Ploidy shapes gemcitabine response through altered potency and delayed cell death

Tumor ploidy is increasingly recognized as a determinant of therapeutic response, but the mechanisms by which ploidy shapes sensitivity to specific cytotoxic agents remain unclear. Here, we investigated the relationship between ploidy and gemcitabine response using pharmacogenomic reanalysis, isogenic cancer cell systems, live-cell imaging, intracellular pharmacokinetic/pharmacodynamic (PKPD) measurements, and mathematical modeling. Across public pharmacogenomic datasets, gemcitabine emerged as a low-ploidy-selective cytotoxic agent. In matched isogenic low- and high-ploidy cell systems, higher-ploidy cells were consistently less sensitive to gemcitabine across multiple lineages. Focused live-cell imaging and PKPD measurements in near-diploid and near-tetraploid SUM-159 cells showed that both states formed intracellular dFdCTP, but high-ploidy cells exhibited weaker and slower treatment responses, with delayed accumulation of cell death. To quantify these differences, we developed a delay-aware live/dead model driven by intracellular dFdCTP exposure. The best-fit model identified both reduced effective gemcitabine potency and a substantially longer delay from intracellular drug action to observed death in high-ploidy cells. Specifically, the inferred mean delay was approximately 17.5 hours in near-diploid cells versus 42.5 hours in near-tetraploid cells. The model also supported a thresholded, nonlinear, and ploidy-dependent mapping from administered gemcitabine dose to effective intracellular drug action. Together, these results establish ploidy as a determinant of both the magnitude and timing of gemcitabine response and provide a quantitative framework for linking intracellular drug exposure to delayed cytotoxic outcomes across ploidy states.