| Objective:Microsatellite instability(MSI)refers to the phenomenon that the length of a microsatellite sequence changes due to insertion or deletion mutations during DNA replication.It is often caused by the afunction of DNA mismatch repair(MMR)system.Solid malignancies which varies by microsatellite status have heterogenous immune response to immune checkpoint inhibitor drugs,suggesting that microsatellite status has an important impact on tumor immune microenvironment.Tumor microenvironment(TME)is composed of tumor cells,host cells that resident and recruited(fibroblasts and immune cells associated with cancer,respectively),products secreted by these cells(such as cytokines and chemokines)and noncellular components in the extracellular matrix.Lymphocytes in the tumor microenvironment are the main force attacking cancer,including T cells,B cells and natural killer cells(NK cells).These cells are also called tumor infiltrating lymphocytes(TILs).Studies have shown that the anti-tumor activity of TILs in the TME is much lower than those far away from the tumor.The reason for this difference is tumor immunosuppressive microenvironment,which not only makes the surrounding cells immune suppressive,but also helps tumor cells escape immune attacks.The tumor immunosuppressive microenvironment is mainly composed of immunosuppressive molecules,matrix components,immunosuppressive cells and related immunosuppressive cytokines produced by tumor cells.The main immunosuppressive cells include tumor-associated macrophages,regulatory T cells,bone marrow-derived suppressor cells and tumor-associated fibroblasts.And immune cytokines mainly include IL-10,TGF-β,PD1/PDL1and other immune checkpoint molecules and exosomes,etc.The studies that focus on TME may provide important clues for explain the interaction between tumor cells and immune cells.Gastric cancer is the fifth most common malignant tumor worldwide and the third leading cause of cancer death.At present,surgical treatment is mainly used,and radiotherapy,chemotherapy,targeting and immunotherapy are also used,but some patients have poor clinical prognosis.Precision therapies based gene have been successful in a variety of cancer treatments,and have attracted widespread attention.As one of the four molecular subtypes,microsatellite unstable(MSI)gastric cancer has a series of unique molecular changes,which makes it unique in terms of pathogenesis,clinical characteristics,treatment response and prognosis.At present,genes related with immune cells in MSI GC were ambiguous,and the interaction between tumor cells and immune cells in the tumor microenvironment has not yet been elucidated.This study aims to investigate the correlations between immune cells and genes and possible regulatory mechanisms in the microenvironment of gastric cancer with different microsatellite status,and provide experimental basis and theoretical reference for explaining the pathogenesis of gastric cancer with different microsatellite status and finding new molecular markers and new drug targets.Methods:Part Ⅰ Integration analysis of infiltrated immune cells and related genes in gastric cancer with different microsatellite status1.Download and integration of data sets:The GC cohort data of mRNA expression profile containing 415 samples,immune cell profile containing 267 samples and molecular typing containing 295 samples were downloaded from TCGA.The ID of the GC sample was used as the unique identification code,the above 3 data sets were integrated and patients without complete information were excluded.This study finally included 267 GC samples,which included 209 MSS GC and 58 MSI GC.2.Differentially immune cells analysis in GC with different microsatellite status:The immune cell data set downloaded from TCGA contains the relative values of 34 types of immune cells.SPSS software was used to compare the differences between MSI GC and MSS GC.3.Screening of differentially expressed genes(DEGs):The edge R method of R package TCGA biolinks was used to analyze differentially expressed genes(DEGs)in GC with different microsatellite status.(FDR<0.01 and log FC absolute value greater than or equal to 2).4.GO and KEGG analyses:GO and KEGG pathway enrichment analyses were performed using KEGG pathway、GO Biological Processes、Reactome gene Sets、Canonical Pathways and CORUM,and then visualization using Cytoscape.5.Protein-protein interaction(PPI)network analysis:The retrieval of Interacting Genes(STRING)database tool(string-db.org)was used to evaluate interactive relationships among the DEGs.Interactions with a combined score>0.4 were considered significant.6.Statistical methods:All statistical analyses were performed using R or SPSS software.For the comparison of the two groups,student t test is used for the normal distribution variable,the Mann Whitney U test is used for the non-normal distribution