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.