A Study Focused On Molecular Regulatory Network Of Hypoxic Microenvironment Based On Single Cell And Tissue RNA-Seq Data In Pan-Cancer

Hypoxia is the main characteristics of the tumor microenvironment(TME),and is associated with impaired metabolic reprogramming,immune responses,increased genomic instability,and other cancer hallmarks,and also related to tumor progression,treatment resistance and poor clinical prognosis.Research on hypoxia based on cell culture experiments and bulk sequencing data provides a theoretical basis for a comprehensive understanding of the molecular mechanism of hypoxia in promoting cancer development.However,in the pancancer landscape,the effects of hypoxia on the biological functions of both malignant and nonmalignant cells in the TME,the molecular map of interactions between different cell types,the further cancer-promoting signal pathways,and the formation of key cell subtypes and mediating their cancer-promoting functions under hypoxic microenvironment have not yet been fully revealed.And there is still lacking of in-depth understanding of the hypoxic microenvironment system.Therefore,this study is based on single-cells RNA sequencing data of 6 cancer types including 424194 single-cells and 25 types of cancer tissue multi-omics sequencing data,using Seurat package,Cell Phone DB tool,Gene Set Variation Analysis(GSVA),Cox proportional risk regression model,difference analysis,correlation analysis and in vitro cell culture and molecular experiments,to identify cell subgroups in across cancer types,to study the response mechanism of each cell types in the hypoxic TME and reveal the interaction between cell types and their specific molecular signaling pathways promoting cancer development.At last,based on the high correlation between hypoxia and glycolysis,using 25 types of cancer tissue multiomics sequencing data to explore the various molecular mechanisms of glycolysis and reveal the downstream regulatory mechanisms of hypoxia.The detailed research contents and results are summarized as follows:(1)Based on the single-cell RNA sequencing data,we distinguish the cell subtypes of the four main cell types: tumor cells,myeloid cells,fibroblasts,and T cells among the six cancers.We identified the cell subtypes that commonly occur among multiple cancers,including cancer cells with high cell cycle and epithelial–mesenchymal transition(EMT)biological process,fibroblasts involving in extracellular matrix(ECM)remodeling,CD4+ Treg cells regulation immunosuppressive environment,SPP1+ tumor-associated macrophages(TAMs)with the highest hypoxia level.The environmental stress in solid tumor,hypoxia,shows different contributions to each cell types in our study,participating in ECM remodeling,cancer cell invasion,M2 TAMs polarization,SPP1+ TAMs expansion,interleukin-10 signal activation and T cell exhaustion.In addition,the hypoxia score has no correlation with cytotoxicity score,but is positively correlated with the score of T cell exhaustion score,and is significantly positively correlated with the T cell inhibitory receptors genes: PDCD1,TIGIT,HAVCR2,LAG3 and CTLA4.Combined with TCGA pan-cancer research,it is found that the cell cycle and EMT biological processes are related to the poor prognosis of certain cancer types and the biological process including cell cycle,ECM remodeling,keratinization,glycolysis,interleukin signaling and collagen degradation is highly enriched in hypoxic samples.(2)The molecular interaction network between various cell subtypes in the pan-cancer tumor microenvironment was constructed.It was found that tumor-associated fibroblasts(CAFs)activated the estrogen receptor signal and promoted cell proliferation of breast cancer and ovarian tumor cells through IGF/IGFR signals;tumor cells activated M2 type macrophage polarization signals through GAS6/AXL,VEGFA/NRP1 and VEGFA/NRP2 signals;SPP1+TAMs activated tumor cell glycolysis and EMT process through SPP1/CD44 signals;TAMs activated Treg cells through TNF/TNFRSF1 B and TNF/FAS signals;TAMs regulate T cell differentiation status through LGALS9/HAVCR2 signals.Combined with the TCGA pancancer sample study,it was found that SPP1 gene expression and the high level of SPP1+TAMs infiltration are related to the poor prognosis of 6 cancer types,including lung cancer;it was found that a positive correlation between SPP1+TAMs molecular signature scores and T cell exhaustion scores;the SPP1 gene was found to be significantly up-regulated in hypoxic samples.Through the A549 lung tumor cell culturing,it was found that the cells stimulated with the human recombinant proteins SPP1 and TNFSF12 showed significant migration and invasion phenotypes and up-regulated the expression of genes related to glycolysis and EMT.(3)Based on the above results that the glycolysis pathway was enriched in hypoxic samples,in-depth revealing multi-omics molecular characteristics related to glycolysis helps to further reveal the downstream regulation of hypoxia.The performance of 22 gene expression to quantify the glycolysis levels was verified robustness using defined glycolysis samples from previous studies.Glycolysis and hypoxia scores are highly positively correlated with a correlation coefficient greater than 0.7 in all cancer samples and lung cancer single cells.The results of survival analysis showed that patients with high glycolysis scores have a poor prognosis in pan-cancers.Glycolytic and hypoxic GSVA scores were higher in tumor samples than normal samples,and hypoxic status and glycolytic activity showed a heterogeneous distribution in different cancer tissues.In most cancer types,high-glycolytic tumor samples were exhibited specific genes copy number variants such as MYC,genes single nucleotide mutations such as TP53 and high tumor mutation burden.TCA cycle,DNA replication,G2 M checkpoint,tumor cell proliferation,nucleotide synthesis,and other cancer-related biological processes were more active in high glycolytic tumors,and the glycolytic score and cell proliferation score had a significant positive correlation.The differential genes in samples with high glycolytic activity were mainly enriched in pathways such as glucose metabolism,extracellular matrix remodeling,and cell cycle.The expression of HSPA8 and P4HA1 were highly correlated with glycolysis score and the expression of multiple glycolysis genes such as LDHA and PGK1,thus they were the potential factors regulating glycolysis.In summary,CAFs,TAMs,T cells,and tumor cell subtypes that are ubiquitous in the pancancer TME and have different response mechanisms under hypoxia stress,but they show the biological characteristics of promoting cancer development;SPP1+TAMs expansion is observed in the hypoxia TME and is related to tumor cell glycolysis,EMT,T cell exhaustion,and poor prognosis in lung cancer and other cancers;the hypoxia degree and glycolytic activity are different among various cancer types;There are similarities and differences between the characteristics of multiple omics molecules related to glycolysis and hypoxia,which further illustrates the high correlation between hypoxia and glycolysis,but also suggests the complexity of the two and related regulation.Therefore,this study provides a molecular map of the impact of hypoxia on the biological processes of various cells and the interaction between cells in the TME,and provides data support for identification of more important cancer-promoting cell subtypes.It provides valuable directions for screening potential targeted hypoxia and crosstalk signals between cells to block pathways that are conducive to cancer development.