variable,and the Kaplan-Meier is used for survival analysis.P<0.05 was considered significant.Part Ⅱ Expression of PD1/PDL1 in gastric cancer at different microsatellite status and its correlation with infiltrating immune cells in tumor microenvironment1.Patients and clinical data collection:215 patients who underwent surgical resection of primary GC at the first hospital of China Medical University and Jinzhou Medical University of Liaoning province between January 2012 and December 2018 were collected retrospectively.Clinicopathological data including age,gender,pathological type and invasion depth,lymph node metastasis,vascular tumor thrombus,and TNM staging were all derived from medical records,pathological reports or discharge summary.The study was approved by the ethics committee of the First Affiliated Hospital of China Medical University and Jinzhou Medical University,respectively.All participants enrolled in the study had signed written informed consent forms.2.Immunohistochemical(IHC)staining:SP method was used for IHC staining.3.Criteria for the determination of IHC staining results:(1)Microsatellite status assessment:Each section was observed under low-power microscope to selecte positive staining areas and then observed under high-power microscope.The evaluation was conducted by two senior pathologists independently.MLH1,PMS2,MSH2 and MSH6 were all located in GC cells nucleus.The tumor cell nucleus with brown yellow or brown granules were determined as positive staining.Absence was determined when tumor cell nucleus failed to be stained.MSS was determined when all the four proteins were positive,and MSI when one or more proteins were absent.(2)PD1 and PDL1 expression evaluation:PD1 and PDL1 were all located in the membrane and/or cytoplasm.PD1 was stained in tumor infiltrating immune cells(labeled as PD1),while PDL1 was stained in GC cells(labeled as PDL1[T])and tumor infiltrating immune cells(labeled as PDL1).Each sample was randomly selected five high-power fields(HPF,x 400,0.025mm~2),100 tumor cells or immune cells were manually counted in each HPF,the percentage of positive staining cells was calculated,and the mean value was adopted as the positive expression rate of PD1,PDL1[T]and PDL1.The positive staining was defined as more than 1%stained cells regardless of cytoplasmic or membrane staining.(3)CD8 and CD68 expression evaluation:We randomly chose five high-power fields(HPF,x 400,0.025mm~2)in tumor center and invasive front respectively for each case,then manually counted and calculated the average number of stained cells of CD8+T cells and CD68+M.According to the median number of stained cells(CD8,72.3/HPF;CD68,43.2/HPF),patients were divided into two groups:high and low density.3.Statistical analysis:All statistical analyses were performed using SPSS software.The analysis between PD1/PDL1 expression or CD8+T/CD68+M cell density and microsatellite status or clinicopathological characteristics was conducted byχ2 test or Fisher’s exact test,and Kaplan-Meier is used for survival analysis.P<0.05 was considered significant.Part Ⅲ Correlation between PMS2 deficiency and clinicopathological characteristics in gastric cancer and its effect on the biological behavior of gastric cancer cells as well as its possible mechanism1.Correlation between PMS2 deficiency and clinicopathological characteristics of gastric cancer:χ2 test or Fisher’s exact test was used.2.PMS2 overexpression on the biological behavior of gastric cancer cells and its possible regulatory mechanism:(1)Real-time PCR and Western blot reluts showed that PMS2 mRNA expression was lower in AGS and HGC27 cells.(2)The plasmid transfection technology was used to construct PMS2 overexpression gastric cancer cells.(3)CCK-8 and real-time PCR was used to detect the effect of PMS2 overexpression on gastric cancer cell proliferation.(4)Real-time PCR was used to detect the effect of PMS2 overexpression on gastric cancer cell apoptosis.(5)Wounding healing was used to detect the effect of PMS2 overexpression on gastric cancer cell migration.(6)Transwell and real-time PCR were used to detect the effect of PMS2 overexpression on gastric cancer cell invasion.(7)Real-time PCR was used to detect the effect of PMS2 overexpression on gastric cancer cell differentiation.Result:Part Ⅰ Integration analysis of infiltrated immune cells and related genes in gastric cancer with different microsatellite status1.Differentially immune cells in gastric cancer with different microsatellite status and their correlation with clinicopathological characteristics:(1)Differentially immune cells in gastric cancer with different microsatellite status:There are 12 differentially immune cells in gastric cancer with different microsatellite status.Compared with MSS gastric cancer,IFN gamma Response,Th2 Cells,Macrophages M1,Mast Cells Activated,NK Cells Activated,T Cells CD4 Memory Activated,and T Cells CD8 were significantly up-regulated in MSI gastric cancer;while TGF beta Response,B Cells Na(?)ve,Mast Cells Resting,Monocytes and Plasma Cells were significantly down-regulated.