Abstract Extrachromosomal DNA (ecDNA) is a key driver of intratumoral heterogeneity and has been linked to therapeutic resistance and poor patient outcomes. However, technical limitations in identifying, isolating, and analyzing ecDNA at the single-cell level have hindered our understanding of its role in tumor development and treatment response. We have developed a novel single-cell multiome (RNA + ATAC) sequencing approach that enables the simultaneous analyses of ecDNA and its gene expression profiles. Initial application of this method to medulloblastoma biopsies revealed distinct transcriptional signatures in ecDNA-positive tumor cells compared to ecDNA-negative tumor and control cells. In addition, we have found significant variations in ecDNA copy number among ecDNA-positive cells. Building on these findings, we performed multiome single-cell sequencing on 12 additional ecDNA-positive medulloblastoma specimens previously identified through bulk whole genome sequencing. Our ongoing analysis revealed that ecDNA-positive tumor cells show up-regulation of DNA replication, recombination, and damage repair pathways, while ecDNA-negative cells demonstrated increased expression of ATP synthesis and oxidative phosphorylation pathways. We are now conducting in vitro pharmacological inhibition studies using cell line models to investigate differential therapeutic sensitivities based on ecDNA status. This work aims to identify conserved transcriptional signatures associated with varying ecDNA levels, ultimately informing future functional genetic and pharmacological studies targeting transcriptional dependencies in ecDNA-driven medulloblastoma tumors.
Hui et al. (Fri,) studied this question.