Background: Bladder cancer is among the most lethal urinary system cancers across the globe. Macrophage 1 and Macrophage 2 play an essential role in the pathogenesis of tumors. Nevertheless, prior studies failed to investigate the implication of the two cells, working in combination, in the development, growth, progression and metastasis of bladder cancer.

Methods: We computed the M1/M2 ratio of the samples retrieved from The Cancer Genome Atlas (TCGA) by using the Cibersortx algorithm and calculated the ratio in 32 patients in our series by employing flow cytometry. SurvivalRandomForest was utilized to reduce the dimension of the list of the M1/M2-related genes, with an aim to obtain the most survival-predictive gene (EMP1) encoding epithelial membrane protein 1 (EMP1). The EMP1 was biologically characterized by using Gene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA), and Gene Ontology (GO). The single-cell transcriptome (sc-RNA) analysis was then applied to further look into the function of EMP1. Finally, Cellchat was employed to examine the interaction between macrophages and epithelium cells.

Results: The results showed that higher M1/M2 ratio was found to be associated with a more favorable prognosis of bladder cancer. EMP1 was identified to be the key gene indicative of M1/M2 ratio and higher EMP1 expression was associated with poor prognosis. Further analyses showed that EMP1 might promote tumor invasion and metastasis via epithelial-mesenchymal transition (EMT) and focal adhesion (FA). Moreover, the expression level of EMP1 could serve as an indicator of immunotherapy efficacy. The scRNA-seq data indicated that EMP1 in cancer cells was strongly associated with tumor proliferation. Finally, the Cellchat results exhibited that EMP1 might promote the interaction between macrophages and cancer cells through the fibronectin 1-syndecan 1 (FN1-SDC1) pathway.

Conclusion: Our study identified EMP1, an M1/M2-related gene, the expression of which may act as a prognostic indicator for the proliferation, metastasis, and response to immunotherapy. EMP1 might be involved in the regulation on M1/M2 ratio.