Abstract 5756: Quantification of SHetA2 in patient-derived ovarian cancer spheroids to study drug uptake and resistance in a physiologically relevant model

Abstract Ovarian cancer presents therapeutic challenges, particularly in advanced stages, where malignant ascites—a buildup of fluid in the abdominal cavity—complicates treatment. Within this ascitic environment, clusters of cancer cells, known as spheroids, form, proliferate, and implant upon various organs, contributing to the metastatic nature of ovarian cancer. As they limit drug penetration and have a hypoxic environment that fosters angiogenesis, spheroids exhibit significant chemoresistance, amplified by the inflammatory and tumor-promoting microenvironment (Peng). Therefore, to reveal the potential mechanisms of chemoresistance in ovarian cancer, directly targeting these spheroids is crucial to develop effective therapies that could help reduce the high mortality rate associated with ovarian cancer. ShetA2 (Sulfur Heteroarotinoid A2) is a novel, promising anticancer drug that exhibits unique potential by inducing apoptosis in cancer cells and disrupting mitochondrial pathways without harming healthy cells (Benbrook). Currently in phase 1 clinical trials, it is vital to understand its efficacy and optimize its therapeutic potential. However, conventional quantification methods for SHetA2 primarily rely on plasma analysis in animal models or human samples, providing only indirect measures and failing to replicate microenvironmental factors in the tumor’s 3D structure that inhibit drug access to spheroids (Sharma et al.). Consequently, a direct approach to measure drug concentration in single spheroids is necessary to understand the pharmacodynamics and pharmacokinetics of anticancer drugs (Peng). To address this gap, we developed a highly sensitive nano-flow liquid chromatography-mass spectrometry (nanoLC-MS) method for quantification of SHetA2 in patient-derived spheroids treated ex vivo with cell free-ascites. First, spheroids are washed and lysed. Second, an isotopically labeled compound d4-SHetA2 with known concentration is added to the lysate as the internal standard. Third, a C18 ZipTip is used to clean and desalt the sample to remove competing ions that could reduce detection sensitivity. Lastly, the prepared samples are analyzed using flow ultra-high-performance liquid chromatography (UHPLC) coupled with a high-resolution Thermo Orbitrap mass spectrometer. This integrated approach will provide highly sensitive and accurate quantification of SHetA2 at low concentrations, offering insights into its distribution, penetration, and activity within single patient-derived spheroids. Furthermore, by enabling a more precise and clinically relevant analysis of SHetA2’s efficacy in targeting spheroids, this approach will help establish robust criteria for dose selection and scheduling intervals in clinical applications. Citation Format: Annelise Huynh, Amit Singh, Zongkai Peng, Doris M. Benbrook, Anthony W. Burgett, Zhibo Yang. Quantification of SHetA2 in patient-derived ovarian cancer spheroids to study drug uptake and resistance in a physiologically relevant model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5756.