LAUSR

The highly restrictive properties of the blood-brain barrier (BBB) pose a critical hurdle in combatting brain cancer. Tumor Treating Fields (TTFields) are a non-invasive treatment modality comprised of alternating electric fields of intermediate frequency (100-500 kHz) and low intensity (1-3 V/cm) that are CE-approved for WHO grade 4 glioma, and FDA-approved for the treatment of glioblastoma. At 100 kHz, TTFields loosen the BBB by reversibly delocalizing various tight junction and associated proteins in vascular brain endothelial cells, allowing drug delivery to peripheral regions of the brain. We investigated the effects of TTFields at 100 kHz and 170 kHz on a 3D human cell-based BBB in vitro model with the goal of improving on the outcomes already demonstrated at 100 kHz. The in vitro BBB model combines human brain microvascular endothelial cells (HBMVEC) grown on glass coverslips and immortalized pericytes in transwell inserts, or vice versa, two crucial cell types responsible for BBB integrity. Cells were treated with TTFields at either 100 kHz or 170 kHz for 72 h, whereas untreated cells served as controls. Change in BBB permeability was assessed by fluorescein permeation assays with dextran molecular weights of 4 kDa, 40 kDa and 70 kDa immediately before and after 72 h of TTFields administration. Effects on a molecular level were investigated by immunofluorescence staining of junctional proteins ZO-1, PECAM-1 and Claudin-5 in HBMVEC after 72 h of treatment with either frequency and their expression was monitored by Western-blotting. Permeability assay of various fluorescein molecules demonstrated consistent increase in HBMVEC permeability among all treated groups compared to untreated cells for molecular weights of 4 kDa and 40 kDa with a significant increase from 100 kHz to 170 kHz, as well as significant increase in permeability for 70 kDa after treatment at 170 kHz. Staining of junctional proteins ZO-1, PECAM-1 and Claudin-5 revealed delocalization from the cell membrane to the cytoplasm in all treated samples, while Western-blot proved that their expression did not change in relation to untreated controls. TTFields with a frequency of 170 kHz showed consistent or improved effects on BBB permeabilization over 100 kHz by rearranging tight junction proteins in HBMVEC in vitro, without impairing tight junction protein expression.