Understanding the Impact of Oncogene Copy Number Amplification on Cellular Drug Response

<span style="font-weight: 400;">Cancer progression and therapeutic failure are driven by heterogeneity within tumor cell populations, where subclones exhibit distinct genetic and phenotypic states that influence survival under stress. Extrachromosomal DNA (ecDNA) is a major source of this heterogeneity. Because ecDNA lacks centromeres, it segregates randomly during cell division, generating variable oncogene copy numbers and enabling rapid diversification of signaling. This creates populations in which subsets of cells may be pre-adapted to withstand therapy. HER2 signal intensity was used as a proxy for ecDNA abundance in the HER2-amplified NCI-H2170 non-small cell lung cancer (NSCLC) model, enabling single-cell measurement of oncogene dosage. Multiplexed iterative indirect immunofluorescence imaging (4i) quantified protein expression across multiple pathways. Cells were treated with neratinib (HER2 inhibition) and JQ1 or ARV-771 (BRD4-targeting transcriptional suppression) to probe how ecDNA interacts with different regulatory layers. Protein data were normalized into composite Z scores for apoptosis, cell-cycle proliferation, and senescence-like signaling. Cells were classified using quantile thresholds and a general trend (positive linear) is indicated in each marker with ecDNA abundance. Results show that ecDNA does not dictate a fixed outcome but shifts the probability of cellular states in a context-dependent manner, supporting a continuum model of cell fate under therapeutic pressure.</span>