(2)The correlations between differentially immune cells and clinicpathological characteristics in gastric cancer with different microsatellite status:The correlation between differentially immune cells and clinicpathological characteristics was seen.In MSI gastric cancer,T Cells CD4 Memory Activated was seen more at patients below 60years old;Macrophages M1 was common in gastric cancer with highly differentiated;IFN-gamma Response and Mast Cells Resting were mostly found in patients without lymph node metastasis,however Mast Cells Activated was common in patients with lymph node metastasis.In MSS gastric cancer,TGF-beta Response,B Cells Na(?)ve,Mast Cells Resting and T Cells CD8 were mostly found in diffuse gastric cancer,however Th2Cells,Mast Cells Activated,Plasma Cells and T Cells CD4 Memory Activated was common in intestinal gastric cancer.Th2 Cells,Mast Cells Activated and T Cells CD4Memory Activated was seen more at highly differentiated gastric cancer,however GF-beta Response,Mast Cells Resting and T Cells CD8 was frequent in poorly differentiated gastric cancer.Mast Cells Resting was mostly found in III+IV gastric cancer or in gastric cancer with lymph node metastasis.TGF-beta Response,IFN-gamma Response,Macrophages M1 and T Cells CD8 is common in T3+T4 gastric cancer.2.DEGs screening in GC with different microsatellite status as well as its GO and KEGG analysis:There are 75 DEGs in gastric cancer with different microsatellite status,including ACTN2,AFP,ALDH1A2,APOH,AQP12B,AQP2,ASCL1,etc.The expression of 75 DEGs mRNA was significantly down-regulated except CD274,MYC,PDCD1,PDCD2,QRSL1,RNASE2,SEMG1 and TRIM69 mRNA expression in MSI GC(Supplement table 1).The above 75 DEGs mainly focused on the following clusters:pancreatic secretion,humoral immune response,regulation of gene expression in endocrine-committed(NEUROG3+)progenitor cells,regulation of hormone levels,protein digestion and absorption,etc3.The correlations between differentially immune cells and DEGs in gastric cancer with different microsatellite status:(1)The correlations between differentially immune cells and DEGs in gastric cancer with different microsatellite status:The correlations were seen between 12 differentially immune cells and 71 DEGs in gastric cancer with different microsatellite status.(2)GO and KEGG analysis of 71 DEGs related to differentially immune cells:The 71DEGs related to differentially immune cells are mainly concentrated in the following clusters:humoral immune response,regulation of hormone levels,chemical synaptic transmission,phospholipase C-activating G protein-coupled receptor signaling pathway and cellular metal ion homeostasis,etc.We also found that not only 71 DEGs but also their enrichment clusters have overlap.(3)PPI network analysis of 71 DEGs:PPI network analysis showed that there were interactions among DEGs related to differentially immune cells coding protein.4.Effects of different microsatellite status on the prognosis of gastric cancer:There was no significant effect of microsatellite status on the prognosis of gastric cancer.Part Ⅱ Expression of PD1/PDL1 in gastric cancer at different microsatellite status and its correlation with infiltrating immune cells in tumor microenvironment1.Judgment of microsatellite status in gastric cancer:Based on the expression of MLH1,PMS2,MSH2 and MSH6,215 cases were divided into two groups:46 MSI and 169MSS.2.CD8+T cells/CD68+M density in MSI and MSS gastric cancer:CD8+T cells density in MSI GC was higher than that in MSS gastric cancer,but without statistical significance(P=0.537).CD68+M density in MSI gastric cancer was significantly higher than that in MSS gastric cancer(P<0.001).3.PD1/PDL1 expression in MSI and MSS gastric cancer:PD1,PDL1[T]and PDL1expression rate were 59.0%,35.0%and 61.0%in 46 MSI GC,as well as 45.0%,22.0%and 40.0%in 169 MSS GC respectively.Statistical analysis showed that PDL1expression in MSI GC was significantly higher than that in MSS GC(P=0.011),while PD1/PDL1[T]expression in MSI GC was also higher than that in MSS GC(P=0.109and P=0.090).4.The correlations between PD1/PDL1 expression and CD8+T cells/CD68+M density in MSI and MSS GC:PD1 expression in CD8+T cells high density was greater than that in low density group in MSI GC but without statistical significance(P=0.058).PDL1expression in CD68+M high density was greater than that in low density group in MSS GC(P=0.013).The results indicated that PD1 expression was correlated with CD8+T cells high density in MSI GC and PDL1 expression was correlated with CD68+M high density in MSS GC.However,PDL1[T]expression had no correlation with CD8+T cells/CD68+M density in MSI or MSS