Gastric Cancer Mesenchymal Stem Cells Derived IL-8 Impaires NK Cells Anti-tumor Immunity And Mechanism

Objective:Natural killer(NK)cells are potent immune cells,which play key roles in immune surveillance against tumor cells.However,tumor-infiltrating NK cells are dysfunctional.This is closely related to tumor microenvironment.Our previous studies have proved that gastric cancer mesenchymal stem cells(GCMSCs)could inhibit NK cells function,but the specific regulatory mechanism needs to be explored.The aim of study is to further investigate the mechanism of GCMSCs inducing NK cells dysfunction and provide a new strategy for NK cell-based immunotherapy.Methods:GCMSCs were isolated from gastric cancer tissues using adherent culture method,then identified using cell morphology,adipogenic and osteogenic differentiation and flow cytometry method.GCMSCs were cultivated for 48 hours in either a normoxic or hypoxic condition.GCMSCs were collected in both normal and hypoxic conditions,as well as the supernatant of GCMSCs which collected from normoxic(GCMSCs-CM)and hypoxic(GCMSCs-HCM)condition were obtained.NK cells from healthy donors were separated by MACS beads and then purity of NK cells was evaluated by flow cytometry.Interleukin-8(IL-8)levels in the GCMSCs-CM and GCMSCs-HCM were measured by ELISA,then co-cultured with NK92 cells for 48 h respectively.Lactic acid production assay and CCK-8 were used to measure the proliferative activity and glycolytic metabolism of NK cells.The immune molecular alterations of NK cells were detected using flow cytometry,the antitumor activity of NK cells pretreated with GCMSCs-CM or GCMSCs-HCM was detected using an LDH kit,and the expression of fructose 1,6-diphosphatase(FBP1)in NK cells was detected using a Western blot.The correlation between IL-8 content and infiltrated NK function in human gastric cancer was analyzed by bioinformatics.Results:GCMSCs were successfully isolated and identified in vitro.Fresh GCMSCs-CM and GCMSCs-HCM were collected and the differential expression of cytokine of both were determined by ELISA.The results showed that the expression levels of IL-8 in GCMSCs-HCM were higher than GCMSCs-CM.Bioinformatics analysis showed that the expression of IL-8 in gastric tumor were positively with MSCs count but negatively correlated with the activity of NK cells.The GCMSCs-HCM and GCMSCs-CM were collected and co-cultured with NK cells separately.CCK-8 assays revealed that NK cell proliferation was greatly lower in GCMSCs-HCM treated group than GCMSCs-CM treated group.Meanwhile,the secretion of perforin and IFN-γ by NK cells decreased significantly.However,the expression of FBP1 in NK92 cells treated with GCMSCs-HCM was increased the expression along with the decreased produciton of lactate.Using Anti-IL-8 and FBP1 inhibitor could restore the expression of immune effector molecules and the production of lactate in NK92 cells.We constructed a xenograft model of human gastric cancer using NXG mice by injecting NK cells from healthy donors.The tumor volume of GCMSCs-HCM treated group were significantly larger and the results of immunohistochemistry showed that the cell proliferation indicator Ki67 were also markedly increased.Conclusion:Hypoxic tumor microenvironment could promote the secretion of IL-8 by GCMSCs.GCMSCs derived IL-8 could act directly on NK cells infiltrating human gastric cancer tissues and upregulated the expression of FBP1 in NK cells,thereby inhibiting the secretion of perforin and IFN-γ in NK cells and promoting immune evasion.Inhibiting IL-8 derived from GCMSCs or blocking the expression of FBP1 in NK cells could partially restore the function of NK cells and inhibit tumor development.This further refined the mechanism of tumor immune evasion by the tumor inflammatory microenvironment and provided a new potential target for tumor immunotherapy based on NK cells.