Pan-Cancer Single-Cell Profiling Uncovers the Biological Characteristics of Cancer-Testis Genes

Cancer-testis genes (CTGs), characterized by their restricted expression in testicular and neoplastic tissues, have emerged as pivotal modulators of tumor progression and immunogenicity. Here, leveraging single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) data from 828 tumor samples spanning 13 cancer types, we systematically identified 407 CTGs based on their expression level in malignant cells. Compared to previously reported CTG sets, our curated genes exhibited broader expression among tumor samples and were predominantly enriched on autosomes. These CTGs displayed pronounced co-expression patterns, with a subset consistently activated in proliferating malignant cells, whereas non–cell cycle CTGs were highly correlated with epigenetic regulatory pathways. Functional analyses pinpointed chromatin-modifying genes, including ARID4B and YEATS2 , as putative upstream regulators, whose knockdown resulted in widespread CTG downregulation. Moreover, we demonstrated that high CTG-count constitutes a robust and scalable feature for malignant cell identification across diverse omics data, including scRNA-seq, single-cell ATAC sequencing, and spatial transcriptomics. By quantifying transcriptomic similarity to a high CTG-count reference population, we developed a streamlined classification framework that enables rapid and accurate annotation of malignant cells in tumor sc/snRNA-seq datasets across diverse cancer types. Clinically, CTG expression levels were associated with patient prognosis and immune microenvironment composition. In hepatocellular carcinoma, elevated CTG expression defined a stem-like, immune-suppressive malignant cell state enriched for regulatory T cells, linked to advanced disease and poor survival. Collectively, our study refines the understanding of CTGs in cancer biology and provides a valuable resource to facilitate future research and therapeutic targeting.