Decipher Cellular Heterogeneity After Bladder Cancer Immunotherapy Based On Single-cell RNA Sequencing Analysis Of Mouse Orthotopic Induced Tumor Model

Bladder cancer（BCa）has a high incidence,ranking fourth among male malignant tumors.Although part of patients can be treated by traditional surgery chemotherapy,the prognosis of bladder cancer has not changed substantially in the past 30 years.It is urgent to improve the clinical treatment effect of bladder cancer.Bladder cancer is one of the earliest tumors to be treated with immunotherapy,which changes the prospect of cancer treatment by triggering long-lasting immune responses mediated by anti-tumor cells.Currently,FDA has approved several immune checkpoint inhibitors for the clinical treatment of bladder cancer that cannot be treated with chemotherapy.However,anti-PD1/anti-PD-L1 is only effective in 15-24%of patients,and the mechanism of immune therapy tolerance in bladder cancer needs to be elucidated.In order to overcome the difficulty of obtaining clinical samples,we constructed a mouse orthotopic induced tumor model and treated it with anti-PD-1.Bladder tissue samples before and after treatment were analyzed by single-cell RNA sequencing（sc RNA-seq）to identify tumor cell groups that were resistant to immunotherapy,and KRT23⁺epithelial cells subgroup that were resistant to immunotherapy was found.Differential genes,transcription factors and signaling pathways in tolerance subgroup were further analyzed to clarify the molecular regulatory mechanism of immunotherapy tolerance.Tolerant tumor cell subgroup（KRT23 positive cells）was enriched in WNT signaling,Reactive oxygen species（ROS）signaling,lipid metabolism and lipid peroxidation signaling pathways.Single-Cell Regulatory Network Inference and Clustering（SCNIC）showed that the transcriptional activity of tumor stem cell related transcription factors,such as KLF5,was upregulated in immunotherapy tolerant subgroup.Differential gene expression analysis showed that Hilpda and Plin2,related to lipid peroxidation process,were highly expressed in the tolerant subgroup,and the expression of these two genes was relatively low in the immunokilling sensitive subgroup.Analysis from promoter analysis tools and public databases showed that both promoter regions of these genes had KLF5 binding sites,suggesting that they may be specifically regulated by this transcription factor.This study generated a single-cell transcriptome landscape of tumor heterogeneity after immunotherapy for bladder cancer,and speculated the possible mechanism of KRT⁺23 EPCs subgroup,thus providing new ideas for clinical treatment of bladder cancer.Bladder cancer has the highest risk of recurrence among all malignant tumors and has become a major public health problem seriously endangering human health.N6-methyladenosine modification（m6A）is one of the most abundant m RNA modification in eukaryotic cells and is involved in the expression regulation of a large number of genes.m6 A is mainly composed of the core catalytic enzyme METTL3（methyltransferase-like）3)The complex composed of METTL14 catalyzed the generation of methyl transfer and regulated the expression of a variety of genes,mediating various pathological processes including tumgenesis and immune escape.In previous studies of our group,we found that METTL3 was overexpressed in bladder cancer and promoted the occurrence and development of bladder cancer by regulating m6 A modification However,The abnormal upregulation mechanism of METTL3 in tumor cells has not been reported.In order to explore The upregulation mechanism of METTL3,we analyzed TCGA（The Cancer Genome Atlas）and GEO（Gene Expression）Transcriptome of bladder cancer patients in Omnibus database showed that JNK/MAPK signal was highly enriched in tumor tissues,which was related to the high expression of METTL3 in tumor tissues.At the same time,the tumor tissues were divided into two groups with high and low METTL3 expression,and the comparative analysis showed that JNK/MAPK signal was also significantly enriched in the samples with high METTL3 expression.These data suggested that the activation of JNK/MAPK signal pathway may be involved in regulating the abnormal METTL3 expression in bladder cancer...