Abstract B056: Near-real-time detection of epithelial killing supports BiTE evaluation directly from fresh NSCLC and breast tumors

Abstract Patient-derived organoids (PDOs) are powerful models for therapeutic response prediction in solid-tumors, but fully autologous immunotherapy testing is limited by the loss of native immune cells, necessitating replacement with exogenous immune cells rather than those from the original tumor. PDO-immune co-culture imaging is further constrained by the lack of epithelial-specific viability markers needed to distinguish true tumor-cell death from activation-induced apoptosis in immune cells. We aimed to develop a workflow enabling bispecific T-cell engager (BiTE) testing directly from patient tumor samples that retain endogenous tumor and immune cells. To do so, we identified a near-real-time epithelial viability method for mixed cell populations and integrated it into a high-throughput imaging workflow using MicroOrganoSpheres® (MOS®), a microdroplet-based 3D culture format compatible with automated fluid handling. With this platform, we show robust BiTE responses in established NSCLC and breast cancer PDOs with exogenous T cells. We extended the workflow to fresh NSCLC and breast tumor samples in MOS that preserve endogenous immune cells and measured clear BiTE-mediated killing within 24 hours. These advances enable fully autologous BiTE efficacy testing from a single tumor sample within 14 days. Using flow cytometry and high-content imaging, an epithelial-specific viability method was validated for selective detection of live and dead epithelial cells and minimal reactivity toward immune and stromal cells. Using longitudinal imaging, this method was applied to MOS from established NSCLC and breast PDOs co-cultured with PBMCs and T cells to assess responses to an EpCAMxCD3 BiTE. This workflow was applied to fresh NSCLC and breast tumor MOS that retain native immune cells. Tumor cell compositions were profiled by flow cytometry before BiTE treatment, and epithelial viability was quantified by longitudinal imaging and flow cytometry. We developed an epithelial-specific viability method that quantifies epithelial lifecycle dynamics within mixed cell populations, shows no reactivity toward non-epithelial cells, and is compatible with flow cytometry and longitudinal imaging. In co-culture studies using established PDOs with exogenous T cells and the EpCAMxCD3 BiTE, measurable epithelial killing was observed within 24 hours. In fresh tumor samples, MOS preserved epithelial and diverse immune populations comparable to the original tumors. Treatment of fresh MOS with the BiTE produced clear epithelial killing within 24 hours along with elevated granzyme B activity, indicating endogenous T cell-mediated cytotoxicity. The entire workflow - from sample receipt to data visualization - was completed within 14 days. This study establishes a method for epithelial-specific viability measurement integrated with MOS technology for evaluating BiTE potency in fully autologous tumor samples. The platform enables functional immunotherapy assessment from a single patient specimen, supporting its use in translational and precision immuno-oncology applications. Citation Format: David M. Graham, Laura Contreras-Ruiz, Madison Rivera, Jacob Layton, Kendra Hightower. Near-real-time detection of epithelial killing supports BiTE evaluation directly from fresh NSCLC and breast tumors [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B056.