Single-cell mapping of extrachromosomal circular DNA architecture and evolution in cutaneous squamous cell carcinoma.

Extrachromosomal circular DNA (eccDNA), characterized by abundant amplicons and high chromatin accessibility, contributes to tumor heterogeneity and transcriptional activity; however, its single-cell dynamics in cutaneous squamous cell carcinoma (CSCC) remain poorly understood. Here, we developed a single-cell framework eccDNAscope that reconstructs eccDNA structures and infers clonal trajectories from scATAC-seq data. Integrative analysis of 154,624 single cells revealed that eccDNAs were broadly distributed across diverse cell types but exhibited striking enrichment at promoter regions (79.94%), where they were associated with elevated expression of cell type-specific eccDNA-associated genes. The framework identified both known oncogenic eccDNAs (MYC) and previously unrecognized eccDNAs (PTMA, NRP2, and KLF7), which were independently validated by DNA fluorescence in situ hybridization (FISH), super-resolution microscopy, Immuno-FISH, and Circle-seq. These eccDNAs displayed enhanced transcriptional activity in the epithelial cell populations in which they were detected. We further identified CSCC-specific eccDNAs that were enriched in cell cycle-related pathways, including CACYBP, which was exclusively detected in CSCC, but was not observed in actinic keratosis (AK) or para-tumor tissues. Finally, eccDNA-based phylogenetic reconstruction identified conserved and tissue-specific eccDNA programs during disease evolution, and highlighted recurrent eccDNAs, particularly PTMA and CACYBP, as potential drivers of CSCC progression. Collectively, our single-cell analysis provides a framework for dissecting eccDNA-mediated oncogenesis and offers insights into therapeutic strategies targeting eccDNA-positive clones in cancer.