INTRODUCTION: Glioblastoma multiforme (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5%). Standard interventions consist of aggressive surgical resection, radiotherapy, and chemotherapy; however, GBM is heterogeneous and present therapies are often ineffective. Novel approaches need consideration. We investigated two such novel approaches: Withaferin A and Tumor Treating Fields (TTFields). Withaferin A is a steroidal lactone originating from extracts of the winter cherry plant (Withania somnifera). Extracts such as AshwaMAX can contain up to 4.3% (w/w) Withaferin A. Recently, the U.S. Food and Drug Administration approved another approach to treating glioblastoma: tumor treating fields (TTFields). TTFields involves the delivery of alternating electric fields to the tumor but mechanisms of action are not fully understood.

METHODS: To investigate these two therapies, we used human glioblastoma cells (GBM2, GBM39, and U87-MG) isolated from primary tumors. Cells were modified to express firefly luciferase. Proliferation was monitored by: bioluminescent imaging, cell counting via hemocytometer, or cell titer blue viability assay. TTFields were imposed on cell cultures by the inovitroTM device from Novocure Ltd.

RESULTS: Both AshwaMAX and Withaferin A inhibited growth of GBM cell lines (IC50 ~0.25 µM). In exploring Withaferin A and TTFields interventions in GBM cell culture, we discovered that the steroid worked synergistically with TTFields (p<0.01, n=3 experiments) to inhibit GBM proliferation. Current theories about TTFields’ action involve it disrupting mitosis through interference with mitotic spindle assembly. In the present studies, we show that TTFields also alters cellular membrane structure thus rendering it more permeant to chemotherapeutics. In GBM, increased permeability was indicated through increased bioluminescence with TTFields exposure (p<0.0001, TTFields vs no TTFields), as well as with increased binding and ingress of membrane-associating reagents such as Dextran-FITC (p<0.0001, TTFields vs no TTFields). Increased permeability was observed and with scanning electron microscopy showing augmented number (54+19 vs. 24+11 per ROI, TTFields vs no TTFields, p=0.0002) and sizes of holes (241+92 vs. 130+32 nm2, TTFields vs no TTFields, p=0.0005) in the GBM cellular membrane. Such phenomena were reversible after cessation of TTFields.

CONCLUSIONS: In summary, increased membrane permeability by TTFields helps to explain previous reports of additive vs. synergistic effects between TTFields and chemotherapies, including Withaferin A. These findings have implications for the design of combination therapies in glioblastoma and other cancers and may significantly alter standard of care strategies.

Citation Format: Edwin Chang, Chirag Patel, Christoph Pohling, Caroline Young, Jonathan Song, Thomas A. Flores, Yitian Zeng, Lydia Marie Joubert, Hamed Arami, Arutselvan Natarajan, Robert Sinclair, Sanjiv S. Gambhir. Tumor treating fields increases membrane permeability in glioblastoma cells [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 250.