Study On The Mechanism Of Gastric Cancer Mesenchymal Stem Cells Derived IL-8 Reducing The Efficacy Of Anti-PD-1 In Gastric Cancer

Objective:Programmed cell death 1（PD-1）inhibits anti-tumor immunity by binding to programmed death-ligand 1（PD-L1）in tumor cells.Immunostimulatory therapies targeting PD-1/PD-L1 can restore anti-tumor immunity.However,clinical trials of anti-PD-1 therapy have not achieved satisfactory outcomes in gastric cancer.Previous studies have shown that gastric cancer mesenchymal stem cells（GCMSCs）promote PD-L1 expression through paracrine interleukin 8（IL-8）and inhibit antitumor immunity.However,the therapy effect and mechanism underlying the contribution of GCMSCs to anti-PD-L1 therapy in gastric cancer remains largely unknown.The purpose of this study was to investigate the effect and mechanism of GCMSCs derived IL-8 on anti-PD-1 antibody therapy in gastric cancer and to identify a combined target to improve the efficacy of anti-PD-1 therapy for gastric cancer.Methods:GCMSCs were isolated from fresh gastric cancer by adherent method.The surface markers were detected by flow cytometry.The multidirectional differentiation ability of GCMSCs was determined by adipogenic and osteogenic differentiation experiments.GCMSCs condition medium（GCMSC-CM）was collected and acted on gastric cancer cells.Human umbilical cord blood derived CD34⁺cells or human PBMCs were used to construct immune system humanizing mice.Gastric cancer xenograft mouse model was constructed by human gastric cancer cells and immune system humanizing mice.Immunohistochemistry was used to detect the proliferation and apoptosis of gastric cancer cells.Immunohistochemistry,HE staining and flow cytometry were used to detect the infiltration and function of immune cells in gastric cancer tissues.Lentiviral vector was used to overexpress PD-L1 in gastric cancer cells,and CCK-8 assay was used to detect the proliferation of gastric cancer cells under conventional culture conditions or ischemia and hypoxia conditions.sh RNA-PD-L1was used to inhibit the expression of PD-L1 in gastric cancer cells,and CCK-8 assay was used to detect the proliferation of gastric cancer cells under conventional culture conditions or ischemia and hypoxia conditions.Reactive oxygen species（ROS）was detected by ROS probe under different culture conditions.The expression of PD-L1,Glucose-6-phosphate dehydrogenase（G6PD）and the activation of Akt signal were detected by western-blot.The activity of G6PD was detected by Native-PAGE.si RNA-HK2 transfection inhibited the expression of hexokinase 2（HK2）in gastric cancer cells and the expression of HK2 and PD-L1 were detected by western-blot.Co-Immunoprecipitation（Co-IP）assay demonstrated the binding of HK2 to hypoxia inducible factor-1α（HIF-1α）.The expression of HK2 in the nucleus and cytoplasm was detected by western-blot.Phosphorylation of HK2 was detected by SDS-PAGE^Phos-tag.The transcriptional activity of HIF-1αwas detected by electrophoresis mobility assay.Lactic acid kits are used to detect lactate content in cell conditioned media and tumor tissues.The expression and correlation of HK2,PD-L1 and other proteins in gastric cancer tissues form patients were analyzed by bioinformatics.Results:GCMSCs were successfully isolated and identified.Immune system humanized mice were successfully constructed using human CD34⁺cells and human PBMCs.Mice administered PD-1 antibody exhibited significantly reduced tumor volumes.However,mice administered GCMSC-CM+PD-1 antibody had remarkably increased tumor volumes compared with those administered PD-1 antibody alone.IHC and HE staining revealed that compared with control,GCMSC-CM treatment resulted in a decrease in GZMB⁺-cells in xenograft tumor tissue.IHC staining showed that PD-1 antibody treatment resulted