Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model

Abstract

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib's effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1-10 µM and 25 mg/kg) and in combination with doxil (10-100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell-cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a - 53% versus - 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib's ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

Keywords: Blood-tumor barrier; Doxil; Endothelial cells; Glioma; Ibrutinib.

Fig. 1

High BTK expression in glioma tumors and vasculature. High BTK expression seen in grade IV gliomas with highest expression in glioblastoma tumors as noted by CGGA (left) and TCGA (right) plotted data (a). Representative low and high BTK expression staining in glioblastoma TMA. Low power magnification of BTK expression seen in tumor cells and high magnification is BTK expression of endothelium. Red arrows denote vascular staining (b). High BTK expression scoring predominately seen in both tissue and vascular staining, with table delineating sole versus dual BTK expression, ****p < 0.001 (c)

Fig. 2

Ibrutinib disrupts brain endothelial integrity and inhibits ABC transporter function. Brain endothelial cell viability is not affected by ibrutinib treatment at varied doses (a). Dose-dependently, ibrutinib decreases brain endothelial cell–cell impedance, significantly 2 h after treatment seen with 5 and 10 µM with subsequent cell index plateau (*p < 0.05, **p < 0.005) (b). Bicellular junction protein ZO-1 and tricellular junction protein MarvelD2 was significantly decreased 2 h after 10 µM ibrutinib treatment (c). Junctional mRNA expression also decreased from 2 to 24 h after 10 µM treatment, without a washout period seen in tjp1 (ZO-1), MarvelD2 (tricellulin), Ocln (occludin), Cldn5 (claudin-5), Lsr (lipolysis stimulated lipoprotein receptor/angulin-1), and cldn3 (claudin-3) (d). High baseline BTK expression seen in both cytoplasm and nucleus of brain endothelial cells. Confirmatory immunostaining of ZO-1 expression demonstrated decreased tight junctional linear staining at 4 h, with rearrangement closer to baseline regarding adhesion expression by 24 h. (e). Silencing of BTK with siBTK results in decreased cell–cell impedance transiently and impaired tight junction gene expression (***p < 0.0005, ****p < 0.0001) (f, g). Ibrutinib dose-dependently inhibited Abcb1 function to increase rhodamine accumulation with higher FITC-H fluorescence measurement causing a shift of amplitude to the right, comparative to valspodar (ABCB1 inhibitor) treated cells (h). Monolayer endothelial cells treated with ibrutinib on transwells resulted in approximately 26% higher sodium fluorescein permeability compared with control treatment 24 h later (****p < 0.0001) (i)

Fig. 2

Ibrutinib disrupts brain endothelial integrity and inhibits ABC transporter function. Brain endothelial cell viability is not affected by ibrutinib treatment at varied doses (a). Dose-dependently, ibrutinib decreases brain endothelial cell–cell impedance, significantly 2 h after treatment seen with 5 and 10 µM with subsequent cell index plateau (*p < 0.05, **p < 0.005) (b). Bicellular junction protein ZO-1 and tricellular junction protein MarvelD2 was significantly decreased 2 h after 10 µM ibrutinib treatment (c). Junctional mRNA expression also decreased from 2 to 24 h after 10 µM treatment, without a washout period seen in tjp1 (ZO-1), MarvelD2 (tricellulin), Ocln (occludin), Cldn5 (claudin-5), Lsr (lipolysis stimulated lipoprotein receptor/angulin-1), and cldn3 (claudin-3) (d). High baseline BTK expression seen in both cytoplasm and nucleus of brain endothelial cells. Confirmatory immunostaining of ZO-1 expression demonstrated decreased tight junctional linear staining at 4 h, with rearrangement closer to baseline regarding adhesion expression by 24 h. (e). Silencing of BTK with siBTK results in decreased cell–cell impedance transiently and impaired tight junction gene expression (***p < 0.0005, ****p < 0.0001) (f, g). Ibrutinib dose-dependently inhibited Abcb1 function to increase rhodamine accumulation with higher FITC-H fluorescence measurement causing a shift of amplitude to the right, comparative to valspodar (ABCB1 inhibitor) treated cells (h). Monolayer endothelial cells treated with ibrutinib on transwells resulted in approximately 26% higher sodium fluorescein permeability compared with control treatment 24 h later (****p < 0.0001) (i)

Fig. 2

Ibrutinib disrupts brain endothelial integrity and inhibits ABC transporter function. Brain endothelial cell viability is not affected by ibrutinib treatment at varied doses (a). Dose-dependently, ibrutinib decreases brain endothelial cell–cell impedance, significantly 2 h after treatment seen with 5 and 10 µM with subsequent cell index plateau (*p < 0.05, **p < 0.005) (b). Bicellular junction protein ZO-1 and tricellular junction protein MarvelD2 was significantly decreased 2 h after 10 µM ibrutinib treatment (c). Junctional mRNA expression also decreased from 2 to 24 h after 10 µM treatment, without a washout period seen in tjp1 (ZO-1), MarvelD2 (tricellulin), Ocln (occludin), Cldn5 (claudin-5), Lsr (lipolysis stimulated lipoprotein receptor/angulin-1), and cldn3 (claudin-3) (d). High baseline BTK expression seen in both cytoplasm and nucleus of brain endothelial cells. Confirmatory immunostaining of ZO-1 expression demonstrated decreased tight junctional linear staining at 4 h, with rearrangement closer to baseline regarding adhesion expression by 24 h. (e). Silencing of BTK with siBTK results in decreased cell–cell impedance transiently and impaired tight junction gene expression (***p < 0.0005, ****p < 0.0001) (f, g). Ibrutinib dose-dependently inhibited Abcb1 function to increase rhodamine accumulation with higher FITC-H fluorescence measurement causing a shift of amplitude to the right, comparative to valspodar (ABCB1 inhibitor) treated cells (h). Monolayer endothelial cells treated with ibrutinib on transwells resulted in approximately 26% higher sodium fluorescein permeability compared with control treatment 24 h later (****p < 0.0001) (i)

