Integration of bulk RNA sequencing data and single-cell RNA sequencing analysis on the heterogeneity in patients with colorectal cancer.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has emerged as a critical innate immune pathway that could virtually impact nearly all aspects of tumorigenesis including colorectal cancer. This work aimed to develop and validate molecular subtypes related to cGAS-STING pathways for colorectal cancer using Bulk RNA-seq and single-cell RNA-seq (scRNA-seq) data. Bulk RNA-seq data were acquired from The Cancer Genome Atlas dataset (training dataset) and Gene Expression Omnibus dataset (validation dataset). Univariate COX survival analysis was utilized to identify prognostic differentially expressed genes (DEGs) from 6 immune pathways related to cGAS-STING. ConsensusClusterPlus package was used to classify different subtypes based on DEGs. scRNA-seq data were used to validate differences in immune status between different subtypes. Two clusters with distinct prognosis were identified based on 27 DEGs. The six cGAS-STING-related pathways had different levels of significance between the two clusters. Clust1 had most number of amplified CNVs and clust2 had the most number of loss CNVs. TP53 was the top mutated gene of which missense mutations contributed the most of single-nucleotide variants. Immune score of clust1 was higher than that in clust2, as reflected in macrophages, T cells, and natural killer cells. Three unfavorable genes and 31 protection factors were screened between the two clusters in three datasets. ScRNA-seq data analysis demonstrated that macrophages were more enriched in clust1, and tumor cells and immune cells had close interaction. We classified two distinct subtypes with different prognosis, mutation landscape, and immune characteristics.