Single-cell data revealed CD14-type and FCGR3A-type macrophages and relevant prognostic factors for predicting immunotherapy and prognosis in stomach adenocarcinoma.

Background: Stomach adenocarcinoma (STAD) exhibits profound tumor heterogeneity and represents a great therapeutic challenge. Single-cell sequencing technology is a powerful tool to identify characteristic cell types. Methods: Single-cell sequencing data (scRNA-seq) GSE167297 and bulk RNA-seq data from TCGA, GTEx, GSE26901 and GSE15459 database were included in this study. By downscaling and annotating the cellular data in scRNA-seq, critical cell types in tumor progression were identified by AUCell score. Relevant gene modules were then identified by weighted gene co-expression network analysis (WGCNA). A prognostic scoring system was constructed by identifying prognostic factors in STAD by Least absolute shrinkage and selection operator (LASSO) COX model. The prognosis and model performance in the RiskScore groups were measured by Kaplan-Meier (K-M) curves and Receiver operating characteristic (ROC) curves. Nomogram was drawn based on RiskScore and prognosis-related clinical factors. In addition, we evaluated patient's feedback on immunotherapy in the RiskScore groups by TIMER, ESTIMATE and TIDE analysis. Finally, the expression levels of prognostic factors were verified in gastric cancer cell lines (MKN7 and MKN28) and human normal gastric mucosal epithelial cells (GES-1), and the effects of prognostic factors on the viability of gastric cancer cells were examined by the CCK8 assay and cell cycle. Results: scRNA-seq analysis revealed that 11 cell types were identified, and macrophages exhibited relatively higher AUCell scores and specifically expressed CD14 and FCGR3A. High macrophage scores worsened the prognosis of STAD patients. We intersected the specifically expressed genes in macrophages subgroups (670) and macrophage module genes (2,360) obtained from WGCNA analysis. Among 86 common genes, seven prognostic factors (RGS2, GNAI2, ANXA5, MARCKS, CD36, NRP1 and PDE4A) were identified and composed a RiskScore model. Patients in low Risk group showed a better survival advantage. Nomogram also provided a favorable prediction for survival at 1, 3 and 5 years in STAD patients. Besides, we found positive feedback to immunotherapy in patients with low RiskScore. The expression tendency of the seven prognostic factors in MKN7 and MKN28 was consistent with that in the RNA-seq data in addition to comparison of protein expression levels in the public HPA (The Human Protein Atlas) database. Further functional exploration disclosed that MARCKS was an important prognostic factor in regulating cell viability in STAD. Conclusion: This study preliminary uncovered a single cell atlas for STAD patients, and Macrophages relevant gene signature and nomogram displayed favorable immunotherapy and prognostic prediction ability. Collectively, our work provides a new insight into the molecular mechanisms and therapeutic approach for LUAD patients.