Classification Of Gastric Cancer Based On Tumor Microenvironments And Mechanism Of Immune Evasion In Gastric Cancer Co-Regulated By PD-L1/PD-1 And MICA/NKG2D

Objective: The aims of this study were to classify gastric cancer subsets based on the tumor microenvironments(TME)immune status according to the expression of PD-L1 and infiltration of CD8+ T-cells,explore how IFN-? works through the JAKs/STATs pathway and regulates the expression of MICA and PD-L1 in gastric cancer cells,and determine the mechanism of immune evasion in gastric cancer co-regulated by PD-L1/PD-1 and MICA.Furthermore,we investigated the MICA and PD-L1 status in patients with gastric carcinoma,and determined the potential relationship between MICA,PD-L1 and clinical outcome of a CIK containing therapy.Methods: Part I: One hundred and eighty-six gastric cancer patients with a curative D2 gastrectomy were enrolled.The PD-L1,MICA and CD8+ T-cell status were evaluated with immunohistochemistry using specific antibodies.The samples were classified into four TME immune types and associated with different clinicopathological features and outcomes.Part II: We use methods including Western blot,RT-q PCR,gene transfection CRISPR-Cas9,and protein inhibitor to explore how IFN-? works through the JAKs/STATs pathway and regulates the expression of MICA and PD-L1 in gastric cancer cell lines;including BGC-823,BCG-803,SGC-7901 and MNK45.Part III: We established different immunophenotype of human gastric cell lines and used antibody blocking to explore how PD-1/PD-L1 and NKG2D/MICA regulate the CD8+ T-cell and NKT cell cytolytic effect against the gastric cancer cells by using the LDH assay.Part IV: We retrospectively studied all the patients with stage II–III gastric carcinoma diagnosed after surgery from January 2009 to March 2012.Ninety-five consecutive patients with gastric cancer after a D2 gastrectomy who received adjuvant chemotherapy combined with CIK cell therapy were enrolled.The MICA and PD-L1 expression of their tumors was determined by IHC according to part I.We investigated the MICA and PD-L1 status in TME by IHC according to part I and explored the potential relationship between MICA,PD-L1 status and clinical outcome of a CIK containing adjuvant therapy.Results: Part I: There was positive PD-L1 expression(TC1/2/3 or IC1/2/3)in 60.3%(112/186)of patients.There was a significant correlation between the PD-L1 expression and the CD8+ T-cell infiltration.TME was divided into four immunological-types.Types I,II,III,and IV were 60.3%,11.8%,0%,and 27.9% of the patients,respectively.The expression of MICA was negatively correlated with the CD8+ T-cell infiltration and PD-L1 status(p=0.081,p=0.027).CD8+ T-cell infiltration was significantly associated with disease-free survival(DFS)and overall survival(OS).The DFS and OS of patients with immune type II tumors was significantly worse compared to immune types I and IV: There was no significant difference in DFS and OS between type I and IV.Part II: Gastric cancer cells were incubated for 0,4,12,and 12 h in the presence of the IFN-?,and Western blot and RT-q PCR analysis revealed that IFN-? mediated a pronounced down-regulation of MICA expression and up-regulation of PD-L1 through the phosphorylation of JAK1/JAK2-STAT1 proteins,which was dependent on the duration of IFN-? treatment.Using JAK1/JAK2 inhibitors and STAT1 gene knockout with CRISPR-Cas9 could inhibit the down-regulation of MICA expression and up-regulation of PD-L1 mediated by IFN-?.Part III: LDH assay analyses showed that CD8+T cell-mediated cytotoxicity to MICAko/PD-L1oe-BGC-823 was much lower than PD-L1ko-BGC-823 and PD-L1oe-BGC-823(p<0.05,p<0.001),CD8+ T-cell mediated cytotoxicity to MICAoe/PD-L1ko-BGC-823 was much higher than MICAoe-BGC-823 and MICAko-BGC-823(p<0.01,p<0.001).NKT cells showed similar killing capacity to PD-L1ko-BGC-823,PD-L1oe-BGC-823 and MICAko/PD-L1oe-BGC-823(p>0.05),but had a higher cytotoxicity against MICAoe/PD-L1ko-BGC-823 and MICAoe-BGC-823 that was in contrast to MICAko-BGC-823(p<0.001,p<0.001).Blocked with NKG2 D monoclonal antibody,the CD8+ T-cell killing of PD-L1oe-BGC-823 is significantly weakened,and CD8+ T-cells jointly blocked by NKG2 D and PD-1 monoclonal antibodies is less threatening to PD-L1oe-BGC-823 than is blockade by PD-1 alone.Part IV: High-expression of MICA protein,with IHC scores of 5-7,was documented in 38 of 95 tumor samples(40.0%)and positive PD-L1 expression(TC1/2/3 or IC1/2/3)was documented in 54 of 95 tumor samples(56.8%).For patients with high MICA expressing tumors the median DFS and OS were longer than for the patients with tumors with low expression of MICA;46.0 months vs.41.0 months(p=0.027),and 48.0 months vs.42.0 months(p=0.031),respectively.PD-L1 status was not associated with DFS and OS.In a multivariate analysis,stage and MICA expression were independent prognostic factors for DFS and OS.Conclusion:Based on our data the PD-L1 expression was significantly correlated with the number of tumor infiltrating CD8+ T-cells.The TME immune type III was absence,and type II patients had a worst prognosis compared with patients with immunological types I and IV.The MICA expression had a significantly negative correlation with PD-L1 expression.2.In vitro,IFN-? mediated a pronounced down-regulation of MICA expression and up-regulation of PD-L1 in gastric cancer cell lines through the phosphorylation of JAK1/JAK2-STAT1 proteins.3.The cytotoxicity of CD8+ T-cells against gastric cancer cells was co-regulated by MICA/NKG2 D and PD-1/PD-L1.Down-regulation for MICA in PD-L1 positive gastric cancer may be a mechanism for primary resistance to anti-PD-1 therapy.4.Patients with gastric cancer who have received a D2 gastrectomy and whose tumors have high expression of MICA seem to derive a clinical benefit from adjuvant chemotherapy plus CIK therapy,and the PD-L1 status was not associated with the outcome in a CIK containing adjuvant therapy.