P10.01.A Reversible blood-brain barrier (BBB) disruption by Tumor Treating Fields (TTFields) in a human 3Din vitro model

Abstract

Abstract Background Drug delivery to the central nervous system (CNS) is often impeded by the restrictive nature of the blood brain barrier (BBB). Since many therapeutic molecules are not able to traverse this barrier, the development of new methods to disrupt the BBB is of paramount importance. Tumor Treating Fields (TTFields) are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-300 kHz), which are approved and effective for the treatment of glioblastoma at a frequency of 200 kHz. We recently demonstrated that TTFields at lower frequencies are able to transiently induce BBB permeability in in vitro and in vivo murine models. Here, we explored whether the transient opening of the BBB by TTFields in our murine systems also translates to a human cell-based 3D model. Material and Methods A three-dimensional BBB model was established by co-culturing primary human brain microvascular endothelial cells (HBMVEC) on a transwell insert together with human pericytes on the bottom of a well-plate. The model was treated with TTFields at 100-300 kHz for 2496 h using the inovitro™ TTFields Lab Bench System (Novocure®). Afterwards, the cells recovered for 24-96 h. In order to analyze the effects of TTFields on barrier integrity and compromise, transendothelial electrical resistance (TEER) of the HBMVEC monolayer was measured before the start of TTFields treatment, immediately after TTFields cessation, as well as 24-96 h after TTFields treatment. Permeability of the barrier was assessed by visualizing the movement of FITC-dextran through the HBMVEC monolayer. In addition, changes in expression and localization of the tight junction protein (TJP) claudin-5 (Cl-5) after application of TTFields were analyzed by fractionated Western-blotting and immunofluorescence (IF) staining, respectively. Results Application of TTFields at all investigated frequencies significantly decreased TEER across the HBMVEC monolayer after as early as 24 h, with the strongest effects seen after 72 h at a TTFields frequency of 100 kHz. TTFields treatment delocalized TJP Cl5 from the cell boundaries to the cytoplasm as evidenced by Western-blots and IF stainings. Restoration of the cell barrier was already measurable as early as 24 h after TTFields cessation and a complete recovery was evident after 48 h. Conclusion These results in a human 3D in vitro model confirm our previous observations from mouse models that TTFields could transiently open the BBB. These fundamental pre-clinical data demonstrate the feasibility of facilitating drug delivery to the CNS via concomitant application of TTFields. This method opens up the prospect of improved drug-based treatment of devastating CNS diseases such as GBM if these results could be translated to the clinical setting in the future.