Colcemid duration effects on extrachromosomal DNA analysis in breast cancer cell line

Extrachromosomal DNA (ecDNA) is a circular form of genetic material that exists outside the chromosomes and plays a significant role in tumour heterogeneity and drug resistance. In this preliminary study, we aimed to optimize the visualization of ecDNA by investigating the effects of varying colcemid treatment durations on metaphase arrest in MDA-MB-231 breast cancer cells. We tested four durations of colcemid treatment: 20 hours, 3 hours, 1 hour, and 30 minutes. Using light microscopy, confocal microscopy, and scanning electron microscopy, we analyzed the morphology and presence of ecDNA. Our results demonstrated that 20-hour colcemid treatment yielded the most well-spread metaphase cells, enabling the optimal observation of ecDNA. These structures appeared as ring-like formations with lower DAPI intensity than chromosomes and were often found in pairs. While our findings contribute valuable insights into the visualization of ecDNA, they also serve as a foundation for future research.