Integrated single-cell and bulk transcriptomic analyses reveal a stem-like epithelial subpopulation in adenocarcinoma of the esophagogastric junction and identify VASN as a novel regulator of tumor stemness.

BACKGROUND

Cancer stem cells (CSCs) are widely recognized as key contributors to tumorigenesis, progression, therapeutic resistance and immune evasion in adenocarcinoma of esophagogastric junction (AEG). Nevertheless, the stem-like epithelial subpopulations that may contribute to immunological niche formation remain poorly characterized, hindering the identification of effective therapeutic targets.

METHODS

Owing to their ability to delineate cellular heterogeneity, single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) analyses were employed to characterize the CSCs-like subpopulation in AEG tumor tissues. Utilizing scRNA and bulk transcriptomics cohorts, this study systematically identified the putative CSC-like malignant epithelial subpopulation, and a series of bioinformatics and machine learning as well as in vitro functional experiments further verified VASN on AEG malignancy.

RESULTS

Six main cell types were identified in AEG tumors, and six clusters of malignant epithelial cells were filtered out via inferCNV analysis. The cluster 2 (C2) subgroup exhibited pronounced elevation of CytoTRACE, epithelial-mesenchymal transition, Wnt signaling and stem cell differentiation scores, trajectory analysis positioned C2 subgroup at the initiation stage of pseudotime, which comprehensively identified C2 as stem-like neoplastic subpopulation. Through the integration of high dimensional weighted gene co-expression network analysis (hdWGCNA), Branch Expression Analysis Modeling (BEAM) and differentially expressed genes, 21 genes were identified as hub genes of C2 cluster. The CSC-related prognostic model was developed via LASSO Cox regression analysis. Random survival forest further identified VASN as candidate CSCs marker of AEG. Additionally, knock down of VASN in AEG cell line reduced migration and invasion abilities as well as CSCs markers expressions. Mechanistically, in vitro and bioinformatics analysis revealed a potential link between VASN and stemness-associated Wnt/β-catenin signaling.

CONCLUSIONS

Our investigation identified a stem-like epithelial subpopulation and established a stem-related gene signature, offering insights into stemness-associated tumor progression and immunological niche dynamics in AEG. Furthermore, we identified VASN as a putative regulator of malignant stem phenotypes that may influence tumor microenvironment remodeling.