BACKGROUND Glioblastoma (GBM) is the most lethal primary brain cancer (median survival: 15-17 months, 5-year survival: 5.6%). Standard interventions consist of aggressive surgical resection, radiotherapy, and chemotherapy; however, GBM is heterogeneous and present therapies are ineffective. Tumor treating fields (TTFields) is a form of alternating electric field therapy that has been shown to prolong survival in patients with newly-diagnosed GBM when combined with standard chemotherapy. The mechanism of TTFields' potentiation of standard chemotherapy against GBM is not well understood. We hypothesized that TTFields increases access of chemotherapy to cancer cells by disrupting the cell membrane.
METHODS Human and murine GBM cells (GBM2, GBM39, U87-MG, KR158B) were isolated from primary gliomas. Cells were engineered to stably express firefly or renilla luciferase (fLuc or rLuc, respectively). Cells were either exposed to TTFields (50-500 kHz, 1-4 V/cm) or control conditions. Proliferation was assessed by bioluminescence imaging (BLI) and cell counting. Dextran-FITC binding and influx of 5-aminolevulinic acid (5-ALA) were also assessed. Scanning electron microscopy (SEM) studies were used to probe effects on cellular membranes. All experiments were performed in at least triplicate and 2-way ANOVA or univariate Mann-Whitney test was performed to compare the groups.
RESULTS TTFields significantly inhibited growth of cells (p≤0.02, no TTFields vs. TTFields). BLI suggested alterations in membrane configuration when cancer cells were exposed to TTFields. This was validated with observations of greater fluorescence of membrane-associating Dextran-FITC to U87-MG cells that were subjected to TTFields (p< 0.01, no TTFields vs. TTFields). In GBM39 cells, the optimal effect by TTFields on enhancing Dextran-FITC binding occurred in the range of 100-300 kHz (p<0.02, no TTFields vs. TTFields). TTFields also enhanced 5-ALA uptake into exposed GBM cells (p<0.001, no TTFields vs. TTFields). SEM revealed significantly greater and larger number of holes on the membrane surface of TTFields-exposed U87-MG cancer cells (53.5±19.1 holes per field of view and mean size=240.6±91.7 nm2) compared to unexposed cells (23.9±11.0 holes per field of view and mean size=129.8±31.9 nm2, p< 0.005: TTFields exposed vs. non-exposed). Morphologically, GBM cells unexposed to TTFields had matted and elongated projections from the cell membrane, which were replaced by short, bulbous, bleb-like structures upon TTFields exposure. All observed effects were reversed upon cessation of TTFields.
CONCLUSION The findings suggest a permeabilization of the GBM cell membrane upon exposure to TTFields. This may explain the previously observed synergy between TTFields and standard (e.g., temozolomide) and emerging (Withaferin A) therapies, whereby increased permeability on membranes confers greater accessibility to drugs. Such a strategy is thus a promising candidate for future clinical translation in glioblastoma.
Citation Format: Edwin Chang, Chirag Patel, Caroline J. Young, Thomas Anthony Flores, Lydia-Marie Joubert, Yitian Zeng, Robert Sinclair, Sanjiv Gambhir. Combining the glioblastoma cell membrane-permeabilizing effect of tumor treating fields with chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6258.