Comprehensive pan-cancer characterization of cancer-testis genes at single-cell resolution

Cancer-testis genes (CTGs) are attractive immunotherapeutic targets owing to their restricted testicular expression and aberrant activation in cancers. However, their regulatory mechanisms, spatial organization, and clinical utility remain incompletely understood. Here, we leveraged large-scale single-cell and spatial transcriptomic data to perform a comprehensive pan-cancer analysis of CTGs. We established a high-confidence pan-cancer CTG catalog and uncovered a heterogeneous epigenetic regulatory landscape in which X-linked CTGs are predominantly governed by DNA methylation, whereas autosomal CTGs are more strongly associated with chromatin regulators. Building on the observation that CTG activation is a robust pan-cancer hallmark of malignancy, we developed a computational framework that enables rapid malignant cell annotation with performance comparable to established copy number variation-based methods. Spatial transcriptomic analyses revealed that CTG expression in head and neck squamous cell carcinoma is preferentially enriched at the invasive tumor front. Clinically, we highlighted the underappreciated therapeutic potential of CTGs and prioritized CT83 and DCAF4L2 as promising candidate targets for T-cell receptor-engineered T-cell therapy in triple-negative breast cancer and liver cancer, respectively. Our study advances mechanistic understanding of CTG biology and provides a valuable resource for the development of CTG-based immunotherapies.