Tumor Treating Fields (TTFields) Reversibly Permeabilize the Blood–Brain Barrier In Vitro and In Vivo

Abstract

Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood–brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB’s integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100–300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.

Tumor Treating Fields (TTFields) Induce Cell Junction Alterations in a Human 3D In Vitro Model of the Blood-Brain Barrier

“…After treatment with 100 kHz TTFields for 72 h, a slight alteration in the distribution and localization of claudin-5 was observed via immunofluorescence staining (Figure 1A). However, compared to what we previously observed in cerebEND, which in itself showed a distinct staining of claudin-5 and a notable delocalization from the cell borders to the cytoplasm [22], distinct staining of claudin-5 in HBMVEC was inferior for both TTFields-treated and untreated conditions. Moreover, comparing HBMVEC and cerebEND using Western blots demonstrated a lower level of claudin-5 expression in the latter (Figure 1B).…”

“…Similarly, as in the case of cerebEND cells [ 22 ], we initially examined the effects of TTFields on HBMVEC as a monoculture. After treatment with 100 kHz TTFields for 72 h, a slight alteration in the distribution and localization of claudin-5 was observed via immunofluorescence staining ( Figure 1 A).…”

“…The cells were set together in high ceramic dishes (Novocure ® , Haifa, Israel) after individual culture in a 37 °C incubator Forma™ Steri-Cult™ 200 (Thermo Fisher Scientific), as described above. Next, the co-cultures were subjected to TTFields application (100–300 kHz frequency) using the inovitro™ TTFields Lab Bench System (Novocure ® ) for 24–96 h, as described previously [ 22 ]. Additionally, following treatment, cells were allowed to recover in the 37 °C incubator for 24–96 h.…”

“…The expression of the TJ proteins claudin-5, ZO-1, occludin, and PECAM-1 was assessed via immunofluorescence staining and Western blotting as described previously [ 22 , 27 ].…”

“…Recently, our report on Tumor Treating Fields (TTFields), an FDA-approved treatment modality for glioblastoma [ 18 , 19 , 20 ] and mesothelioma [ 21 ], stated that there could be an increase in the permeability of the BBB in vitro and in vivo, an effect regulated by a Rho kinase-mediated claudin-5 phosphorylation pathway [ 22 ]. TTFields are electric fields of low intensity (1–3 V/cm) and intermediate frequency (100–500 kHz).…”