| BackgroundsIn 2022,92,900 new cases of bladder tumors in China,ranking eleventh among all tumor types and second within urogenital malignancies.The 5-year survival rate for metastatic bladder cancer is a mere 27.7%,and pelvic lymph nodes are the most common site of metastasis but often present with occult features: approximately 25% of cases were found lymph node positive after radical cystectomy,even in patients initially diagnosed with organ-confined disease.However,the mechanisms driving early occult metastasis in bladder cancer remain incompletely understood.Current advancements in treating metastatic bladder cancer include immune checkpoint inhibitors(ICIs)targeting adaptive cell immunity and antibody-drug conjugates(ADCs)targeting tumor cells,which have significantly improved current first-line treatment strategies based on cis-platinum.Despite these advances,strategies targeting tumor stroma components,like anti-angiogenic therapies,have yet to yield optimistic outcomes,while treatments targeting fibroblasts are still in their infancy,which should partly be attributed to the complex tumor microenvironment in metastatic bladder cancer inadequately characterized nowadays.Bulk RNA-seq and molecular subtyping studies based on large bladder cancer cohorts have contributed significantly to precision therapy.Nonetheless,bulk RNA-seq is susceptible to tumor purity bias during sampling,providing an overall transcriptional profile of the tumor sample,leading to significant influence by stromal components and inability to further characterize intra-tumoral heterogeneity.Single-cell RNA sequencing(sc RNA-seq)technology overcomes these limitations by analyzing multiple cellular phenotypes and abundances within tumor tissues in an unbiased way.Current sc RNA-seq research is profoundly unraveling microenvironmental features in various tumors and translating into novel precision therapeutic strategies.Although initial sc RNA-seq studies in bladder cancer have revealed microenvironmental heterogeneity in primary tumors,no study has simultaneously performed paired analysis on both primary and metastatic lesions in metastatic bladder cancer.Therefore,the microenvironmental characteristics of metastatic bladder cancer,particularly in metastatic lesions,largely remain unexplored.Moreover,paired analysis of primary and metastatic lesions can provide critical lineage information,where lineage analyses using copy number variations(CNVs)and mitochondrial mutations(MSM)have successfully identified highly metastatic heterogeneous clones in metastatic tumors,which is not achievable through analyzing bulk RNA-seq data or sc RNA-seq data of primary lesions alone.ObjectivesThis study aimed to elucidate the microenvironmental composition and characteristics in metastatic bladder cancer by analyzing sc RNA-seq data from both primary and metastatic tumors,along with paired single-cell immune repertoire sequencing(sc IR-seq)and spatial transcriptomics(ST-seq)data.We aimed to identify key microenvironmental factors crucial for improving existing therapeutic strategies for metastatic disease and to discover pivotal genes driving lymph node metastasis in bladder cancer.MethodsIn this project,fresh tissue samples,comprising bladder and lymph node specimens,were collected from nine patients undergoing radical cystectomy and pelvic lymphadenectomy.sc RNA-seq and sc IR-seq were conducted on the lymph node metastases,matched primary bladder tumors,normal non-metastatic lymph nodes,and adjacent normal urothelial tissue.Additionally,ST-seq was performed on one pair of primary and metastatic bladder cancer samples.Furthermore,the study also analyzed sc RNA-seq data from 8primary bladder cancer and 3 adjacent normal urothelium samples from the SRA database,bulk RNA-seq data from 405 primary bladder cancers and 19 adjacent normal urothelium samples from the TCGA-BLCA cohort,bulk RNA-seq data from 348 primary bladder cancers from the Im Vigor210 clinical trial,and bulk RNA-seq data from 22 pairs of primary bladder cancers and adjacent normal urothelium from the GEO database.Integrative multiomics bioinformatics analysis was conducted to identify cell subtypes in the microenvironment of metastatic bladder cancer,understand differences in proportions,phenotypes,and histological localization across different tissues,and clarify the impact of cell subtype abundance on bladder cancer prognosis and treatment responsiveness.Clonal evolution analysis and lineage tracing were carried out using information from tumor cell origin,CNV features,and MSM traits to pinpoint heterogeneous clones with high metastatic potential and determine their associated metastatic phenotypes.Experimental validation was conducted on important cell subtypes and molecules identified during the analysis.ResultsBased on scRNA-seq data from 24 samples encompassing 180,906 cells,this study constructed a single-cell transcriptomic atlas of lymph node metastatic bladder cancer,identifying 39 distinct microenvironmental cell subtypes apart from normal urothelial cells,malignant epithelial cells,and cycling cells,revealing significant microenvironmental remodeling in metastatic bladder cancer.Analysis of TCR sequences from 62,341 CD4+/CD8+ T cells uncovered multiple effector cell subsets displaying tissue-specific expansion and migration patterns.Three cell subtypes underwent tumor-specific differentiation(SPP1+ macrophages,CXCL10+macrophages,OLFML2A+ pericytes).This study is the first to report and identify an OLFML2 A positive pericyte subset(PVL＿OLFML2A)in bladder cancer,which is significantly associated with poor prognosis in metastatic disease.Lineage tracing identified54 high-and 91 low-metastatic rate genes,including VEGFB,SPINT1,CXCL8,and KRT17,previously reported to be closely related to tumor metastasis.This study discovered and verified for the first time that loss of expression of the high metastatic rate gene CLIC3 affects the metastatic capability of bladder cancer.ConclusionsBy means of single-cell sequencing and multi-omics analysis,this study provides a detailed characterization of the microenvironmental landscape in lymph node metastatic bladder cancer and reveals cross-regional microenvironmental remodeling and migratory features.Multiple cell types,including the PVL＿OLFML2A pericyte subset and tumor cells expressing high levels of CLIC3,collectively contribute to changes in the bladder cancer microenvironment,influencing tumor metastasis and prognosis.This study offers profound insights into the exploration of targeted therapeutic strategies for bladder cancer lymph node metastases and deeper understanding of the driving forces behind tumor metastasis. |
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