The Investigations Of The Immune Responses Of Colorectal Cancer With NOTCH Mutations And The Differences Of The Single Cell Atlas Between Primary And Metastatic Tumors

Part ?: The investigation of the immune responses of colorectal cancer with NOTCH mutationsThe NOTCH signaling pathway is evolutionarily conserved and can modulate a series of fundamental cellular functions,including cell differentiation,maintenance of stemness,proliferation,and apoptosis.The NOTCH signaling pathway is reported to be dysregulated in CRC,with upregulated expression of NOTCH1 in tumor tissue,which correlates with poor prognosis of CRC.However,the role of NOTCH signaling pathway mutations in shaping the CRC immunophenotype was rarely reported.We analyzed the association of NOTCH signaling pathway mutations with tumor immune microenvironment using two CRC datasets,as well as the influence of mutations on immunotherapy.The results are as follows.(1)According to the analysis of GSE108989 dataset,CD8+ T cells in NOTCH signaling pathway mutation group had higher expression levels of cell-killing molecules and higher cytotoxicity associated genes.The ratio of Treg subset with higher suppressive capability in the mutation group was lower compared to the non-mutation group.(2)According to the results of TCGA dataset,NOTCH signaling pathway mutations were associated with higher expression levels of cell-killing molecules,immune checkpoints,chemokines and chemokine receptors,leading to the activation of anti-tumor immunity.(3)NOTCH signaling pathway mutations were associated with earlier disease stages and lower rates of lymph node and distant metastasis.Mutations did not affect disease free survival and overall survival of CRC patients,but can affect the differentiation of CRC.(4)Immunotherapy can prolong the overall survival of NOTCH signaling mutation CRC patients as well as non-small cell lung cancer patients.However,immunotherapy did not affect the overall survival of bladder cancer,breast cancer,renal cell cancer,esophagogastric cancer,glioma,head and neck cancer and melanoma,which implied the effects of Notch signaling pathway mutations on immunotherapy efficacy varied in different cancers.In summary,we found that NOTCH signaling pathway mutations were associated with higher anti-tumor immune response in CRC with checkpoint molecules up-regulation and benefited from immunotherapy,suggesting that NOTCH signaling pathway mutations may be a promising biomarker for immune checkpoint blockade therapy.This finding may provide preciser treatment for NOTCH signaling pathway mutation CRC patients.Part ?: The investigation of the differences of the single cell atlas between primary and metastatic tumors of colorectal cancerMetastasis is the terminal stage of tumor development and has always been the main reason for the death of cancer patients.Liver is the most common organ of CRC distant metastasis takes place.The gene expression differences between primary tumor and liver metastasis in CRC detemine the treatment efficacy and prognosis.Studies have reported the differences between the primary and metastatic tumors by bulk RNA sequencing.However,tumor microenvironment consists of different components,including cancer cells,fibroblasts,endothelial cells,and immune cells.It is hard to dissect the transcriptional heterogeneity of each component by bulk RNA sequencing.In this study,we conducted single cell RNA sequencing to examine the heterogeneities of the cell atlas in primary and liver metastatic tumors of CRC.The results are as follows.(1)According to the gene expression,we identified non-immune cells and immune cells in the tumor microenvironment.Non-immune cells consisted of cancer cells,fibroblasts,and endothelial cells,which were made up of heterogeneous subclusters.The ratios of each subcluster in the primary and metastatic sites were different.Immune cells consisted of T/natrual killer cells,B/plasma cells,monocytes/macrophages,dendritic cells and mast cells.(2)The cancer cells recapitulated the multilineage differentiation processes of normal colon epithelia and can modulate immune system by secreting chemokines.Compared with those in the primary site,the cancer cells in the liver metastasis had higher adhesiveness and stemness.Fibroblasts in the primary and metastatic sites can produce different inflammatory factors to modulate immune system.(3)Compared with paracancer control,the ratios of macrophage subsets TAM_APOE and TAM_SPP1 increased in the tumor microenvironment.The TAM_APOE subset had higher antigen presenting ability and the TAM_SPP1 subset had higher expression levels of chemokines.(4)NOTCH signaling pathway can modulate the exhaustion of CD8+ T cells and the generation of follicular helper CD4+ T cells.In summary,we analyzed the transcriptional differences of cell atlas between primary and liver metastatic tumors of CRC by single cell RNA sequencing,and dissected the heterogeneity of each cell cluster,providing important clues for precise treatment of CRC.