# Integrated Weighted Gene Co-Expression Network and Single-Cell RNA Sequencing Analyses Reveal the Prognostic Significance of Hypoxia in Gastric Cancer

## TL;DR

This study identifies a hypoxia-related gene signature in gastric cancer that predicts worse survival and links it to an immunosuppressive tumor environment.

## Contribution

The novel hypoxia-related prognostic signature (HYS) comprising SPARC, AXL, NRP1, and VCAN is newly established for gastric cancer prognosis.

## Key findings

- Elevated hypoxia levels correlate with worse survival in gastric cancer patients.
- The HYS signature is associated with increased infiltration of immunosuppressive immune cells and molecules.
- Signature genes are predominantly expressed in cancer-associated fibroblasts and confirmed in tumor tissues.

## Abstract

Background: Hypoxia is a key driver of cancer progression. However, its specific prognostic significance in gastric cancer (GC) remains insufficiently characterized. Methods: Single-sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), univariate Cox regression, and least absolute shrinkage and selection operator (LASSO) regression were employed to identify a hypoxia-related prognostic signature. Subsequently, immune microenvironment profiling and single-cell RNA sequencing analyses were employed to further characterize the biological characteristics of the signature. In addition, quantitative real-time polymerase chain reaction (qPCR) was used to validate the expression levels of key hypoxia-associated genes in human GC tissues. Results: Elevated hypoxia levels were linked to worse survival outcomes in GC patients. Through integrated WGCNA, Cox, and LASSO analyses, a hypoxia-related prognostic signature (HYS) consisting of four genes—SPARC, AXL, NRP1, and VCAN—was established. Patients in the HYS-high group exhibited markedly poorer overall survival than their HYS-low counterparts [p = 0.000126, hazard ratio (HR) = 1.936]. Moreover, the HYS-high group exhibited increased infiltration of resting CD4+ memory T cells, monocytes, M2 macrophages, and resting mast cells, as well as elevated expression of immunosuppressive molecules, including PDCD1LG2 and HAVCR2. Single-cell RNA sequencing analysis revealed that the signature genes were predominantly expressed in cancer-associated fibroblasts. Consistently, qPCR analysis in five paired GC and para-carcinoma tissues confirmed higher expression of these genes in tumor samples (p < 0.01). Conclusions: Our findings indicate that hypoxia is a critical determinant of prognosis in GC and is closely associated with an immunosuppressive tumor microenvironment, highlighting its potential value as a prognostic biomarker and therapeutic target.