Study On Immuno-subtyping In 11 Types Of Hypermutated Tumors By Non-negative Matrix Factorization

BackgroundThe treatment of cancer with immune checkpoint inhibitors(ICI)has grown rapidly over the past decade.Whether it is solid tumors such as melanoma,head and neck cancer,gastric cancer,colorectal cancer,or hematologic tumors such as B-cell lymphoma and non-Hodgkin’s lymphoma,immunotherapy has brought significant benefits to patients in the field of advanced cancer treatment.Tumor mutational burden(TMB)refers to the total number of replacement and insertion/deletion mutations in the coding region of gene exons in tumor cells.There are huge differences in tumor mutation burden between different tumors.Melanoma has the highest overall TMB level,followed by non-small cell lung cancer and other squamous epithelial carcinomas,while leukemia and pediatric tumors have the lowest TMB levels.Elevated TMB may lead to an increase in the number of new antigens,thereby recruiting effector T cells to the tumor area,but the tumor itself will also adapt to the changing microenvironment and cause immune escape.Therefore,the application of ICI to relieve the inhibitory state of effector T cells will activate the anti-tumor immune response of T cells,thereby achieving the effect of attacking and killing tumors.Therefore,tumor TMB detection can help predict the efficacy of immune checkpoint inhibitors.The tumor mutation load of hypermutated tumor(HM,>10 mutation/million bases,mut/Mb)is very high,and it produces more tumor neoantigen,which are good markers for immunotherapy.However,based on the evaluation of the response rate of existing clinical trials,less than half of the hypermutant tumors were completely remitted after immune checkpoint inhibitor treatment,indicating that the heterogeneity of HM tumors is very obvious.Hence,there is an urgent need to distinguish the unknown intrinsic HM tumor subtypes and find out the subtypes with better benefits in immunotherapy to achieve better targeted therapeutic effects.MethodsThe somatic mutation data from 5,519 tumor samples for 11 types of solid tumors including bladder urothelial carcinoma,breast invasive carcinoma,cervical squamous cell carcinoma and endocervical adenocarcinoma,colorectal cancer,head and neck squamous cell carcinoma,lung adenocarcinoma,lung squamous cell carcinoma,ovarian serous adenocarcinoma,skin cutaneous melanoma,stomach adenocarcinoma,and uterine corpus endometrial carcinoma were obtained from The Cancer Genome Atlas(TCGA).In addition,we also envoled 338 colorectal cancer samples obtaind from our cohort.All the somatic mutation data were screened,gene expression data processed and normalized.Based on 1,836 immune-related genes,a total of 1,040 samples across11 types of HM tumors with corresponding transcriptomes were clustered by nonnegative matrix factorization(NMF).Use Gene Ontology(GO)feature enrichment analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and Gene set enrichment analysis(GSEA)was used to compare the enrichment characteristic pathways of different subtypes,and the Kaplan-Meier method was used to analyze and compare the survival differences between different immune subtypes.CIBERSORT and x Cell were used to calculate the difference in the proportion of lymphocytes in different subtypes.Finally,a co-culture model of T cells,MSI-H colon cancer cell lines(a subset of HM tumors)and M2 macrophages was constructed in vitro to explore the changes in the ability of T cells to kill tumors under co-culture,and to evaluate whether M2 macrophages could promote the invasion and metastasis of intestinal cancer cells by inhibiting the function of effector T cells,and explore to restore the killing ability of effector T cells to tumors by inhibiting the IL-6/JAK/STAT3 pathway of M2 type macrophages.ResultsWe demostrate for the first time that 1040 HM samples across these 11 types of solid tumors can be subdivided into three different immune subtypes: HM1,HM2 and HM3.The enrichment analysis results of GO,KEGG and GSEA suggest that the extracellular matrix tissue related processes,epithelial-mesenchymal transition,and TGF-β signaling pathways are significantly enriched in HM2 tumors.The positive regulation of T cell proliferation,activation and migration are the most significantly enriched biological process in HM3 tumors,and the immune-related pathways in HM3 tumors are significantly enriched,and the most obvious enrichment signatures in HM1 tumors are E2 F and MYC targets.However,the differences in tumor mutation burden,intratumoral heterogeneity and related neoantigens between the three subtypes are not significant.Kaplan-Meier survival analysis showed that HM3 tumors had the best prognosis among the three types.The median overall survival time of HM1,HM2,and HM3 tumors were 64 months,50 months,and 70 months,respectively.The results of tumor lymphocyte infiltration analysis based on CIBERSORT and x Cell show that there are more CD8+ T cell infiltrations in HM3 tumors than in HM1 and HM2 tumors,and the expression level of chemokine CCL5,CXCL9 is highly relevant with these increased CD8+ T cells.CD8 A and CCL5 immunofluorescence experiments on colorectal cancer samples of HM1 and HM3 tumors in our group also suggest that the expression of CD8 A and CCL5 in HM3 tumors is much higher than that in HM1 tumors.In view of the fact that the gene Homeobox C6(HOXC6)is highly expressed in microsatellite instability-high colorectal cancer(a subset of HM tumors)based on the TCGA cohort.HOXC6 is significantly negatively correlated with MLH1 expression,and is associated with poor prognosis.The group with higher expression level of HOXC6 has higher TMB,and in that group the infiltration ratio of M2 macrophages is significantly increased.Therefore,we used in vitro co-culture model to construct HOXC6 overexpression and knockdown colorectal cancer cell lines,and found that the invasion and migration ability of colorectal cancer cells was enhanced after HOXC6 overexpression,and the invasion and migration ability of colorectal cancer cells was weakened while HOXC6 was knocked down.After co-culture of M2 macrophages,T cells and cancer cells,the expression level of related costimulatory molecules and common immune checkpoints of T cells were significantly reduced,especially the expression level of 4-1BB decreased most significantly.After inhibiting the IL-6/JAK/STAT3 pathway of M2 macrophages,the 4-1BB expression level of effector T cells was restored,and the secretion of IFN-γ and TNF-α also increased,and the tumor killing capacity of T cells had also been restored.ConclusionThe application of non-negative matrix factorization can subdivide HM tumors into three immune subtypes,and these three subtypes have significantly different immune characteristics and prognosis,which suggests that high TMB tumors themselves are also highly heterogeneous,The response to ICI treatment of the three subtypes may be different.HM3 tumors may be suitable for conventional single immune checkpoint inhibitor or ICI synergistic immunotherapy,while for HM2 tumors,targeting TGF-β or a combination of EMT and ICI may be more promising treatment regimens.The high heterogeneity of the HM cohort demonstrated in this study will help us to carry out follow-up targeted research on different molecular subtypes of pan-cancer and provide new ideas for the existing HM tumor treatment.