GC.5.The correlations between PD1/PDL1 expression and CD8+T cells/CD68+M density at different locations in MSI and MSS GC:PD1 expression in CD8+T cells high density was significantly more than that in the low density group at invasion front of MSI GC(P=0.031).PDL1 expression in CD68+M high density was significantly more than that in the low density group in tumor center and invasion front of MSS GC(P=0.001 and P=0.014,respectively).However,PDL1[T]expression had no correlation with CD8+T cells/CD68+M density in tumor center and invasion front of MSI or MSS GC.6.The correlations between CD8+T cells/CD68+M density or PD1/PDL1 expression and clinicopathological characteristics in gastric cancer:CD68+M high density was more common in elderly patients(>60 years old),and increased with the increase of TNM stage;PDL1[T]and PDL1 expression were all more common in patients with T3+T4infiltration,lymph node metastasis and vascular carcinoma embolism.However,CD8+T cells high density and PD1 expression had no correlation with clinicopathological parameters.7.The correlations between CD8+T cells/CD68+M density or PD1/PDL1 expression and prognosis in gastric cancer:Gastric cancer patients with MSI,PD1 positive,PDL1[T]negative and PDL1 negative had better prognosis.Multivariate analysis showed that MSI,CD8+T cells density,PDL1[T],TNM stage,infiltration depth and vascular thrombi were all independent prognostic factors for gastric cancer.Part Ⅲ The correlations between PMS2 deficiency and clinicopathological characteristics in gastric cancer and its effect on the biological behavior of gastric cancer cells as well as its possible mechanism1.The correlations between PMS2 deficiency and clinical pathological characteristics as well as prognosis in gastric cancer:(1)The correlations between PMS2 deficiency and clinicopathological characteristics in gastric cancer:PMS2 deficiency was more seen in patients with women,aging(>60years),TNM stage(stage Ⅰ+Ⅱ),no lymph node metastasis and no vascular cancer thrombus.(2)The correlation between PMS2 deficiency and prognosis in gastric cancer:The patients with PMS2 deficiency have better overall survival(p=0.069).2.The effect of PMS2 overexpression on the biological behavior of gastric cancer cells and its possible regulatory mechanism:(1)Real-time PCR and Western blot relsuts showed that PMS2 mRNA expression was lower in AGS and HGC27 cells.(2)Real-time PCR and Western blot relsuts confirmed that PMS2 mRNA and protein expression were higher in gastric cancer cells with PMS2 overexpression.(3)CCK-8 and real-time PCR relsuts showed that PMS2 overexpression affected the proliferation in gastric cancer cells.(4)Real-time PCR relsuts showed that PMS2 overexpression significantly affected the apoptosis in gastric cancer cells.(5)Wounding healing and real-time PCR relsuts showed that PMS2 overexpression did not affect the migration in gastric cancer cells.(6)Transwell method and real-time PCR relsuts showed that PMS2 overexpression did not affect the invasion in gastric cancer cells.(7)Real-time PCR relsuts showed that PMS2 overexpression significantly affected the level of SOX2 mRNA in AGS and HGC27 cells.Conclusions:1.The immune cells and genes are significantly different in the microenvironment of gastric cancer with different microsatellite status;differentially immune cells are not only related to different clinical pathological characteristics,but also related to differentially expressed genes;DEGs related to differentially immune cells are concentrated in different biological processes and pathways,and there is overlap and interaction between them.DEGs in gastric cancer with different microsatellite status may affect the clinical biological behavior of gastric cancer cells by affecting the type and function of immune cells in the microenvironment,and ultimately lead to different clinical outcomes.2.PD1/PDL1 expression and CD8+T cells/CD68+M density were higher in MSI GC than those in MSS GC,suggesting that PD1/PDL1 expression and the immune response varied from microsatellite status in GC.Moreover,we found a significant correlation between PD1/PDL1 expression and CD8+T cells/CD68+M high density,especially at invasion front in MSI or MSS GC,suggesting that they may participate in immune escape and affect the efficacy of immunotherapy.3.PMS2 deficiency was significantly associated with women,aging(>60 years),TNM stage(stage Ⅰ+Ⅱ),no lymph node metastasis and no vascular cancer thrombus;patients with PMS2 deficiency have better overall survival.after PMS2 overexpression in gastric cancer cells,apoptosis is significantly increased,proliferation is decreased;there was no significant change in migration and invasion;the results suggested that PMS2 may affect the development of gastric cancer by changing of proliferation or apoptosis. |
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