Category: Assay Development and Screening
Immunotherapies such as checkpoint inhibitors, CAR-Ts and immune-targeting Abs have great promise for cancer treatment. Tumor spheroid models offer more translational biological insight than 2D cell models and provide a more physiologically relevant model for testing immunotherapy biologics in vitro. In this study, we describe a novel, medium throughput immune cell-killing assay using automated live-cell analysis of 3D tumor spheroids. Single spheroids were formed using common tumor cell lines stably expressing red fluorescent protein (RFP) in round bottom, ultra-low attachment 96-well plates. Following spheroid formation (1-2 days), PBMCs were added and tumor spheroid viability was assessed over 10 days by measuring the loss of RFP fluorescence. This approach was validated using biological cocktails that generically activate sub-populations of immune cells from PBMCs, e.g. T cells and Natural Killer (NK) cells. Specifically, T cells were activated using anti-CD3 antibody in combination with IL-2, or natural killer cells were activated using a cocktail of IL-12 and IL-2. Significant loss in spheroid size and fluorescence intensity were observed in a time- and concentration-dependent following activation of either T cells or NK cells. In addition, we observed a significant increase in the magnitude and rate of spheroid cytotoxicity with increasing effector-to-target cell ratios. We also show that this approach can be applied to a model of antibody-dependent cell-mediated cytotoxicity (ADCC). These data show Herceptin, an Ab-based biologic directed towards cells expressing Her-2, induced a concentration-dependent specific killing of Her-2 expressing tumor cells (SKOV-3). Interestingly, higher concentrations of Herceptin were required in 3D tumor spheroids compared to a 2D monolayer cultures. These data demonstrate the ability to kinetically visualize and quantify 3D immune cell killing and ADCC assays, and illustrate how these assays can be extended from traditional 2D cultures to 3D. These assays will be highly valuable in the search for novel immuno-modulators.
Daniel Appledorn– Director of R&D, Essen BioScience, Ann Arbor, MI
Director of R&D
Ann Arbor, MI
Dan is the Director of US Biology R&D at Essen BioScience. Dan joined Essen in the fall of 2010 and is leading a team of scientists to develop new applications, biological reagents and instruments for drug discovery with a focus on building assays for quantitative live cell analysis using the IncuCyte system.
Prior to joining Essen, Dan was a postdoctoral research fellow at Michigan State University where his primary focus was investigating the interaction between adenovirus vectors and the innate and adaptive immune systems in mouse models with the goal of developing gene therapy and/or vaccine vectors for the treatment of human disease.
Over the course of his 4 year postdoc, Dan’s pioneering work in this field resulted in several patents and 20+ publications describing the utility of Ad vectors in both gene therapy and vaccine platforms.