Tumor treating fields (TTFields) are a clinically applied antineoplastic treatment modality delivered via noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. The antimitotic 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. Murine Lewis lung carcinoma (LLC-1), CT-26 colon carcinoma, and ovarian surface epithelial (MOSE) cells were treated with TTFields using the inovitro 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. We demonstrate that cancer cells that die under TTFields application exhibit release of HMGB1, ATP depletion from cells, and ER stress leading to CRT translocation to the cell surface, all of which are cardinal signs of immunogenic cell death (ICD). Moreover, we show that TTFields-treated cells promote in vitro phagocytosis by DCs and DC maturation as well as initiate inflammation in vivo. We also show that the combined treatment of lung tumor-bearing mice with TTFields plus the immune checkpoint inhibitor anti-PD-1 led to a significant decrease in tumor volume compared to anti-PD-1 alone or to the control group. Significant increases in CD45+ tumor-infiltrating cells were 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. Our results demonstrate the potential of TTFields therapy to induce ICD. We also demonstrate robust efficacy of concurrent application of TTFields and anti-PD-1 therapy in a mouse model of lung cancer. These data suggest that combining TTFields with anti-PD-1 might achieve tumor control by further enhancing antitumor immunity.
Citation Format: Tali Voloshin, Shiri Davidi, Yaara Porat, Anna Shteingauz, Mijal Munster, Noa Kaynan, Rosa S Schneiderman, Catherine Tempel Brami, Yaniv Alon, Einav Zeevi, Karnit Gotlib, Roni Blat, Shay Cahal, Aviran Itzhaki, Moshe Giladi, Eilon D. Kirson, Uri Weinberg, Adrian Kinzel, Yoram Palti. Immunomodulatory effect of tumor treating fields (TTFields) results in enhanced antitumor efficacy when combined with anti-PD-1 therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B14.