Abstract Despite decades of progress in ovarian cancer treatment, therapeutic resistance remains a persistent and lethal challenge. Emerging evidence suggests polyploid giant cancer cells (PGCCs) as key drivers of this resistance. PGCCs are distinguished by multinucleation or a single oversized nucleus containing multiple chromosome sets. Polyploid cell populations are present in premalignant lesions and expand with disease progression and therapeutic stress. PGCCs demonstrate heightened resistance to treatment relative to non-PGCCs and can regenerate non-PGCCs via budding once therapeutic pressure is alleviated, leading to relapse. Supported by extensive preclinical and clinical evidence, we hypothesize that elimination of PGCCs may reduce therapeutic resistance in ovarian cancer. Given their significant role in resistance and relapse, PGCCs remain largely untargeted due to a lack of scalable detection methods. Standard viability assays such as MTT, XTT, and ATP-based screens fail to capture the rare but critical PGCC subpopulation. Current gold-standard methods, fluorescence-activated cell sorting (FACS) coupled with manual microscopy, while accurate, are time-intensive and unsuitable for high-throughput drug screening. Thus, a rapid and accurate technique for studying PGCCs is essential. In this pilot study, we have developed a high-throughput single-cell morphological analysis pipeline capable of precisely quantifying PGCC numbers and proportions. The pipeline accurately quantifies PGCCs based on nuclear and cellular features, including size and DNA content. Leveraging this platform, we screened a library of 2, 726 FDA-approved compounds to identify agents that effectively kill PGCCs. Hits from this screen were validated in 3D spheroid cultures, revealing compounds with potent anti-PGCC activity. Complementary transcriptomic analysis (RNA-Seq) of flow-sorted PGCCs versus non-PGCCs unveiled profound dysregulation of the FOXM1 signaling axis, implicating this pathway as a key regulator of PGCC formation and survival. Consistent with this, the high-throughput screen identified Thiostrepton, a known FOXM1 inhibitor, as a selective anti-PGCC agent. Across multiple ovarian cancer cell lines, FOXM1 inhibitors outperformed standard-of-care agents (Paclitaxel, Cisplatin, Olaparib) in eliminating PGCCs. siRNA knockdown of FOXM1 significantly reduced PGCC numbers, particularly under Paclitaxel stress. Moreover, FOXM1 inhibition triggered apoptosis in PGCCs, an effect reversible with the pan-caspase inhibitor Z-VAD-FMK. Together, these findings underscore PGCCs as a therapeutically actionable cell state driving resistance in ovarian cancer. Our integrative single-cell morphological and transcriptomic platform enables rapid discovery of anti-PGCC agents and establishes FOXM1 inhibition as a compelling strategy to eradicate this resilient subpopulation. This pipeline offers broad utility for overcoming therapy resistance across diverse malignancies. Citation Format: Hsiao-Chun Chen, Yuan Zhang, Yushu Ma, Yu-Chih Chen. Combating ovarian cancer resistance via polyploid cancer cell targeted screening abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl): Abstract nr A023.
Chen et al. (Fri,) studied this question.