Exploration On Microenvironmental Heterogeneity Of Muscle Invasive Bladder Cancer Based On Single Cell Proteomics Techniques

Muscle invasive bladder cancer(MIBC)is a common malignant tumor in urinary system,which is characterized by high recurrence rate,poor prognosis and high heterogeneity.Numerous evidences indicate that tumor microenvironment is involved in tumor genesis and development in many aspects,which is a major cause of tumor heterogeneity.This study aims to investigate the phenotypic diversity and spatial heterogeneity of MIBC tumor microenvironment by single cell mass cytometry and imaging mass cytometry combined with whole transcriptome sequencing,to reveal the heterogeneity of MIBC tumor microenvironment and explore the potential regulatory mechanism.Part Ⅰ Tumor cell heterogeneity and tumor stem like cells in muscle invasive bladder cancerObjective:MIBC is a molecularly heterogeneous disease of urinary system with a high recurrence rate and poor prognosis.The diversity phenotypes of tumor cells are the key causes of tumor heterogeneity and the culprits of tumor recurrence,metastasis and drug resistance.Therefore,revealing the heterogeneity of MIBC tumor cells and the regulatory mechanism will help to find potential diagnostic and therapeutic targets for MIBC.Methods:Forty-four MIBC tumor tissues and 35 para-cancer tissues were collected;Part of the tissues were used for whole transcriptome sequencing,and the other part were prepared into single-cell suspensions for mass cytometry detection;Paraffin sections of 44 MIBC tissues were examined by image mass cytometry to explore the spatial characteristics of MIBC tumor cells in situ.Results:1.Compared with para-cancer tissues,the tumor microenvironment of tumor tissues has changed;2.Cell clusters of MIBC tumor microenvironment showed diverse phenotypes,some tumor cell clusters highly expressed tumor stem cell markers and characterized by phenotypes of tumor stem like cells;3.We found that a previously unreported tumor stem like cell cluster ALDH~+PD-L1~+ER-β~-was associated with poor prognosis of MIBC;4.The tumor stem like cell cluster ALDH~+PD-L1~+ER-β~-was closely related to neuro-related pathways and might be regulated by glycosphingolipid synthase B4GALNT1;5.B4GALNT1 might induce extracellular matrix remodeling and simultaneously activate exosomes to promote intercellular crosstalk.Conclusion:The tumor microenvironment of MIBC is heterogeneous,and tumor cells have diverse phenotypes;Specific tumor stem like cell cluster ALDH~+PD-L1~+ER-β~-is associated with poor prognosis;B4GALNT1 may regulate this tumor stem like cell cluster by influencing tumor microenvironment.Part Ⅱ Heterogeneity of immune microenvironment exists in muscle invasive bladder cancerObjective: The emergence of immunotherapy has opened a new chapter in tumor treatment and brought new hopes to tumor patients.However,MIBC patients have a low response to immunotherapy,and only a small number of patients can benefit from immunotherapy.Therefore,it is of great significance to explore the diversity of phenotypes in MIBC immune microenvironment at single cell level for the development of new immunotherapy regiments.Methods: Single-cell suspensions from 44 MIBC tumor tissues and 35 paracancer tissues were detected by mass cytometry;According to the characters of immune cells with the expression of CD45,gating immune cells on Cytobank analysis platform(https://premium.cytobank.org),then extracted and downloaded the data of immune cells of each sample;Through data dimension reduction and clustering,the immune cells were classified,and then the immune cell clusters were accurately analyzed according to the expression levels of specific markers.Results: 1.Cancer tissues were more immunosuppressive than para-cancer tissues;2.There was a group of immune cell cluster with an absolute superiority in number in each sample;3.T cell clusters expressed immune checkpoints,coinhibitory receptors and co-activated receptors to varying degrees,showing immunosuppressive or immune-depletion phenotypes;4.Immunosuppressive T cell clusters were recruited by ER-β~+ tumor cells.Conclusion: Immune cells in the immune microenvironment of MIBC have heterogeneous phenotypes,and the T cell clusters are characterized by different degrees of immunosuppression or immune exhaustion.Part Ⅲ Maping spatial landscape of tumor microenvironment in muscle invasive bladder cancer by high-throughput imaging mass cytometryObjective: The spatial distribution,phenotypic diversity and number of components of tumor microenvironment affected tumor progression.Single cell RNA sequencing has enhanced our understanding of the phenotypes,numbers,and their potential functions of the cell clusters in the tumor microenvironment,but it cannot analyze the spatial information of the tumor microenvironment and has certain limitations.Therefore,in this study,imaging mass cytometry was used to depict the spatial landscape of MIBC tumor microenvironment,revealing the spatial heterogeneity of it.Methods: The spatial phenotypes of tumor cells,immune cells,blood vessels and collagen in MIBC were accurately analyzed by imaging mass cytometry.Results: 1.There were three tumor cell phenotypes in MIBC tumor microenvironment: differentiated,dedifferentiated and budding;2.The immune cells of MIBC exhibited three spatial distribution patterns: rejection,infiltration and depletion;3.MIBC tumor microenvironment existed four collagen morphologies: curved,parallel edge,directional distribution and chaotic;4.MIBC tumor microenvironment existed two types of blood vessels: interstitial blood vessel and tumor blood vessel.The activity of interstitial blood vessel was higher than tumor blood vessel.Conclusion: The tumor cells,immune cells,blood vessels and collagen in MIBC tumor microenvironment have multiple spatial phenotypes,which contribute to the complexity of MIBC tumor microenvironment.Part Ⅳ A preliminary study of tumor microenvironment heterogeneity in muscle invasive bladder cancer molecular subtypesObjective: The establishment of MIBC molecular subtype classifiers provides a new strategy to study the heterogeneity of MIBC.However,the characteristics of tumor microenvironment of each molecular subtype are unclear and need to be clarified.Therefore,in this study,we combined mass cytometry,imaging mass cytometry and whole transcriptome sequencing to preliminarily explore the heterogeneity of tumor microenvironment of MIBC molecular subtypes.Methods: MIBC patients were divided into six molecular subtypes using the consensus molecular subtype classifier;The transcriptome and histological characteristics of each subtype were analyzed by RNA sequencing and hematoxylin-eosin staining;The frequency of infiltrating immune cells and the related immune functions of the six MIBC subtypes were evaluated based on 29 immune-related signatures;The tumor microenvironment heterogeneities of the six MIBC subtypes were analyzed by mass cytometry and imaging mass cytometry.Results: 1.The six molecular subtypes of MIBC had different molecular characteristics;2.The six molecular subtypes of MIBC had different histological characteristics;3.The frequency of infiltrating immune cells and the related immune functions of the six MIBC subtypes were different;4.Some specific cell cluster had a tendency to enrich in specific MIBC molecular subtypes;5.The tumor microenvironment of the Ba/Sq subtype recruited a variety of immunosuppressive immune cells.Conclusion: The tumor microenvironment of the six MIBC molecular subtypes is highly heterogeneous,and the cell cluster tended to be enriched in specific molecular subtypes;The immunosuppressive phenotype of Ba/Sq subtype is more prominent than other subtypes,and the Ba/Sq subtype patients are more suitable for immunotherapy.