82 Advancing cancer therapy: high-content imaging for precise tumor cell killing analysis in patient-derived tumoroids

<h3>Background</h3> Advancing cancer research and therapeutic development relies on effective tumor cell killing (TCK) analysis, particularly during the critical preclinical phase where accurate evaluation platforms are indispensable. Conventional methods, such as cell lines or primary cultures, often fail to replicate the complex 3D structures of patient-derived tumors. Nilogen Oncosystems’ tumoroids precisely mimic the tumor microenvironment and are vital for precise drug response assessments. To overcome current challenges in detecting tumor cell killing with the existing image analysis platforms, Nilogen is presenting a high-content imaging-based assay which integrates confocal microscopy with unique cell identifier dyes and a robust data analysis pipeline to capture detailed 3D segmentation of each tumoroid. This approach ensures rapid and accurate detection with quantification of tumor and immune cell responses, significantly enhancing accuracy and reproducibility compared to existing methods. <h3>Methods</h3> Post-treatment interactions were dynamically captured via time-lapse imaging, followed by staining with assay and cell feature identifiers at endpoint. High-content confocal imaging was then conducted to capture z-series image sets of all tumoroids within each replicate well. Advanced image processing techniques were employed to segment cell populations and assess viability using 3D objects derived from the z-stacks. Rigorous pre-processing ensured the reliability of data, which were integrated into a TCK calculation algorithm. <h3>Results</h3> Our workflow facilitated precise detection and analysis of tumoroid features, including diverse cellular subsets within the tumoroid microenvironment. The use of a set of dyes enabled identification and analysis of individual tumor cells and the immune microenvironment within each tumoroid. Advanced segmentation techniques and meticulous pre-processing ensured reliable TCK analysis across varied treatment conditions. <h3>Conclusions</h3> Our high-content imaging-based TCK assay addresses the limitations of traditional methods by providing a solution for an unmet scientific need. This unique approach greatly enhances the reliability and reproducibility of TCK analysis in our <i>ex-vivo</i> platforms, resulting in efficient examination of effective future cancer treatment strategies. <h3>Ethics Approval</h3> Ethics approval was obtained through Chesapeak IRB (Pro00014313) which determined ‘Using the Department of Health and Human Services regulations at 45 CFR 46, the IRB determined that this research project does not constitute human subject research and, therefore, does not require IRB oversight.’ Full informed consent was obtained for each tissue used in this study.