Extrachromosomal DNA Detection and Ultrastructural Profiling in Lung and Colorectal Cancer Models

Lung and colorectal cancers are malignancies ranked as the leading cause of cancer-related mortality worldwide. Their high mortality rate is closely associated with drug resistance driven by cancer cell heterogeneity. One of the major contributors to this heterogeneity is the presence of extrachromosomal DNA (ecDNA), circular DNA fragments located outside linear chromosomes and generated through DNA damage. ecDNA is found exclusively in cancer cells, frequently carries highly amplified oncogenes, and contributes to cancer aggressiveness. To date, no study has characterized ecDNA in A549 and HT-29 cells, representative lung and colorectal cancer cell lines. This study aimed to detect and characterize ecDNA in A549 and HT-29 cells using Giemsa staining, DAPI staining, and scanning electron microscopy (SEM). Our findings revealed the presence of ecDNA, with the percentage of 15% in A549 and 10% of HT-29 cells from the total of 180 metaphase spread chromosomes. Among these, some spreads contained a single ecDNA and two ecDNA elements, either solitary or paired. The ecDNA area ranged from 0.537 μm² to 1.162 μm², with an average of 0.85 μm² in A549 cells. The fluorescence intensity of ecDNA ranged from 152.204 A.U. to 196.109 A.U., with an average of 179.847 A.U. While the ecDNA of HT-29 exhibited an average area of 0.455 μm² with an average DAPI fluorescence intensity of 102.747 A.U. SEM imaging further revealed ring-like circular structures with an area consistent with previously reported ecDNA ultrastructural characteristics. These results suggest that ecDNA is present across different cancer cell lines and may serve as a potential biomarker as well as a promising target for future cancer therapies.