in significantly increased percentages of cleaved caspase-3⁺-,GZMB⁺-and CD8⁺-cells in tumor tissue,compared with control.However,GCMSC-CM almost completely reversed the effects of PD-1 antibody treatment on caspase-3,GZMB,and CD8 expression.Meanwhile,GCMSC-CM treatment dramatically increased the percentage of Ki67⁺-cells in tumor tissue,regardless of the presence or absence of PD-1 antibody.IHC and flow cytometry analyses consistently showed that GCMSC-CM treatment remarkably elevated PD-L1protein levels in gastric tumor tissue samples of mice.In normal medium,PD-L1overexpression did not affect gastric cancer cell proliferation compared with control.However,under serum deprivation and hypoxia,PD-L1 overexpression significantly promoted gastric cancer cell proliferation in a time-dependent manner.GCMSC-CM treatment significantly promoted gastric cancer cell proliferation compared with control,whereas PD-L1 silencing partially but significantly attenuated GCMSC-CM-induced cell proliferation under serum deprivation and hypoxia.HK2silencing completely blocked GCMSC-CM-induced PD-L1 upregulation.Co-IP analysis showed that HK2 bound to HIF-1αin HGC-27 and MGC-803 cells.GCMSC-CM treatment increased the nuclear expression of HK2 whereas IL-8neutralizing antibody or AKT inhibitor MK2206 reduced the nuclear expression of HK2 in MGC-803 cells.SDS-PAGE^Phos-tagshowed that both phosphorylated-and nonphosphorylated-HK2 were detectable in the cytoplasm whereas only phosphorylated-HK2 was detectable in the nucleus of gastric cancer cells.HK2silencing significantly reduced the levels of HIF-1αnuclear protein complex in HGC-27 and MGC-803 cells.GCMSC-CM-treated gastric cancer cells had significantly increased levels of HIF-1αnuclear protein complex compared with control cells,which was reversed by IL-8 neutralizing antibody or MK2206.GCMSC-CM treatment significantly upregulated HK2 protein expression and lactate production in xenograft tumors.IL-8 was required for HK2 upregulation and lactate overproduction induced by GCMSC-CM in gastric cancer cells in vitro,and HK2m RNA levels were positively correlated with IL-8 levels.Lactate exposure significantly reduced IFN-γproduction in CD8⁺T cells in a dose-dependent manner.However,lactate showed no effect on GZMB production in CD8⁺T cells.IHC staining demonstrated that lactate content was significantly increased in xenograft gastric cancer and human gastric cancer tissue samples with high HK2 expression but decreased in those with high CD8,GZMB and IFN-γexpression.Compared with wildtype control,CXCR1/2 depletion completely reversed GCMSC-CM-induced tumor growth and entirely restored the antitumor effect of PD-1 antibody suppressed by GCMSC-CM in mice.In addition,CXCR1/2 depletion effectively reduced lactate level while elevating CD8⁺T cell infiltration in tumor tissue.IHC staining showed that CXCR1/2 depletion effectively enhanced protein expression of CD8,IFN-γand GZMB in tumor tissue.In contrast,CXCR1/2 depletion resulted in decreases in the protein levels of HK2,PD-L1 and Ki67 in tumor tissue.Conclusions:GCMSC-CM promotes cell proliferation while inhibiting cell apoptosis and immune response in gastric tumor tissue,thereby attenuating the inhibitory effect of PD-1 antibody on gastric tumor growth.Both PD-L1 upregulation and lactate overproduction induced by GCMSC-derived IL-8-AKT-HK2 signal contribute to the attenuated anti-tumor immunity and anti-tumor effect of anti-PD-1 therapy.Blocking GCMSC-derived IL-8 signaling by CXCR1/2 depletion can effectively maintain the antitumor effect of anti-PD-1 antibody,thereby inhibiting tumor growth.This study provides a theoretical basis for blocking IL-8 signal combined with anti-PD-1antibody therapy to improve the effective of immunotherapy for gastric cancer.