(1348-A) Multiplexed drug and immune cell screening in 3D patient-derived tumoroid models

Abstract: Patient-derived 3D cancer models (tumoroids or cancer organoids) better recapitulate properties of patient tumors compared to immortalized in vitro 2D cell lines. However, labor-intensive media preparation and scale up requirements and differences in handling 3D cell cultures compared to traditional 2D cell lines have limited adoption of 3D techniques in compound and cell therapy screening experiments. To facilitate the use of patient-derived 3D tumoroids, we have recently developed Gibco™ OncoPro™ Tumoroid Culture Medium, a serum-free, conditioned medium-free tumoroid culture medium. Here, we demonstrate how tumoroids derived in OncoPro™ medium can be scaled and used in drug and immune cell screening assays. OncoPro™ medium is compatible with a suspension culture approach for easier handling compared to traditional embedded culture formats, enabling expansion for downstream assays. In our testing, tumoroid growth rates and morphologies were consistent across non-tissue culture treated flask sizes. In some cases, as many as 100x106 dissociated tumoroid cells could be recovered from a single Nunc™ TripleFlask™ after a week of growth. After scaling up, tumoroids were dissociated and seeded in multiwell plates to test multiple screening conditions (compound identity, compound concentration) in parallel. Dissociated cells were seeded in 96-well plates using both manual and automated liquid handling. Optimization of cell seeding protocols led to standard deviations in tumoroid metabolic activity, number, and size that were comparable to manual seeding, with a coefficient of variation of less than 3% between wells for multiple tumoroid lines when automation was implemented. We further demonstrate the heterogeneous response of a panel of colorectal cancer tumoroids to staurosporine via multiplexed measurements of the reducing power, ATP content, and release of lactate dehydrogenase upon treatment. Furthermore, we show how patient-derived 3D models established in OncoPro™ medium can be engineered for more complex co-culture assays with natural killer (NK) cells. Engineered tumoroids exhibited identical (Pearson r>0.99) gene expression profiles and heterogeneity compared to the parental cells. The killing efficiency of NK cells increased with increasing with effector (E) to target (T) ratios, as measured by both a decrease in GFP signal from the tumoroid cells and an increase in caspase-3/7 signal. NK cells derived from different sources showed markedly different killing efficiencies and cytokine release profiles, highlighting the potential of this platform for screening different immune cell engineering constructs or donors. Results here present optimized multiplexed assays for screening drugs and immunotherapies using 3D patient-derived cancer models. In combination with OncoPro™ Tumoroid Culture Medium for the derivation and expansion of tumoroid models, this workflow provides a platform for large-scale drug discovery and immunotherapy screens.