Xenograft tumour cells with higher ecDNA copy numbers showed higher Ki67 expression, indicating a strong association between increased ecDNA with proliferative fitness and suggesting a new optimal copy number in vivo that only ecDNA(+) cells can shift to by virtue of uneven inheritance. 10/13

We found that while HSR(+) cells divide more uniformly and outcompete ecDNA(+) cells in culture, ecDNA(+) cells far outcompete HSR(+) cells upon injection into mice. The ecDNA(+) cell population in vivo exhibited a two-fold higher copy number than cells in culture. 9/13

We reasoned that this optimal copy number is determined by the balance of promoting oncogenic activity while minimizing cellular stress, and this balance can be perturbed upon drug treatment or change in environment. 8/13

Very interestingly, ecDNA(+) cells also exhibited increased time to next division heterogeneity compared to HSR(+) cells. We found that there is a middle copy number range associated with the shortest time to division. 7/13

We engineered an isogenic cancer cell line pair with fluorescently tagged 𝘔𝘠𝘊 amplicons encoded either on ecDNA or chromosomally as a homogeneously staining region (HSR). From long-term live-cell imaging, we found that 𝘔𝘠𝘊 copy number heterogeneity was generated rapidly in our ecDNA(+) cells. 6/13

We found that ecDNA copy number is positively correlated with downstream oncogenic activity as well as cellular stressors, including the DNA damage response and the unfolded protein response. 5/13

ecDNAs are circular DNA particles typically harboring oncogenes and prevalent across cancers. EcDNAs lack centromeres, so they are unevenly inherited during cell division, subsequently leading to immense copy number heterogeneity among cancer cells. 2/13