(T0930-03-17) Targeted Chemoprevention of Ovarian Cancer Using a Redox-Sensitive PCQ-Gemcitabine Conjugate

Purpose: High-grade serous ovarian cancer (HGSOC), a common subtype of ovarian cancer, remains the most lethal gynecologic malignancy, largely due to late-stage diagnosis and a lack of effective prevention strategies [1-3]. Mutations in tumor suppressor genes such as TP53 are nearly widespread in HGSOC, and individuals with such genetic alterations are at increased risk of developing OC [4, 5]. Despite advances in surgical and chemotherapeutic approaches, recurrence is common, and long-term survival remains poor [6, 7]. Most women are diagnosed at advanced stages when tumors have already seeded the peritoneal cavity [8, 9]. There is an urgent need for novel interventions that can either prevent or delay tumor establishment in genetically predisposed individuals. Gemcitabine (GEM), a cytotoxic nucleoside analog, shows anti-tumor activity in ovarian cancer but suffers from rapid clearance, systemic toxicity, and non-specific activation [10]. To address this gap, we developed a redox-responsive polymer-drug conjugate (PCQ-SS-GEM) that combines a hydroxychloroquine-derived polymer (PCQ) with GEM via a disulfide linker. This design enables selective intracellular GEM release in the glutathione (GSH)-rich tumor environment and allows PCQ to inhibit epithelial-to-mesenchymal transition (EMT) and migration. We hypothesized that PCQ-SS-GEM, a redox-responsive polymer-drug conjugate, will suppress early ovarian cancer progression by simultaneously inhibiting cell migration and releasing cytotoxic GEM selectively within tumor cells, thereby reducing peritoneal tumor establishment in high-risk settings, providing a foundation for its development as a chemopreventive agent in ovarian cancer.

Methods: We evaluated the conjugate using both in vitro functional assays and an in vivo syngeneic mouse model of HGSOC. For in vitro testing, ID8 p53⁻/⁻ cells were treated with PCQ-SS-GEM, GEM alone, PCQ+GEM, or HCQ+GEM at equivalent GEM doses (~75 µM). Cell viability was assessed using the CellTiter-Blue (CTB) assay, and migration was evaluated using the transwell migration assay. For in vivo studies, 9 female C57BL/6 mice inoculated intraperitoneally with luciferase-tagged ID8 p53⁻/⁻ cells were stratified by weight and randomized into three groups (n = 3 each)- Placebo, HCQ+GEM, PCQ-SS-GEM. Mice received 10 intraperitoneal doses of 10 mg/kg GEM equivalent on a regular schedule (every 3rd day). In Vivo Imaging System (IVIS) imaging was performed after the 1st, 3rd, 7th, and 10th doses (corresponding to day 20, 26, 38, and 49 post-inoculation, respectively) to assess tumor burden. At necropsy (day 51 post-inoculation), disease severity was scored using a standardized peritoneal carcinomatosis index (PCI).

Results: In vitro, PCQ-SS-GEM treatment significantly reduced cell viability compared to GEM alone or combination controls (IC50 for PCQ-SS-GEM, PCQ+GEM, and GEM: 640.5, 74.18, and 112.5, respectively). In migration assays, PCQ-SS-GEM (107.87 µg/mL) nearly suppressed cell migration within 48 hours, indicating potent inhibition of migratory capacity. HCQ+GEM, and PCQ+GEM groups demonstrated only partial suppression of migration. These findings suggest dual functional activity of the conjugate- cytotoxicity via targeted GEM release and anti-migratory effects mediated by the PCQ backbone. In vivo, PCQ-SS-GEM treatment group (PCQ-SS-GEM vs. Placebo: p = 0.013) exhibited markedly reduced bioluminescent signals on IVIS imaging after the 10th dose compared to the Placebo and HCQ+GEM groups (HCQ+GEM vs. Placebo: p = 0.157), indicating reduced tumor burden (Figure 1 A and B). These imaging findings were corroborated by terminal peritoneal carcinomatosis index (PCI) scores. Mice in the Placebo group showed high PCI scores (16.3 ± 1.5, mean ± SEM), reflecting extensive tumor dissemination throughout the peritoneal cavity. The HCQ+GEM group demonstrated a modest reduction in tumor burden with PCI scores of 5.3 ± 1.5 (mean ± SEM) (HCQ+GEM vs. Placebo: p = 0.0002). In contrast, mice treated with PCQ-SS-GEM displayed a substantial suppression of tumor spread, with a lower PCI score (2.6 ± 0.5) (mean ± SEM) (PCQ-SS-GEM vs. Placebo: p < 0.0001) (Figure 2). Although the differences between the HCQ+GEM and PCQ-SS-GEM groups were not statistically significant, the PCQ-SS-GEM treatments consistently demonstrated a strong trend toward reduced tumor burden, as reflected by both IVIS imaging and PCI scores.

Conclusion: This study demonstrates that PCQ-SS-GEM, a redox-sensitive polymer-drug conjugate, effectively inhibits ovarian cancer cell migration and viability in vitro and significantly reduces peritoneal tumor burden in a syngeneic, immunocompetent ovarian cancer mouse model. The conjugate’s dual functionality- tumor-selective gemcitabine (GEM) release and suppression of early metastatic behavior, offers a compelling chemopreventive strategy for individuals at high risk for ovarian cancer. Both IVIS imaging and PCI scoring confirmed the conjugate’s superior efficacy over GEM combination treatments. This work introduces PCQ-SS-GEM as a first-in-class, dual-function prodrug polymer, specifically engineered to address a critical gap in the early prevention and treatment of high-grade serous ovarian cancer (HGSOC). By combining redox-triggered intracellular GEM activation with anti-migratory effects mediated by the hydroxychloroquine-derived backbone, the conjugate exerts targeted cytotoxicity while minimizing off-target toxicity. Unlike conventional chemotherapeutics, this approach strategically leverages the tumor-associated redox environment to control drug activation, making it a promising platform for non-surgical, mechanism-driven interventions in ovarian and other peritoneal malignancies characterized by early dissemination and recurrence.

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Acknowledgements: We are grateful to Dr. Adam Karpf at the University of Nebraska Medical Center for generously providing the ID8 p53⁻/⁻ cell line used in this study. We also acknowledge the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska Medical Center (UNMC) for their continued support and oversight of animal care and welfare throughout this project.

Figure 1. The conjugate reduces tumor burden in vivo in a murine model of ovarian cancer. (a) Representative IVIS bioluminescence images of ID8 p53⁻/⁻ +Luc tumor-bearing C57BL/6 mice treated intraperitoneally with placebo (PBS), HCQ+GEM, or PCQ-SS-GEM. Images indicated time points reflect changes in tumor burden across treatment groups. (b) Quantification of total bioluminescence signal (photons/sec) from individual mice over the treatment period. Data represent mean ± SEM; n = 3 per group.


Figure 2. PCQ-SS-GEM treatment significantly reduces peritoneal tumor burden in vivo. Peritoneal Carcinomatosis Index (PCI) scores were assessed at necropsy on day 51 in tumor-bearing mice treated with Placebo, HCQ+GEM, or PCQ-SS-GEM