Abstract

Antigen recognition by T cells induces powerful effects, including infiltration and targeted killing, which aid in the elimination of cancer. Typically, therapeutic effects on T cell response have been evaluated using 2D cell models, which bear limited resemblance to the complex physiology of the in vivo tumor microenvironment. Here we provide two 3D spheroid-based solutions for monitoring immune-cancer cell interactions: immune cell killing (ICK) and tumor infiltrating lymphocytes (TILs).

Single spheroids were co-cultured with PBMCs and CD3/CD28 Dynabeads. To measure the rate of ICK, labeled tumor cells were imaged using the Incucyte® Live-Cell Analysis System and quantified for spheroid fluorescence over time. For the TILs assay, spheroids were washed to separate non-infiltrated immune cells from the TILs. Both assays required endpoint spheroid dissociation prior to analysis of cell subsets and cytokines using the iQue® Advanced Flow Cytometer and T cell characterization kits.

Separation of the TILs and non-infiltrated T cells enabled comparison of their activation status. Infiltrated cells maintained high CD69 expression (67.1 ± 0.6%) regardless of the number of Dynabeads present. Non-infiltrated cells were more sensitive to external stimuli with CD69 expression increasing from 2.3 ± 0.2% to 86.9 ± 0.7% with increasing Dynabead density. The effects of Dynabead activation were further profiled using the ICK assay. Incucyte data showed a 98.4% reduction in spheroid fluorescence in the presence of 50K Dynabeads compared to the non-activated control on day 4. Spheroid killing was accompanied by an increase in T cell activation and exhaustion marker expression (CD25 and PD-1) and cytokine production (IFNγ). These data demonstrate the study of complex 3D tumor models using advanced flow cytometry and live-cell analysis as a translational approach to in vitro profiling of immunotherapeutics.