TNFRSF12A expression in stomach adenocarcinoma and its preliminary role in predicting immunotherapy response

Abstract

Background: TNFRSF12A is abnormally expressed in various malignancies, especially in stomach adenocarcinoma (STAD), which is related to tumor invasiveness and prognosis of patients. This study examined the expression pattern of TNFRSF12A in STAD and predicted immunotherapy response.

Methods: Data were derived from The Cancer Gene Atlas (TCGA), Gene Expression Omnibus (GEO), and Gene Expression Profiling Interactive Analysis (GEPIA) to analyze the expression pattern of TNFRSF12A in pan-cancer and STAD, as well as its correlation with clinical features. Biological pathways involved in TNFRSF12A were analyzed by "clusterProfiler" package. Immune cell infiltration was evaluated by "GSVA" and "CIBERSORT" packages. Immunotherapy response was assessed by TIDE score and tumor mutation burden (TMB) level. Expression level of TNFRSF12A in the single cell of STAD was analyzed by scRNA-seq. Finally, in vitro test detected the mRNA expression of TNFRSF12A in STAD cells, Wound healing and Transwell assays were performed to measure the capabilities of STAD cell to migrate and invade.

Results: TNFRSF12A was highly expressed in STAD. However, TNFRSF12A expression did not shown significant difference in relation to clinical features. TNFRSF12A exhibited notably positive correlation with many carcinogenic signaling pathways and immune cells infiltration such as T cells and macrophages. High TNFRSF12A expression group showed a higher TIDE score, Exclusion score, and TMB level than the low TNFRSF12A expression group, which indicated that STAD patients with high TNFRSF12A expression responded more poorly to immunotherapy. TNFRSF12A showed a positive relation with most of immune checkpoint genes. By scRNA-seq analysis, TNFRSF12A was chiefly expressed in Fibroblasts and Mast cells of STAD. Further, in vitro assays verified the high expression of TNFRSF12A in STAD cells, and the migration and invasion capabilities of STAD cells were notably suppressed by TNFRSF12A silencing (p<0.05).

Conclusion: The present study not only reveals the potential of TNFRSF12A as a therapeutic target for STAD, but also explores its great potential in STAD immunotherapy. This finding opens up a new way of thinking for the personalized treatment of STAD.

Keywords: TNFRSF12A; immunotherapy; single-cell RNA sequencing; stomach adenocarcinoma; tumor microenvironment.

Figure 1

Expression of TNFRSF12A in pan-cancer and its correlation with clinical features in STAD. (A) Expression levels of TNFRSF12A in pan-cancer; (B) Kaplan-Meier (K-M) curve of overall survival (OS) for high and low TNFRSF12A expression groups in STAD; (C) K-M curve of progression-free survival (PFS) for high and low TNFRSF12A expression groups in STAD; (D) Relationship between TNFRSF12A expression and T stages in STAD; (E) Relationship between TNFRSF12A expression and N stages in STAD; (F) Relationship between TNFRSF12A expression and M stages in STAD; (G) Relationship between TNFRSF12A expression and Stage in STAD; (H) Relationship between TNFRSF12A expression and Grade in STAD.

Figure 2

TNFRSF12A expression and its relationship with clinical features in GSE66229 dataset. (A) Expression level of TNFRSF12A in STAD tissue and normal gastric tissue; (B) K-M curve of disease-free survival (DFS) for high and low TNFRSF12A expression groups; (C) Correlation between TNFRSF12A expression and Stage; (D) Correlation between TNFRSF12A expression and T stage; (E) Correlation between TNFRSF12A expression and N stage; (F) Correlation between TNFRSF12A expression and M stage.

Figure 3

Analysis of biological pathways involved in TNFRSF12A in TCGA-STAD cohort. (A) KEGG enrichment pathways for high TNFRSF12A expression group; (B) KEGG enrichment pathways for low TNFRSF12A expression group; (C) Correlation between TNFRSF12A expression and HALLMARK pathways.

Figure 4

Correlation analysis of TNFRSF12A expression and immune cell infiltration. (A) Correlation between TNFRSF12A and 28 tumor-infiltrating lymphocytes (TILs) assessed by ssGSEA; (B) Correlation between TNFRSF12A and 22 immune cells evaluated by CIBERSORT.

Figure 5

Prediction of immunotherapy response between high and low TNFRSF12A expression groups. (A) TIDE score in high and low TNFRSF12A expression groups; (B) Correlation between TNFRSF12A and TIDE score; (C) Exclusion score in high and low TNFRSF12A expression groups; (D) Correlation between TNFRSF12A and Exclusion score; (E) TMB in high and low TNFRSF12A expression groups; (F) Correlation between TNFRSF12A and immune checkpoint genes; *** means p<0.001, ** means p<0.01, * means p<0.05.

Figure 6

Expression of TNFRSF12A in the single cell of STAD. (A) The tSNE plot of cell clusters in STAD; (B) The tSNE plot of cell types in STAD; (C) Bubble diagram of marker genes expression in each cell type; (D) Expression levels of TNFRSF12A in each cell type.

Figure 7

In vitro verification utilizing STAD cells. (A) Relative mRNA expression levels of TNFRSF12A, TPSAB1, and DCN in human gastric mucosa epithelial cell line GES-1 and STAD cell line AGS detected by RT−qPCR; (B) RT-qPCR to verify the success of TNFRSF12A transfection; (C) Cell migration of TNFRSF12A-silenced AGS assessed by Wound healing assay; (D) Cell invasion of TNFRSF12A-silenced AGS evaluated by Transwell assay. And ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.