| BackgroundGastric cancer is one of the major fatal diseases in the world.Due to the lack of typical symptoms in the early stage of gastric cancer and the fact that most patients do not have the concept of early diagnosis,80%of patients are in advanced or late stages when diagnosed.Currently,the comprehensive treatments based on surgery are mostly used for locally advanced gastric cancer.As one of the means of comprehensive treatments,neoadjuvant chemotherapy refers to systemic chemotherapy applied before surgery for malignant tumors that can be surgically resected,which can reduce tumor size and clinical stage,help to preserve organs,increase surgical resection rate,and prolong patients’ survival.In recent years,studies have shown that chemotherapy can not only inhibit tumor cell growth,but also achieve anti-tumor effects by activating the immune system.Neoadjuvant chemotherapy can obtain paired tumor tissues at diagnosis and after chemotherapy,which is a good clinical model to evaluate the effect of chemotherapy on the tumor immune microenvironment.The tumor immune microenvironment refers to all immune components in tumor tissues,including innate immune cells,adaptive immune cells,extracellular immune factors and cell surface molecules,which are closely related to the occurrence,recurrence and metastasis of tumors.It is also an important determinant of the response rate and prognosis of patients.Therapy targeting the immune microenvironment has also developed into an important antitumor tool.Therefore,a comprehensive understanding of the remodeling effect of neoadjuvant chemotherapy on the immune microenvironment of gastric cancer can help provide strategies and theoretical basis for the combined therapy of neoadjuvant chemotherapy and immunotherapy.Objective1.To explore the effect of neoadjuvant chemotherapy on the tumor immune microenvironment of patients with gastric cancer;2.To investigate the effect of oxaliplatin and S-1 combination regimen on the tumor immune microenvironment of mice with gastric cancer;3.To explore the molecular mechanism by which oxaliplatin and S-1 combination regimen reshapes the tumor immune microenvironment of gastric cancer.Methods1.Patients with gastric cancer who received neoadjuvant chemotherapy in the Oncology Department of Shanghai Changhai Hospital from January 2015 to June 2021 were retrospectively analyzed.The effect of neoadjuvant chemotherapy on the infiltration of immune cells in gastric cancer tissue was explored by immunohistochemical staining and immunohistochemical fluorescence staining of gastric cancer tissue paraffin sections before and after neoadjuvant chemotherapy,and the correlation between immune cell infiltration and tumor regression grade and clinical response was analyzed.2.A mouse subcutaneous transplant model of gastric cancer was established,and the effect of oxaliplatin and S-1 combination therapy on gastric cancer tissue TIME and peripheral blood immune cells in mice was detected by flow cytometry.The transcriptome sequencing data was used to detect differential genes and enriched signaling pathways after drug treatment,and the changes of various immune cells and immune factors in the TIME were further analyzed by deconvolution.3.Tumor cell culture supernatant was collected after gastric cancer cells were treated by oxaliplatin and 5-FU combined therapy.The recruitment effect of tumor cell culture supernatant on NK92 cells was detected by Transwell assay.Chemokines related to immune cell recruitment were detected by cytokine chip.The mRNA expression level of chemokines recruiting NK cells was detected by RT-qPCR.After neutralizing CCL8 in tumor cell culture supernatant with neutralizing antibody,the recruitment change of tumor cell culture supernatant to NK92 cells were detected.After inhibition of CCL8 in mice,the anti-tumor effect of oxaliplatin and S-1 combination therapy and the infiltration of NK cells in gastric cancer tissue were measured.4.After gastric cancer cells were treated by oxaliplatin and 5-FU combined therapy,RTqPCR was used to detect the mRNA expression change of NK cell-killing ligands on tumor cells.After gastric cancer cells were treated by oxaliplatin and 5-FU combined therapy for 48h,NK92 cells were added for