1012 Priming of tumor spheroids with IFN-gamma increases efficacy of T cell mediated killing with PD-1 blockade

Background

T cell checkpoint inhibition has emerged as a valuable tool in cancer treatment, enhancing T cell responsiveness within the tumor microenvironment. Traditional in vitro testing has relied on mixed leukocyte reactions to demonstrate improved T cell function through cytokine production. Our approach, however, aimed to develop an in vitro tumor model that highlights active tumor cell killing modulated by checkpoint blockade.

Methods

To mimic the cellular and molecular characteristics of in vivo tumors, we generated spheroids using a lung carcinoma cell line (A549) and normal lung fibroblasts (MRC5), and exposed them to human PBMCs activated with an anti-CD3 antibody. A549 cells were transduced with red fluorescent protein for real-time tracking of spheroid growth using the Incucyte S3 platform. We treated the cultures with nivolumab, an anti-PD-1 antibody, or a relevant isotype control, with or without IFN-γ treatment, known to increase levels of PD-L1, a PD-1 ligand.

Results

We demonstrated that A549-MRC5 spheroids self-assembled within 72 hours and maintained growth over a 7-day period. The addition of non-activated PBMCs had no effect on spheroid growth. However, reduced spheroid volume was observed in the presence of anti-CD3 activated PBMCs, indicating T cell activation and tumor-targeting cytotoxicity. Analysis of spheroid cellular composition revealed the presence of immune cells, predominantly T cells, within the spheroids. Notably, PD-1 blockade did not enhance tumor cell killing in spheroids not previously exposed to IFN-γ. Conversely, spheroids exposed to IFN-γ showed significantly enhanced tumor cell killing in the presence of anti-PD-1 compared to isotype control. IFN-γ treatment increased PD-L1 expression on tumor cells, enabling the demonstration of anti-PD-1 efficacy. The expression of the relevant ligands and receptors is necessary to observe changes following the administration of checkpoint inhibitors.

Conclusions

The tumor spheroid assay is a valuable in vitro model for demonstrating the efficacy of various immunotherapy products, including checkpoint inhibitors. The assay system can be easily manipulated to capture the key molecular characteristics of different tumor types, making it a powerful tool for testing novel or improved anti-cancer therapies using tumor cell killing as a readout, thereby advancing immunotherapy projects.