Fig. 3

Ibrutinib hinders glioma cell migration and Abcb1 efflux and viability in combination with doxil. Varied ibrutinib dosing does not influence S635 rat glioma cell viability after 24 h (a). Glioma cell migration was influenced greatest by 10 µM ibrutinib 36–48 h after treatment compared with control and 1 µM therapy as evidenced by decreased cell migration to serum containing fetal bovine-serum (*p < 0.05) (b). Rhodamine efflux as a measure of Abcb1 function demonstrates both 10 µM ibrutinib effectively decreased efflux akin to known inhibitor valspodar (c). Combination therapy of ibrutinib with doxil found dose-dependent cooperation to hinder cell viability after 48 h exposure to therapy (***p < 0.001****p < 0.0001) (d). Combined ibrutinib (10 µM) and doxil (10 and 100 µM) resulted in increased caspase3/7 apoptosis activity compared with single therapy (****p < 0.0001) (e)

Fig. 4

Combination ibrutinib with doxil impairs glioma model growth and prolongs survival. Treatment schema denoting repeat drug therapies, image timing, doxil blood/tissue concentrations and survival studies (a). Repeat ibrutinib with doxil therapy does no influence doxil plasma concentrations yet increases brain tumor doxil concentrations by approximately 32% (*p < 0.05) (b). Brain MRIs reflect additive ibrutinib effect of ibrutinib compared to vehicle (left panel) doxil alone (left middle panel), ibrutinib alone (right middle panel) or ibrutinib + doxil (right panel) as seen by statistically significant volumetric decrease as denoted via graphed values (*p < 0.05) (c). H/E staining demonstrates the lessened tumor size with doxil treatment alone and with combination therapy (d). 3kD dextran extravasation studies revealed ibrutinib can increase the blood-tumor barrier to larger agents, specifically within the peritumoral tissue region (*p < 0.01) (e). Co-staining of CD31/Claudin-5 on tumor, peritumoral and distant sites areas did not demonstrate any statistical differences in co-localized expression. (f). Prolonged survival seen with combination therapy with a median survival of 27 days versus 16 days for control therapy (****p < 0.0001) (*p < 0.05) (g)

Fig. 4

Combination ibrutinib with doxil impairs glioma model growth and prolongs survival. Treatment schema denoting repeat drug therapies, image timing, doxil blood/tissue concentrations and survival studies (a). Repeat ibrutinib with doxil therapy does no influence doxil plasma concentrations yet increases brain tumor doxil concentrations by approximately 32% (*p < 0.05) (b). Brain MRIs reflect additive ibrutinib effect of ibrutinib compared to vehicle (left panel) doxil alone (left middle panel), ibrutinib alone (right middle panel) or ibrutinib + doxil (right panel) as seen by statistically significant volumetric decrease as denoted via graphed values (*p < 0.05) (c). H/E staining demonstrates the lessened tumor size with doxil treatment alone and with combination therapy (d). 3kD dextran extravasation studies revealed ibrutinib can increase the blood-tumor barrier to larger agents, specifically within the peritumoral tissue region (*p < 0.01) (e). Co-staining of CD31/Claudin-5 on tumor, peritumoral and distant sites areas did not demonstrate any statistical differences in co-localized expression. (f). Prolonged survival seen with combination therapy with a median survival of 27 days versus 16 days for control therapy (****p < 0.0001) (*p < 0.05) (g)

Fig. 4

Combination ibrutinib with doxil impairs glioma model growth and prolongs survival. Treatment schema denoting repeat drug therapies, image timing, doxil blood/tissue concentrations and survival studies (a). Repeat ibrutinib with doxil therapy does no influence doxil plasma concentrations yet increases brain tumor doxil concentrations by approximately 32% (*p < 0.05) (b). Brain MRIs reflect additive ibrutinib effect of ibrutinib compared to vehicle (left panel) doxil alone (left middle panel), ibrutinib alone (right middle panel) or ibrutinib + doxil (right panel) as seen by statistically significant volumetric decrease as denoted via graphed values (*p < 0.05) (c). H/E staining demonstrates the lessened tumor size with doxil treatment alone and with combination therapy (d). 3kD dextran extravasation studies revealed ibrutinib can increase the blood-tumor barrier to larger agents, specifically within the peritumoral tissue region (*p < 0.01) (e). Co-staining of CD31/Claudin-5 on tumor, peritumoral and distant sites areas did not demonstrate any statistical differences in co-localized expression. (f). Prolonged survival seen with combination therapy with a median survival of 27 days versus 16 days for control therapy (****p < 0.0001) (*p < 0.05) (g)

Fig. 5

Ibrutinib aids doxil entry to hinder glioma progression. An intact blood–brain barrier limits doxil permeability through brain endothelial tight junction integrity and ABCB1 efflux activity. In the context of the blood-tumor barrier treated with ibrutinib, junctional expression is decreased, ABCB1 function is inhibited and doxil therapy entry is enhanced to delay glioma migration and growth for prolonged survival. Images created via Biorender