Summary Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional condensates, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harboring distinct ecDNA species ( EGFR, MYC, PDGFR ). We found no evidence that ecDNA cluster with one another or closely interact with transcriptional condensates. Cells with EGFR -containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy was similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with the cellular transcriptional machinery.

Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering