Tumor Treating Fields (TTFields) Induce Immunogenic Cell Death Resulting in Enhanced Antitumor Efficacy When Combined with Anti-PD-1 Therapy

Introduction

Tumor Treating Fields (TTFields) are a clinically applied anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. The anti-mitotic effects of TTFields include disruption of the mitotic spindle and abnormal chromosome segregation, which trigger different forms of cell death. In this study we evaluated whether TTFields-induced cell death can be perceived as immunogenic by the immune system.

Material and method

Murine Lewis lung carcinoma (LLC), CT-26 colon carcinoma, and ovarian surface epithelial (MOSE) cells were treated with TTFields using the inovitro^TM system. Levels of calreticulin (CRT) on the surface of treated cells and intracellular ATP levels were evaluated using flow cytometry. High mobility group box 1 (HMGB1) secretion was measured using an ELISA assay. For detection of ER stress, phosphorylation of the translation initiation factor eIF2α was evaluated using immunoblot analysis. TTFields induced upregulation of autophagy was evaluated using immunoblot analysis and immunofluorescence evaluation of LC3. Bone marrow derived dendritic cells (DCs) were co-incubated with TTFields treated LLC-1 cells and phagocytosis by DCs and DCs maturation were evaluated using flow cytometry. Mice orthotopically implanted with LLC cells were treated with TTFields, the immune checkpoint inhibitor anti-PD-1 or a combination of the two modalities. Tumor volume was monitored and flow cytometry analysis was performed for phenotypic characterization of infiltrating immune cells.

Results and discussion

We demonstrate that cancer cells which die during TTFields application exhibit ER stress leading to CRT translocation to the cell surface, as well as release of damage-associated molecular patterns including the chromatin-binding protein HMGB1 and ATP. TTFields also promote upregulation of autophagy which could explain the depletion of ATP following treatment. Moreover, we show that TTFields-treated cells promote phagocytosis by DCs and DCs maturation under co-culture conditions. In vivo, the combined treatment of lung tumor-bearing mice with TTFields in combination with anti-PD-1, significantly improved therapeutic efficacy compared to the control group or TTFields and anti-PD-1 alone. Significant increase in the number of tumor infiltrating immune cells was observed in the TTFields plus anti-PD-1 group. These infiltrating cells, specifically macrophages and DCs, demonstrated upregulation of surface PD-L1 expression. Correspondingly, cytotoxic T-cells isolated from these tumors have shown higher levels of IFN-γ production relative to untreated mice.

Conclusion

Collectively, our results suggest that TTFields application induces both ER stress and autophagy, resulting in immunogenic cell death. Combining TTFields with anti-PD-1 might therefore achieve tumor control by further enhancing antitumor immunity.