To Explore The Biological Characteristics And Heterogeneity Of Macrophages In Human Bladder Cancer Microenvironment At The Single-Cell Level

Objectives: Macrophages are a common cell type in myeloid cells.They play an important role in regulating tumor inflammation and angiogenesis,and participate in the process of tumorigenesis and development.Flow cytometry provides a basis for analyzing cell heterogeneity,but because it is limited by the known cell surface markers and the number,the recognition of differences within cell subpopulations is limited.With the rapid development of single-cell transcriptome sequencing technology,more and more cell types and their functional characteristics have been revealed,providing new method and mean for analyzing the myeloid cell map in the bladder cancer microenvironment,especially the heterogeneity of macrophages.To compare the biological characteristics and heterogeneity of macrophages in the microenvironment of muscular-infiltrating bladder cancer(MIBC)and non-muscular-infiltrating bladder cancer(NMIBC)at the single-cell level,and to explore the interaction between them and other myeloid cells,and to reveal the subsets of macrophages that may interact with T cells.Methods: Twelve patients with bladder cancer were enrolled,including 10 males and 2 females.A total of 7 MIBC tumor tissue samples,3 NMIBC tumor tissue samples,4 PBMC samples,and 3 lymph node tissue samples were obtained.The tissue was digested by enzyme to obtain a single cell suspension,and the blood sample was obtained by ficoll density gradient centrifugation to obtain PBMC.The oil in water was prepared on 10 X Genomics platform,and then amplified by reverse transcription.The library was prepared and sequenced.Finally,the data of all samples were merged to extract macrophages and other myeloid cells for re-clustering analysis.Visual analysis revealed the heterogeneity and interaction between myeloid cell clusters.Immunohistochemistry revealed the infiltration of macrophages in MIBC and NMIBC,and immunofluorescence verified the resident macrophages,macrophage immune checkpoint ligands and the relationship with T cells.Results: 1.We found 11 groups of mono-macrophages,including 3 groups of monocytes and 8 groups of macrophages.Macrophage were more likely to infiltrate the tumor tissue,and the MIBC and NMIBC localized the tumor region and the stroma-tumor margin region,respectively.2.The two populations of macrophages enriched in MIBC are Mφ-c1-CXCL9 and Mφ-c4-ADAMDEC1,both of which are M2-like phenotypes.The macrophages enriched in NMIBC are Mφ-c7-HLA-DQA2 and Mφ-c8-HTRA1,the former shows an M2-like phenotype,the latter shows an M1-like phenotype.3.There is also a certain connection between the cell subsets enriched in MIBC and NMIBC.Mφ-c4-ADAMDEC1 can be transformed from Mφ-c1-CXCL9 and Mφ-c7-HLA-DQA2,and Mφ-c8-HTRA1 can be transformed into Mφ-c1-CXCL9,which can differentiate into Mφ-c7-HLA-DQA2.4.A group of macrophages may interact with T cells in both MIBC and NMIBC.Mφ-C1-CXCL9 and Mφ-C7-HLA-DQA2 express ligand binding T cell immune checkpoint,which may interact with depleted T cells to form an immunosuppressive microenvironment in bladder cancer.5.Cell Chat analysis revealed the correlation between myeloid cells in MIBC and NMIBC respectively,and found that there were differences in the signaling pathways between myeloid cells in MIBC and NMIBC.MC3 and Eso2 may activate the macrophages enriched in MIBC to M2 phenotype through IL4 and IL13,while Mφ-c1-CXCL9,Mφ-c6-MKI67,Mφ-c7-DQA2 in NMIBC can activate Mφ-c7-DQA2 to differentiate into M2 phenotype through IL10.6.Key chemokines and key cell subsets that chemotactic myeloid cells to tumor regions in the two types of tumors were compared.Conclusions: In general,there are differences in the localization and ratio of TAMs in MIBC and NMIBC.M1 related genes and M2 related genes are coexpressed in the same macrophage,especially the subgroups enriched in MIBC.The enriched taxa in NMIBC showed M1 or M2 phenotypes,respectively.However,the phenotype of TAMs in MIBC and NMIBC expresses different M2 genes,which further proves the limitations of the in vitro M1/M2 polarization model in bladder cancer.In addition,it has also been found that the enriched cell subsets between MIBC and NMIBC are also related to a certain extent,which may be related to the difference in the prognosis and recurrence of these two tumor types.The phenotypes of neutrophils,mast cells and dendritic cells all exist to varying degrees in bladder cancer.It is found that the pathways related to the differentiation of TAMs and the key cell subgroups driven by MIBC and NMIBC are also different.Therefore,immunotherapy should distinguish the different microenvironments of MIBC and NMIBC in order to target targeted therapy.