(F-206) Multiplexed, high-throughput drug and immune cell screening in 3D patient-derived tumoroid models

Introduction:: Patient-derived 3D cancer models (tumoroids or cancer organoids) better recapitulate clinical samples compared to in vitro 2D models. Several studies have indicated that the response of patient-derived models to treatment reflects the response of patients from which the models were derived, suggesting their utility in screening potential therapies for drug development, immunotherapy evaluation, and personalized medicine. Here, we present the development of a high-throughput, multiplexed drug and immunotherapy screening assays using 3D patient-derived tumoroids. The effects of dimensionality (2D vs. 3D) on drug response was assessed, and tumoroid response was compared to that of an immortalized cancer cell line. Additionally, the efficiency of NK cell killing was quantified across effector to target cell ratios and NK cell sources.

Materials and Methods:: Patient-derived tumoroids were cultured in OncoPro™ Tumoroid Culture Medium before being scaled down for drug and immunotherapy screening. For drug screening assays, colorectal cancer tumoroid lines from three unique donors and the immortalized HCT-116 colorectal cancer cell line were cultured in 3D in 96-well plates prior to exposure to pharmacological compounds for three days. The same tumoroid and cell lines were cultured and treated in 2D as an experimental control. The drug response readout was multiplexed using three different plate reader-based assays measuring the reducing power, ATP, and the release of lactate dehydrogenase from treated cells from each well, and IC50s were calculated. For select lines, the drug screening workflow was performed in parallel using an automated liquid handler to demonstrate the compatibility of our approach with high-throughput screening. For immunotherapy screening workflows, a patient-derived colorectal tumoroid model was engineered using an EmGFP lentivirus to generate a reporter pool. Transduced pools were characterized using bulk and single-cell RNAseq for comparison to the non-engineered parental line. Natural killer (NK) cells were cultured as established lines (NK-92 cells) or expanded from leukopaks prior to co-culture with EmGFP-expressing tumoroids in various effector-to-target ratios and in a 1:1 mixture of tumoroid and NK cell media. Cells were dynamically monitored using a live cell imaging platform for detection of EmGFP (tumoroid signal) and caspage-3/7 (apoptotic cells). After 3 days of co-culture, cytokine release profiling was used to further characterize immune cell activity in response to patient-derived 3D tumoroids.

Results, Conclusions, and Discussions::

Comparable drug responses were observed from three different viability/cytotoxicity assays that were multiplexed from single experimental replicates to gain a holistic view of cell health and viability. Both patient-derived and cancer cell lines cultured in 3D models showed markedly increased resistance to drug treatments compared with 2D adherent culture across assay readouts (Figure 1A). Differential response was also observed between HCT-116 immortalized cells vs patient-derived lines, with the tumoroid models exhibiting greater sensitivity to staurosporine and comparable sensitivity to gefitinib. Similar results were obtained when using an automated liquid handler for select experiments. Importantly, patient-derived 3D models established in OncoPro could also be engineered for more complex co-culture assays. Engineered tumoroids exhibiting identical (Pearson r>0.99) gene expression profiles and heterogeneity compared to the parental cells. The killing efficiency of natural killer immune 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 (Figure 1B). NK cells derived from different sources showed markedly different killing efficiencies (Figure 1B) and cytokine release profiles, highlighting the potential of this platform for screening different immune cell engineering constructs or donors. This platform can be used in a variety of immune cell workflows, providing a method that can predict tissue-specific responses and evaluate solid tumor immunotherapies in high throughput cell-based assays.  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.

Acknowledgements (Optional): : We thank Discovery Life Sciences for providing dissociated tumor cells to generate data in this study.

References (Optional): :