co-culturing.LDH releasing experiment was used to detect the killing function of NK92 cells on gastric cancer cells.5.After gastric cancer cells were treated by oxaliplatin and 5-FU combined therapy,the phosphorylation level of ERK1/2,JNK and p38 protein in the MAPK signaling pathway were detected by western blot.After inhibiting the activation of ERK1/2,JNK and p38 proteins in MAPK signaling pathway by kinase inhibitors,the secretion level of CCL8 by tumor cells was detected.Results1.Neoadjuvant chemotherapy can reshape the immune microenvironment of gastric cancer.After neoadjuvant,the infiltration of CD4+T cells,CD8+T cells,B cells,NK cells,macrophages and dendritic cells in human gastric cancer tissues increased.The infiltration of CD8+T cells and NK cells after neoadjuvant chemotherapy were associated with better clinical response.2.The SOX regimen reshapes the tumor immune microenvironment and peripheral immunity of mice with gastric cancer,and promotes the recruitment of NK cells and the expression of chemokines related to NK cell recruitment.SOX regimen can inhibit the growth of gastric cancer in mice,promote the infiltration of CD4+T cells,CD8+T cells,B cells,NK cells,macrophages and dendritic cells into gastric cancer tissues in mice,and reduce Tregs infiltration.The proportions of CD4+T cells and CD8+T cells in peripheral blood of mice with gastric cancer increased,and the proportions of MDSCs,macrophages and neutrophils decreased.Transcriptomic sequencing data and deconvolution analysis showed that after treated with SOX chemotherapy,the differential genes in the cytokine activation response and interaction signaling pathway upregulated,the number of NK cells infiltrated by mouse gastric cancer tissues increased,and the chemokines that recruited NK cells(CCL2,CCL7,CCL8 and CCL11)increased.3.Combination therapy with oxaliplatin and 5-FU can promote the secretion of CCL8 by gastric cancer cells,thereby promoting the recruitment of NK cells.Transwell results showed that after treated with oxaliplatin and 5-FU combined therapy,the recruitment of tumor cell culture supernatant to NK92 cells was enhanced.Cytokine microarray results showed that after treated with oxaliplatin and 5-FU combined therapy,chemokines recruiting NK cells(CCL7,CCL8,CCL13 and CCL16)in gastric cancer cells had an increase in protein levels.RT-qPCR results showed that the mRNA expression of the chemokines recruiting NK cells(CCL2,CCL7,CCL8 and CCL11)increased in gastric cancer cells after oxaliplatin combined with 5-FU treatment.Transwell results indicated that after neutralizing CCL8 in the tumor cell culture supernatant,the recruitment of NK92 cells decreased.After inhibiting CCL8 in mice,NK cells infiltrated in mouse gastric cancer tissue decreased,and the antitumor effect of oxaliplatin and 5-FU combination therapy was weakened.4.The combination of oxaliplatin and 5-FU enhances the sensitivity of gastric cancer cells to NK cell killing.RT-qPCR results showed that the expression of NK cell-killing ligands on gastric cancer cells increased after oxaliplatin combined with 5-FU treatment.LDH release experiment showed that the killing effect of NK92 cells was enhanced after oxaliplatin combined with 5-FU treatment.5.The combination of oxaliplatin and 5-FU can activate the MAPK signaling pathway and promote the secretion of CCL8 by gastric cancer cells,thereby promoting the recruitment of NK cells.Western blot results showed that the combined treatment of oxaliplatin and 5-FU enhanced the phosphorylation of ERK1/2,JNK and p38 proteins of MAPK signaling pathway in gastric cancer cells.Western blot and RT-qPCR results showed that the level of CCL8 secreted by gastric cancer cells decreased in both mRNA and protein levels after inhibiting the activation of ERK1/2,JNK and p38.Conclusion1.Neoadjuvant chemotherapy can reshape the immune microenvironment of gastric cancer,promote the recruitment of anti-tumor immune cells and reduce the infiltration of inhibitory immune cells;2.Neoadjuvant chemotherapy can promote the secretion of CCL8 by gastric cancer cells via activating MAPK signaling pathway,thus promoting the recruitment of NK cells,and ultimately exerting anti-tumor immunity. |
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