The construction of CD8+ T cell-associated molecular subtypes in esophageal squamous cell carcinoma reveals tumor heterogeneity, tumor microenvironment, and immunotherapy.

Esophageal squamous cell carcinoma (ESCC) is a cancer that is common worldwide. Its morbidity and mortality rates remain high, seriously threatening human health. In the tumor microenvironment (TME), CD8+ T cells undergo a series of dynamic changes that have important implications for tumor progression and the efficacy of immunotherapy. Our research aims to construct novel molecular subtypes through the gene expression profiling of CD8+ T cells during differentiation and to predict the prognostic and therapeutic effects in ESCC patients. In the single-cell sequencing (scRNA-seq) analysis, we clustered and visualized cell subsets using the Seurat package, removed batch effects between samples using the Harmony package, and performed pseudo-time analysis of CD8+ T cells using the Monocle2 package. In the bulk-RNA analysis, non-negative matrix factorization (NMF) was applied to construct molecular subtypes, and the unique expression of molecular subtypes in prognostic assessment, biological processes, genomic variation, and immune microenvironment composition was explored. The drug sensitivity of different subtypes was further studied. The causal relationship between gene expression during CD8+ T cell differentiation and ESCC occurrence was inferred using Mendelian Randomization (MR) analysis. In addition, the biological functions of key genes were further explored by in vitro cellular experiments. Based on the genes during CD8+ T cell differentiation, we identified three molecular subtypes with different clinical outcomes. To ensure the robustness and reproducibility of the molecular subtypes, we used the GSE53625 cohort for validation. Among them, the C1 subtype showed higher genomic variation, and its patients had significantly worse prognoses than the C2 and C3 subtypes. It is worth noting that the C3 subtype exhibits an "immune-heat" phenotype, which is accompanied by a large number of immune cell infiltrates, up-regulated immunological checkpoint molecules, and greater sensitivity to immune therapy. In addition, we have screened a series of potential therapeutic drugs, which provides strong support for future clinical translational research. Finally, the results of Mendelian randomization analysis indicated that the RHOB gene could increase the risk of ESCC development. In addition, in vitro cellular assays such as CCK-8, scratch assay and Transwell verified that RHOB could promote the proliferation, migration and invasion of ESCC cells in vitro. This study constructed molecular subtypes related to CD8+ T cells and analyzed in depth the biological characteristics, genomic variation, and role of these three molecular subtypes in the tumor microenvironment and immunotherapy. These molecular subtypes allow us to more accurately identify patient groups and thus provide more effective treatment strategies for ESCC patients. In addition, this study identified RHOB as a novel biomarker for esophageal squamous cell carcinoma and verified that RHOB was associated with proliferation, migration and invasion of ESCC by in vitro cellular assays.