype and function of BM-MSCs in vitro.The exosomes in the supernatant of gastric cancer cells were separated and extracted by ultracentrifugation,and the exosomes were identified by transmission electron microscopy,nanoparticle analyzer,Western blot and flow cytometry.To analyze and compare the effects of lymph node metastatic gastric cancer cell supernatant,exosomes and exosomes-free supernatant on the phenotype and function of BM-MSCs,and further combine the lymphatic metastasis model of nude mice to compare the effects of lymph node metastatic gastric cancer cell exosomes and non-metastatic gastric cancer cell line exosomes on the phenotype and function of BM-MSCs in vivo and in vitro.Proteinase K was used alone or in combination with Triton-100 to remove exosomes protein components,and the effects of exosomes on BM-MSCs before and after treatment were observed.Label-free method was used to detect and confirm differential protein in exosomes of lymph node metastatic gastric cancer and non-metastatic gastric cancer cells.For the verified proteins,RNA interference and gene overexpression techniques were used to intervene to observe the effects of exosomes on BM-MSCs before and after intervention.Serum samples from patients with clinical lymph node metastasis,non-metastatic gastric cancer and healthy subjects were collected,and the corresponding exosomes were isolated and identified.Western blot was used to compare the difference in protein content in serum exosomes.BM-MSCs were treated with serum exosomes,and the effects of serum exosomes on the phenotype and function of BM-MSCs were analyzed.Results: Compared with the supernatant of non-metastatic gastric cancer cells,the expression of a-SMA and migration ability of BM-MSCs was significantly increased after treated with supernatant of lymph node metastatic gastric cancer cells.The supernatant of BM-MSCs significantly promoted the migration of gastric cancer cells.Lymph node metastatic gastric cancer cells exosomes can reproduce the effect of cell supernatant on the phenotype and function of BM-MSCs,and removal of exosomes supernatant can eliminate the effect of supernatant on BM-MSCs.Compared with non-lymph node metastatic gastric cancer exosomes,lymph node metastatic exosomes can induce BM-MSCs tumor-promoting phenotype and enhance supernatant to promote gastric cancer metastasis in vitro and in vivo.Removal of exosomes protein can significantly inhibit the effect of lymph node metastatic gastric cancer cells exosomes on BM-MSCs.Proteomic detection and validation found that lymph node metastatic gastric cancer exosomes is more enriched in Wnt5 a than non-lymph node metastatic exosomes.Knockdown of Wnt5 a in lymph node metastatic gastric cancer cell exosomes,which can significantly inhibit the effect on phenotype and function of BM-MSCs.Non-metastatic gastric cancer cells were infected with Wnt5 a overexpresses lentivirus and its exosomes enriched in Wnt5 a highly.It can obtain the similar effect on BM-MSCs phenotype and function induced by lymph node metastatic gastric cancer cells exosomes.The content of Wnt5 a in serum exosomes of patients with clinical lymph node metastasis was significantly higher than that serum exosomes of patients with non-metastatic gastric cancer and healthy subjects.There was no significant difference between the latter two.Serum exosomes in gastric cancer patients with regional lymph node metastasis have stronger induction of phenotype and function of BM-MSCs than serum exosome in patients with non-metastatic gastric cancer and healthy subjects.Conclusions: Regional lymph node metastatic gastric cancer exosomes can be significantly remodel BM-MSCs than non-metastatic gastric cancer exosomes.Wnt5 a is highly enriched in lymph node metastatic gastric cancer exosomes,and is a key protein for remodeling BM-MSCs in lymph node metastatic gastric cancer exosomes.This study will elucidate the new molecular mechanism of malignant progression of regional lymph node metastasis gastric cancer though remodeling microenvironment cells,and it is expected to provide potential molecular targets for the diagnosis and treatment of clinical gastric cancer patients with lymph node metastasis.