Abstract 2713: Modulation of the blood-tumor barrier by 6-bromoindirubin acetoxime increases intra-tumoral drug accumulation and extends survival in murine glioblastoma models
Authors: Macias Jorge L. Jimenez 
Published: April 2023

Abstract
Introduction: Glioblastoma (GBM) is the most frequent primary malignant adult brain tumor. Median survival is ~15 months after diagnosis. Therapies to effectively manage glioblastoma are hindered due to the presence of the blood-tumor barrier (BTB). Indirubins are 3,2’-bisindole compounds of the Indigo naturalis used in traditional Chinese medicine to treat proliferative diseases. Herein, we demonstrate that systemic delivery of 6-bromoindirubin-3’-acetoxime (BIA), an indirubin derivative, increases cisplatin delivery to GBM tumors through alteration of the BTB integrity and synergy of BIA with platinum therapeutics to potentiate anti-tumor killing.
Methods: Pre-clinical studies involved G9-pCDH glioblastoma cell line implantation in naïve nude mice. Cisplatin uptake 24 hours after BIA tail-vein administration into healthy brain and GBM murine tumors was assessed via inductively-coupled plasma mass spectrometry (ICP-MS). DNA-damage was assessed by γH2AX immunofluorescence. BIA combination with cisplatin in vitro studies were performed via luminescent viability assays. Treatment of human cerebrovascular endothelial cells (HCMEC/D3) with BIA for BBB studies in vitro involved trans-endothelial electrical resistance (TEER) measurement, transcriptome analysis, VE-cadherin western blotting and immunofluorescence.
Results: Combined addition of cisplatin and BIA showed enhanced anti-GBM killing effects than cisplatin alone in G9-pCDH (IC50 cisplatin=1, BIA+cisplatin=0.2), G34-pCDH (IC50 cisplatin=3, BIA+cisplatin=1.7) and G30-LRP (IC50 cisplatin=1, BIA+cisplatin=0.3) lines. Efficacy studies using the G9-pCDH xenograft murine model showed extended survival when using cisplatin in combination with BIA (median 27d, p=0.0016) when compared to cisplatin (18.5d), BIA (21.5d) and control (18d). Increased intra-tumoral accumulation of cisplatin was observed when used in combination with BIA (mean 100 pg/mg, p=0.012) rather than cisplatin alone (5 pg/mg). No increased platinum uptake was seen in healthy brain tissue under any conditions. γH2AX foci in tumor were more frequent in the BIA and cisplatin combination arm (p=0.0086) than each single agent and control groups. Evaluation of HCMEC/D3 cells treated with BIA showed a decrease in TEER levels (p&lt;0.001) and transcriptomic dysregulation of members of the WNT pathway and downregulation of VE-cadherin (~4-fold). BIA exerted cytoskeleton changes in HCMEC/D3 cells and decreased VE-cadherin expression at the protein level and its distribution on cellular surface, suggesting loss of adherens junctions as a potential mechanism for BIA-induced permeability of the BTB.
Conclusion: Overall, our data support the applicability of BIA for modulating the BTB permeability and increasing intra-tumoral drug accumulation for improving GBM therapeutic efficacy.
Citation Format: Jorge L. Jimenez Macias. Modulation of the blood-tumor barrier by 6-bromoindirubin acetoxime increases intra-tumoral drug accumulation and extends survival in murine glioblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2713.