Single-cell transcriptomic analysis uncovers metabolic reprogramming and tumor–immune–stroma interactions in testicular mixed germ cell tumors

Testicular mixed germ cell tumors (TMGCTs) are a rare but increasingly prevalent subtype of testicular germ cell tumors, yet their heterogeneity and microenvironment remain poorly understood. This study presents a comprehensive single-cell transcriptomic atlas of TMGCTs, identifying 13 major cell types, marked immune infiltration, and loss of spermatogenic lineages. Malignant cells were subdivided into three distinct subclusters with unique transcriptional programs, and pseudotime trajectory analysis suggested a developmental hierarchy supported by the dynamic expression of pluripotency factors like POU5F1. We further uncovered a significant upregulation of N6-methyladenosine (m6A) RNA modification machinery in tumor cells, with METTL16 and POU5F1 showing prognostic relevance. Within the tumor microenvironment, we found a predominance of MRC1 + macrophages with pro-angiogenic features and distinct T cell subsets, including the presence of more effector T cells (Teff), naïve T cells (Tnai) and Treg cells. Notably, elevated expression of the Treg marker FOXP3 was significantly associated with poor overall survival. Additionally, tumor endothelial cells were enriched in Tip_cells and Veins, and exhibited high expression of angiogenesis-related genes and genes involved in collagen formation. Specially, high COL4A1 expression is significantly associated with poorer overall and recurrence-free survival. Collectively, this comprehensive single-cell transcriptomic atlas reveals cellular ecosystems, transcriptomic drivers, and potential biomarkers and therapeutic targets in TMGCTs.