Introduction

Glioblastoma (GBM) is the most common and deadliest of malignant primary brain tumors affecting humans. Extensive local invasion by the primary tumor and its location behind the blood brain barrier (BBB) prevents adequate delivery of most chemotherapy agents. H-FIRE represents a novel non-thermal ablation method for safe and potentially effective treatment of GBM. H-FIRE transiently disrupts the BBB, allowing for electrochemoablation of microscopic tumor cells located within the tumor penumbra. Here, we characterize the mechanisms of H-FIRE mediated BBB disruption (BBBD) in normal rat brain.

Methods

Intracranial H-FIRE was delivered to Fischer rats prior to sacrifice at predetermined time points. Western blotting and immunoprecipitation were performed on brain lysates to detect tight junction (TJ) proteins in their native and ubiquitinated forms, and cytoskeletal proteins. TJ proteins were also evaluated with immunofluorescence. RNA isolates from regions of BBBD were pooled for gene expression and pathway analysis, completed using commercially available SuperArray plates containing genes associated with TJs. Data was analyzed using Qiagen Ingenuity Pathway Analysis (IPA) software. Significant results were confirmed using qRT-PCR.

Results

TJ protein expression decreased following H-FIRE, with gradual recovery over time. Increased ubiquitination of claudin-5 and occludin was apparent 1–48 hours post-H-FIRE compared to controls. F-actin/total actin and F/G actin ratios were significantly decreased 1-hour post-H-FIRE compared to all other treatment groups. An increase in ZO-1 and claudin-5 gene expression was observed over time relative to controls, consistent with BBB recovery. Relative expression of occludin was decreased at all time points. IPA analysis revealed significant dysregulation of claudin genes, centered around claudin-6 and peaking at 72 hours post-H-FIRE, which was further supported by qRT- PCR.

Conclusion

In conclusion, H-FIRE transiently permeates the BBB via cytoskeletal remodeling, altered TJ gene expression and subsequent protein assembly, and increased TJ protein degradation.

Funding Information

Internal Research Competition Grant
RO1CA213423, NIH/NCI
PO1CA207206, NIH/NCI