Abstract 3961: Alternating electric fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy
Authors: Voloshin Tali , Kaynan Noa , Davidi Shiri , Porat Yaara , Shteingauz Anna , Munster Mijal , Schneiderman Rosa S. , Brami Catherine Tempel , Zeevi Einav , Gotlib Karnit , Cahal Shay , Itzhaki Aviran , Giladi Moshe , Kirson Eilon D. , Weinberg Uri , Kinzel Adrian , Palti Yoram 
Published: August 2019

Abstract
Tumor Treating Fields (TTFields) are a clinically applied anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. In this study we evaluated whether TTFields-induced cell death is immunogenic. For evaluation of immunogenic cell death (ICD), cultured murine cells were treated with TTFields using the inovitro system. ICD was characterized by the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, secretion of adenosine triphosphate (ATP), and release of the chromatin-binding protein high mobility group B1 (HMGB1). For detection of ER stress, phosphorylation of the translation initiation factor eIF2α was assessed. TTFields effect on autophagy was evaluated using electron microscopy and immunoblot and immunofluorescence evaluation of LC3. For evaluation of the effect of TTFields on dendritic cells (DCs), bone marrow derived dendritic cells were co-incubated with TTFields treated LLC-1 cells and phagocytosis by DCs and DCs maturation were evaluated using flow cytometry. For in-vivo studies, 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. Moreover, we show that TTFields treated cells promote phagocytosis by DCs, DC maturation in vitro, and 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, Noa Kaynan, Shiri Davidi, Yaara Porat, Anna Shteingauz, Mijal Munster, Rosa S. Schneiderman, Catherine Tempel Brami, Einav Zeevi, Karnit Gotlib, Shay Cahal, Aviran Itzhaki, Moshe Giladi, Eilon D. Kirson, Uri Weinberg, Adrian Kinzel, Yoram Palti. Alternating electric fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3961.