Prognostic prediction using a gene signature developed based on exhausted T cells for liver cancer patients

Abstract

Background

Liver hepatocellular carcinoma (LIHC) is a solid primary malignancy with poor prognosis. This study discovered key prognostic genes based on T cell exhaustion and used them to develop a prognostic prediction model for LIHC.

Methods

SingleR's annotations combined with Seurat was used to automatically annotate the single-cell clustering results of the LIHC dataset GSE166635 downloaded from the Gene Expression Omnibus (GEO) database and to identify clusters related to exhausted T cells. Patients were classified using ConsensusClusterPlus package. Next, weighted gene co-expression network analysis (WGCNA) package was employed to distinguish key gene module, based on which least absolute shrinkage and selection operator (Lasso) and multi/univariate cox analysis were performed to construct a RiskScore system. Kaplan-Meier (KM) analysis and receiver operating characteristic curve (ROC) were employed to evaluate the efficacy of the model. To further optimize the risk model, a nomogram capable of predicting immune infiltration and immunotherapy sensitivity in different risk groups was developed. Expressions of genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence and Cell Counting Kit-8 (CCK-8) were performed for analyzing cell functions.

Results

We obtained 18,413 cells and clustered them into 7 immune and non-immune cell subpopulations. Based on highly variable genes among T cell exhaustion clusters, 3 molecular subtypes (C1, C2 and C3) of LIHC were defined, with C3 subtype showing the highest score of exhausted T cells and a poor prognosis. The Lasso and multivariate cox analysis selected 7 risk genes from the green module, which were closely associated with the C3 subtype. All the patients were divided into low- and high-risk groups based on the medium value of RiskScore, and we found that high-risk patients had higher immune infiltration and immune escape and poorer prognosis. The nomogram exhibited a strong performance for predicting long-term LIHC prognosis. In vitro experiments revealed that the 7 risk genes all had a higher expression in HCC cells, and that both liver HCC cell numbers and cell viability were reduced by knocking down MMP-9.

Conclusion

We developed a RiskScore model for predicting LIHC prognosis based on the scRNA-seq and RNA-seq data. The RiskScore as an independent prognostic factor could improve the clinical treatment for LIHC patients.