The Effect And Mechanism Of Cancer Associated Fibroblasts On The Molecular Classification Of Gastric Cancer

Objective Gastric cancer(GC)is the third primary cause of cancer-associated death in the world.Cancer associated fibroblast(CAFs),the most common stroma cells in tumor environment(TME),plays a pivotal role in cancer progression.Recent study found that CAFs was a highly heterogenous cell population in some cancer types,but it was still unclear which role it plays in gastric CAFs.As we known,the clinicopathological and prognostic significance in diffuse-type gastric cancer is very different from that in intestial-type.So whether there is phenotypical and functional difference between intestinal-and diffuse-type CAFs? Recent proposed molecular classifications unveiled intrinsic pathological alterations of GC.In this study,simplified molecular classification was used to catagrize GC patients in Chinese population.The relationship between molecular subypes and PD-L1 expression or amplification was further observed to clarify the different clinical and prognostic difference between intestinal-and diffuse-type GC.Moreover,the comparison of phenotype markers and secretion ability between intestinal-and diffuse-type CAFs was investigated.The effect of the principal factor secreted by CAFs on GC cells was also explored.Thus,this study might provide a new theoretical basis for the confirmation of heterogenous GC.Methods 1.The simplified molecular classification using IHC and ISH was implemend in 165 GC cases.The PD-L1 expression and amplification was detected on 165 GC cases by IHC and FISH respectively.The clinicopathological significance of PD-L1-positive as well as its associtation with molecular classfication was analysed.MMR protein and MSI status was evaluated on 279 GC cases.The molecular classification under different Lauren background was analysed in 165 GC cases.2.The 3 paired intestinal-and diffuse-type CAFs & NFs were directly isolated from human GC patients.The expression of FAP and SMA in each CAFs & NFs wasdeteted by Western blotting.The secreted protein of intestinal-and diffuseCAFs-CM was tested by HPLC-MS/MS.The discrepant cytokines between CAFs and NFs were detected by cytokines chip.Among them,the most distinct cytokine was then verified by Western blotting and ELISA assay.The ability of this aberrant expressed cytokine(OPN)on proliferation,apoptosis,migration and invasion was confirmed by CCK8 assay,colone formation assay,flow cytometry and transwell assay.Results 1.The simplified molecular classification showed similar clinical and prognosis characteristics as previous study.PD-L1 expression was strongly associated with d-MMR GC.Expressed PD-L1 on immune cells had the inclination to a good prognosis.PD-L1 amplification just appeared in PD-L1-positive cases with an extremely low proportion.The high conformity of MMR-IHC and MSI-PCR was confirmed(98.5%).The d-MMR/MSI-H GC had a good survival than p-MMR/MSS,but not an indepentend prognostic factor.2.The clinicopathological and prognostic significance in molecular subtypes of intestinal-type GC was very different from that in diffuse-type.3.Western blotting showed the expression of FAP and SMA in intestinal-CAFs & NFs was obviously different from that in diffuse type.FAP was higer in intestinal CAFs than NFs,but not in diffuse.SMA was higer in diffuse CAFs than NFs,but not in instesinal.That secretory ablility of diffuse CAFs was tronger than intestinal CAFs by analysis of HPLC-MS/MS.OPN was detected as the primary discriminate factor between CAFs and NFs by cytokines chip,Western blotting and ELISA assay.OPN promoted the ability of migration and invasion,but not the proliferation and apoptosis on GC cells in vitro.Conclusions 1.The simplified molecular classification could be used as a surrogate for molecular classification in clinlic practice.PD-L1 expression is associated with d-MMR GC.PD-L1 expressed on immune cells inclined to have a good prognosis.PD-L1 amplification just appeared in PD-L1 expressed GC with a low proportion.MMRprotein and MSI testing had a high conformity.2.The clinicopathological and prognostic significance of molecular subtypes in intestinal-type GC were different from that in diffuse-type.3.The pheonotype and secretion of diffuse-type CAFs is very different from intestinal-type.CAFs secreted OPN promoted the ability of migration and invasion,but not proliferation and apoptosis on GC cells.