LAUSR

Tumor treating fields (TTFields) are an approved treatment modality for patients with glioblastoma. TTFields are delivered via noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. Previous studies have shown that TTFields lead to increased granularity which is often associated with autophagy. In this study we evaluated the effect of TTFields on the induction of autophagy in glioma and NSCLC cells. Cells were treated with TTFields using the inovitro system. Cellular granularity was evaluated using flow cytometry. Autophagy was monitored by quantifying levels of lipidated Microtubule Associated Protein Light Chain 3 (LC3-II), in the presence and absence of the lysomotropic agent and autophagy inhibitor chloroquine (CQ), using immunoblotting and immunofluorescence microscopy. Transmission Electron Microscopy (TEM) was used to visualize autophagosome-like structures. Western blot analysis was utilized to evaluate autophagy regulatory activity of the mammalian target of rapamycin (mTOR) through p70 S6 kinase1 (S6K1) and AMP activated protein kinase (AMPK) and its downstream target ULK-1. To determine if AMPK is responsible for TTFields stimulated autophagy pool of siRNAs was used to deplete siAMPK from U87-MG cells. To evaluate involvement of autophagy in cell fate after TTFields treatment we produced glioma cell lines depleted from ATG7 by shATG7 infection. Flow cytometry analysis demonstrated that TTFields application leads to a significant increase in cellular granularity in all tested cell lines. Significant elevation in LC3-II levels was observed in treated cells using fluorescence microscopy, where punctate distribution of LC3-II was observed. TEM micrographs demonstrated the presence of autophagy typical, autophagosome-like structures, in TTFields treated cells. . Evidence of increased autophagic flux following TTFields application was also detected using immunoblotting analysis in the presence of CQ. Western blot analysis of cells after TTFields treatment revealed stimulation of AMPK signaling as well as activation of p70. Depletion of AMPK from U87-MG cells resulted in reduction of autophagy as reflected by LC3-II levels and enhancement of TTFields cytotoxicity. ER stress in treated cells has been evident by immunoblotting, showing increased levels of ER stress marker GRP78. Combination of TTFields with CQ resulted in a significant dose dependent reduction of cell growth compared with TTFields treatment alone. Cells with ATG7 depletion showed similar results. Combined, these results suggest that cells upregulate autophagy in response to ER stress induced by TTFields application and that AMPK may serve as a key regulator of this process. Citation Format: Anna Shteingauz, Yaara Porat, Moshe Giladi, Roza Schneiderman, Tali Voloshin, Mijal Munster, Eilon Kirson, Uri Weinberg, Yoram Palti. Induction of autophagy following TTFields application serves as a survival mechanism mediated by AMPK activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1343.