DDEL-18. OPTIMIZED METHOD TO QUANTIFY TTFIELDS-INDUCED PERMEABILIZATION OF CELL MEMBRANES

Abstract Tumor Treating Fields (TTFields) therapy is FDA-approved for glioblastoma. Using plate-based readouts of FITC-dextran fluorescent probes in U87 human glioblastoma cells, we previously reported increased uptake of FITC-dextrans (4-20kDa) post-TTFields exposure. However, this approach is unable to distinguish FITC-dextran uptake in live-vs-dead cells post-TTFields exposure. We demonstrate flow cytometry (FC) as a more robust and economical method to quantify the amount of FITC-dextrans entering the cells. We first identified the optimal [FITC-dextran] for the FC-based approach (titration 0-6.5mg/mL). Cells were then seeded onto coverslips and exposed to 200kHz TTFields, 1-3V/cm peak-to-peak (inovitro systemTM, Novocure), or no-TTFields (control), for 24-72h. Cells were harvested and stained for 1h using 4kDa or 20kDa FITC-dextrans. Cells were washed and analyzed by FC for mean±SD fluorescence intensity, and compared by unpaired t-test. Dead cells were excluded using propidium iodide staining. In the control vs. TTFields group, 4kDa FITC-dextrans had significantly increased uptake at 24h (4.9±0.3 vs. 8.0±1.7, p=0.01), 48h (6.5±0.2 vs. 8.3±1.0, p=0.01), and 72h (5.7±0.3 vs. 8.8±1.0, p=0.001). Likewise, in the TTFields group, the 20kDa FITC-dextrans had significantly increased uptake at 24h (5.6±0.3 vs. 7.5±0.9, p=0.006), 48h (4.8±0.3 vs. 6.6±0.8, p=0.006), and 72h (5.3±0.2 vs. 11.3±2.6, p=0.004). In the TTFields group, the 20kDa FITC-dextrans had significantly increased uptake between the 24h and 72h timepoints (p=0.03). The FC-based method utilized 0.36mg of FITC-dextrans/sample, much less compared to the 1-3mg recommended for plate-based methods. Another advantage of the FC-based method was exclusion of the dead cell population after TTFields exposure. We validated a FC-based-method that is economical and can reproducibly quantify the membrane permeabilization induced by TTFields. Compared to control conditions, TTFields increase membrane permeabilization of cells that remain alive after TTFields exposure, in a time-dependent manner for the 20kDa probe.