Ferguson Dominique, Kwak Minhye, Lim Sanghee, Cesaire Melissa, Mills Jatia, Dalmage Mahalia, Jones Jane, Tarasov Sergey, Dyba Marzena, Robey Rob, Yang Yanbo, Simpson Shae K, Karim Baktiar, Butcher Donna, Gartrell Robyn, Gottesman Michael, Jackson Sadhana
Surgical Neurology Branch, National Institute of Neurologic Disorders and Stroke, Bethesda, MD.
CCR Protein Expression Laboratory, Frederick, MD.
bioRxiv. 2025 Aug 2:2025.07.31.667901. doi: 10.1101/2025.07.31.667901.
The blood-tumor barrier (BTB) prevents effective central nervous system (CNS) drug delivery, especially in malignant gliomas. Brain endothelium predominates the BTB and connects through bicellular and tricellular tight junctions (TJ). Angulin-1/LSR, is a highly expressed endothelial tricellular TJ. Our studies explore the role of Angubindin-1, an Angulin-1/LSR binder, to disrupt tricellular TJ integrity, increase drug entry and hamper glioma progression. Using rat brain endothelial cells (RBMVEC) we tracked Angulin-1/LSR localization and expression to the membrane; binding tightest to Angubindin-1 2-8 hours post-treatment ( < 0.05). Angubindin-1 dose-dependently reduced bicellular and tricellular TJs 1-4 hours post treatment ( < 0.05), returning to baseline by 24 hours ( < 0.05). In human and rat-derived glioma cells, Angubindin-1 transiently reduced Angulin-1/LSR expression between 2-8 hours ( < 0.05), with return to baseline by 24 hours ( < 0.001). Silenced Angulin-1/LSR expression on endothelium resulted in decreased mRNA levels of bicellular (occludin, claudin-5, ZO-1) and tricellular (tricellulin/MARVELD2, angulin-1/LSR) TJs compared to control ( < 0.01). Angubindin-1 treatment also inhibited efflux transporter P-gp in both RBMVECs and glioma cells with high P-gp expression only. Orthotopic rat glioma models were treated with Doxil (3 mg/kg), Angubindin-1 (10 mg/kg), or combination to evaluate BTB permeability/drug accumulation, and overall survival. Combination therapy enhanced Doxil tumor accumulation by 20% ( < 0.001), reduced tumor volume by day 14 (77.5% vs. 81.6%, < 0.05), and significantly extended survival compared to Doxil alone (24 days vs. 18 days, < 0.0001). These findings demonstrate the effects of tricellular tight junction inhibition on disrupting the BTB, enhancing CNS drug delivery, and improving rodent glioma survival.
血脑肿瘤屏障（BTB）阻碍了中枢神经系统（CNS）药物的有效递送，尤其是在恶性胶质瘤中。脑内皮细胞在BTB中占主导地位，并通过双细胞和三细胞紧密连接（TJ）相连。血管生成素-1/LSR是一种在内皮细胞三细胞紧密连接中高度表达的蛋白。我们的研究探讨了血管生成素-1结合蛋白-1（一种血管生成素-1/LSR结合剂）在破坏三细胞紧密连接完整性、增加药物进入以及抑制胶质瘤进展方面的作用。我们使用大鼠脑内皮细胞（RBMVEC）追踪血管生成素-1/LSR在细胞膜上的定位和表达；在处理后2 - 8小时与血管生成素-1结合蛋白-1的结合最为紧密（<0.05）。血管生成素-1结合蛋白-1在处理后1 - 4小时剂量依赖性地减少双细胞和三细胞紧密连接（<0.05），到24小时恢复至基线水平（<0.05）。在人源和大鼠源胶质瘤细胞中，血管生成素-1结合蛋白-1在2 - 8小时内短暂降低血管生成素-1/LSR的表达（<0.05），到24小时恢复至基线水平（<0.001）。与对照组相比，内皮细胞中血管生成素-1/LSR表达沉默导致双细胞紧密连接（闭合蛋白、claudin - 5、ZO - 1）和三细胞紧密连接（三细胞ulin/MARVELD2、血管生成素-1/LSR）的mRNA水平降低（<0.01）。血管生成素-1结合蛋白-1处理还仅在RBMVEC和具有高P - gp表达的胶质瘤细胞中抑制了外排转运蛋白P - gp。用阿霉素（3 mg/kg）、血管生成素-1结合蛋白-1（10 mg/kg）或联合用药处理原位大鼠胶质瘤模型，以评估BTB通透性/药物蓄积以及总体生存期。联合治疗使阿霉素在肿瘤中的蓄积增加了20%（<0.001），到第14天时肿瘤体积减小（77.5%对81.6%，<0.05），与单独使用阿霉素相比显著延长了生存期（24天对18天，<0.0001）。这些发现证明了三细胞紧密连接抑制在破坏血脑肿瘤屏障、增强中枢神经系统药物递送以及改善啮齿动物胶质瘤生存期方面的作用。
