Abstract 5194: Establishment and characterization of tumoroids from patient derived xenograft tissue for in vitro applications

Abstract Lung cancer remained the most frequently diagnosed cancer in 2022 and the leading cause of cancer-related deaths worldwide. Oncology drug development attrition is highest compared with other therapeutic areas, and success rates in clinical development range from 3.4-5%. Tumoroids as sophisticated three-dimensional in vitro cultures serve as an advanced near-physiological model but lung cancer tumoroid development remains challenging. The treatment strategy for lung cancer patients is currently determined based on histology, stage, and biomarker status, thus we aimed to establish a panel of complex in vitro tumoroids covering a broad range of different subtypes of the disease. Two methods of primary cell isolations, five different media formulations and two types of culture methods were assessed to find optimal conditions for tumoroids. Tumoroids were kept in culture for up to 10 passages including freeze-thaw tests. Characterization involved determination of morphology by brightfield microscopy, histology (H&E staining), biomarker expression using multiplex IHC staining and QuantiGene for murine stroma content assessment. Multiple commercial systems that allow uniform organoid formation using microwell technologies were compared. The models in question were mostly of lung adenocarcinoma origin, either “hot” or “cold” in terms of immune cell infiltration status. Tumoroid growth was dependent on the niche growth factor formulation. The highest success rates were achieved with a custom medium formulation (7/8 models). Feasibility of biobanking the organoids either as single cell suspension or whole organoids was confirmed. Different media formulations led to distinct phenotypes and retention of biomarkers in each model. Tumoroids showed signs of cellular plasticity by epithelial to mesenchymal transition characterized by simultaneous expression of EPCAM and aSMA in a subpopulation of cells. The process of de-differentiation during the culture phase has been explored in detail. After model establishment, the models will be tested in a viability assay with several SoC small molecules and in an immune cell co-culture with activated PBMCs and checkpoint inhibitors, to fully establish a platform for drug-screening and evaluation of immunotherapies. This study showcases a complete workflow in tumoroid establishment, highlighting the importance of adequate quality control and validation. Substantial variations of histological characteristics and biomarker expression depending on the culture condition, show the lineage-specific differences and the heterogeneity of the tumoroid. Organoid equivalence to the parental tissue is rarely addressed and this work highlights the impact of standardized culture conditions and thorough characterization of tumoroids to unfold their full potential as drug development platform. Citation Format: Jannik Scherer, Kanstantsin Lashuk, Julia Thiel, Meng Dong, Julia B. Schueler. Establishment and characterization of tumoroids from patient derived xenograft tissue for in vitro applications [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 5194.