Abstract 1228: High throughput phenotyping and drug discovery using the TumorGraft3D co-culture platform and flow cytometry

Abstract Many anticancer drugs fail in clinical trials due to issues resulting from inadequate efficacy and/or high toxicity emphasizing the need for improving clinically relevant, preclinical models for high throughput drug screening. Currently, 3D models have emerged as a highly promising tool for initial drug screening that closely mimics in vivo tumors through preserved morphology and molecular mechanisms. To further recapitulate the in vivo models, interactions between immune and tumor cells can be performed via co-culture models. As the complexity of the models has increased, the analytical methods have had to evolve as well. In this work, we have developed a high throughput flow cytometry (FlowHT) assay using Champions' TumorGraft3D (CTG3D) models. CTG3D are 3D organoids derived from our highly characterized and clinically annotated Patient Derived Xenograft (PDX) models. We have developed a method of TumorGraft3D dissociation which preserves cell viability and epitope stability. We further optimized a robust FlowHT workflow that allows for the detection of low-frequency targets and the characterization of unique subpopulations in modular multicolor panels to distinguish immune cells and CTG3D in mixed populations. Using this method, we have validated that the CTG3D models retain their original phenotype with minimal differences observed between source PDX tumors and expanded ex-vivo 3D models. At co-culture endpoints, our assay was highly sensitive to immune cell-mediated tumor killing as well as SOC drug responses. Interrogation in an allogenic PBMC coculture revealed the ability to detect cytotoxic T cells (Tc) and helper T cells (Th) activation and immune checkpoint markers such as CD69, TIM-3, and PD-1 among others. Collectively, we have developed a quantitative, sensitive, and robust assay that can be utilized as an analytical endpoint for both monoculture and coculture studies to enhance the interrogation of immune-oncology agents for tumor killing, ADCC effects, increasing T cell activation, reversal of T cell exhaustion and identifying specific markers across multiple cell types. Citation Format: Sumanun Suwunnakorn, Fu-Ju Chou, Elena Bruni, Karolina Pilipow, Xiangyang Zhang, Chelsea Riveley, Mara Gilardi, Stefano Cairo, Brandon Walling. High throughput phenotyping and drug discovery using the TumorGraft3D co-culture platform and flow cytometry [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 1228.