Identification and validation of biomarkers for epithelial-mesenchymal transition-related cells to estimate the prognosis and immune microenvironment in primary gastric cancer by the integrated analysis of single-cell and bulk RNA sequencing data.

Abstract

The epithelial-mesenchymal transition (EMT) is associated with gastric cancer (GC) progression and immune microenvironment. To better understand the heterogeneity underlying EMT, we integrated single-cell RNA-sequencing (scRNA-seq) data and bulk sequencing data from GC patients to evaluate the prognostic utility of biomarkers for EMT-related cells (ERCs), namely, cancer-associated fibroblasts (CAFs) and epithelial cells (ECs). scRNA-seq data from primary GC tumor samples were obtained from the Gene Expression Omnibus (GEO) database to identify ERC marker genes. Bulk GC datasets from the Cancer Genome Atlas (TCGA) and GEO were used as training and validation sets, respectively. Differentially expressed markers were identified from the TCGA database. Univariate Cox, least-absolute shrinkage, and selection operator regression analyses were performed to identify EMT-related cell-prognostic genes (ERCPGs). Kaplan-Meier, Cox regression, and receiver-operating characteristic (ROC) curve analyses were adopted to evaluate the prognostic utility of the ERCPG signature. An ERCPG-based nomogram was constructed by integrating independent prognostic factors. Finally, we evaluated the correlations between the ERCPG signature and immune-cell infiltration and verified the expression of ERCPG prognostic signature genes by in vitro cellular assays. The ERCPG signature was comprised of seven genes (COL4A1, F2R, MMP11, CAV1, VCAN, FKBP10, and APOD). Patients were divided into high- and low-risk groups based on the ERCPG risk scores. Patients in the high-risk group showed a poor prognosis. ROC and calibration curves suggested that the ERCPG signature and nomogram had a good prognostic utility. An immune cell-infiltration analysis suggested that the abnormal expression of ERCPGs induced the formation of an unfavorable tumor immune microenvironment. In vitro cellular assays showed that ERCPGs were more abundantly expressed in GC cell lines compared to normal gastric tissue cell lines. We constructed and validated an ERCPG signature using scRNA-seq and bulk sequencing data from ERCs of GC patients. Our findings support the estimation of patient prognosis and tumor treatment in future clinical practice.