Immune Landscape Of Mismatch Repair-proficient Colorectal Cancer By Multiplex Immunohistochemistry Revealed Distinctive Tumor Microenvironment

Background:Colorectal cancer is one of the most common causes of cancer death.To date,surgery remains the major approaches in cancer therapy,assisted with chemotherapy or chemoradiotherapy,but some patients still present with a poor prognosis.Immunotherapy has been a successful treatment in various cancers,its benefits in colorectal cancer are limited to a small part of patients with microsatellite unstable colorectal cancer,while the benefits in mismatch repair proficient or microsatellite stable colorectal cancer have not been determined.It is crucial to reveal the distinctive tumor immune microenvironment of mismatch repair proficient colorectal cancer and find out the targets of immunotherapy,and then improve the clinical outcomes of the most patients with colorectal cancer.Objective:To analyze the distinctive tumor immune microenvironment of mismatch repair proficient colorectal cancer by multiplex immunohistochemistry technology,comprehensively reveal the spatial associations between immune populations in the tumor microenvironment and the association with clinicopathological parameters and overall survival.To explore the potential targets for immunotherapy.Methods:Four kinds of mismatch repair proteins were detected in patients with colorectal cancer and then we screened out the patients with mismatch repair proficient colorectal cancer.The clinicopathological variables of them were retrospectively analyzed.HE staining was used for the preliminary analysis about the infiltration of immune cells in the tumor microenvironment of colorectal cancer.Multiplex immunohistochemistry was used to stain antibodies on the single pathological paraffin-embedded sections repeatedly.The experiment was divided into two groups,with 81 and 107 patients,respectively.The immune landscape of mismatch repair proficient colorectal cancer was investigated and the correlation between spatial orientation of immune cells and clinical pathological parameters and overall survival was revealed.Furthermore,the immunological factors associated with the clinical outcome in the tumor microenvironment of MMR-proficient colorectal cancer were explored.Results:Lymphocyte phenotypes included:CD8~+T cells and CD4~+T cells,CD8~+GzmB~+CTLs and CD4~+GzmB~+CTLs,CD8~+CD103~+TRM cells,CD4~+CD103~+TRM cells and B cells.Myeloid cells included granulocytes and macrophages.CD8~+T cells(P=0.0017),CD4~+T cells(P<0.0001),CD8~+TRM cells(P=0.0001),CD4~+TRM cells(P<0.0001),CD4~+GzmB~+ CTLs(P=0.0321)and B cells(P=0.0085)showed more in CT than that in IM.In any region of the tumor microenvironment,granulocytes were the main cells compared with B cells and macrophages(P<0.05).The CD8~+T cells,CD4~+T cells,CD4~+CTLs,CD8~+CTLs and CD8~+TRM cells in the particular region of the patients with early clinical stage were more than those in the advanced patients(P<0.05).When tumor progression or metastasis was described,most of immune cells,especially CD8~+TRm cells,showed the decrease in the particular regions(P<0.05).When distant metastasis occurred,we found that macrophages infiltrated in CT increased(P=0.0089).The density of CD8~+TRM cells in the particular regions decreased when lymphovascular invasion or perineural invasion was detected,and infiltration of B cells in the particular regions of the tumors with perineural invasion increased(P<0.05).Cytotoxic T cells infiltrated in the particular regions of rectal cancer were more than colon cancer(P<0.05).Intraepithelial infiltration of the CD4~+TM cells(P=0.0473)was associated with prolonged overall survival.Patients with colorectal cancer with high-density CD4~+CTLs infiltration in the IM region(P=0.0158),especially in the IMs region(P=0.0158),had a longer overall survival time.High-density intraepithelial granulocytes(P=0.0496)was associated with prolonged overall survival of patients with colorectal cancer.Conclusions:1.Multiplexed immunohistochemical consecutive staining on single slide can provide a synthetic method of visualizing the immune landscape in situ at the same cell level.The information to be gained from this detection is more comprehensive and accurate.2.The spatial orientation of immune cells in the tumor microenvironment of MMR-proficient colorectal cancer is heterogeneous and is affected by clinical stage,TNM stage,lymphovascular invasion,perineural invasion,and so on.Patients with early stage or with rectal cancer may be the key population for the immunotherapy of patients with MMR-proficient colorectal cancer.3.CD8~+TRM cells and CD4~+CTLs can be regarded as the potential targets for immunotherapy possibly.