Changing Bood-brain Tumor Barrier Permeability For Enhanced Anti-glioma Therapy

BackgroundMalignant glioma has a high recurrence and mortality rate.Current strategy contains surgical debulking,radiation therapy and chemotherapy.Despite the increase in therapy methods,the median survival of patients with glioblastoma multiforme(GBM)remains 12-14 months.GBM is associated with high levels of angiogenesis and has increased expression of angiogenic factors and irregular extensive vascular proliferation.Vascular endothelial growth factor(VEGF)is the central factor in regulating GBM neovascularization.High VEGF expression could cause proliferation and growth of endothelial cells,which is associated with hypoxia and necrosis in glioma.And these factors can promote tumor angiogenesis and progression.Antiangiogenic therapy is the focus of glioma therapeutic development in recent years.Strategies that inhibit VEGF have been explored the most.Bevacizumab is a humanized monoclonal antibody and it could specifically bind to the VEGF-A isoform.Although bevacizumab was initially approved by the FDA for treatment of metastatic colorectal cancer,its potential for widespread use in other tumor types still need further investigation.Bevacizumab have been approved for use in recurrent and newly diagnosed GBM and promising results have been obtained with the treatment of brain tumor patients.However,bevacizumab could reduce vascular permeability and temporally reverse abnormal capillary leakage.The transient vascular normalization restricts further drugs penetration into brain parenchyma and reduces therapy effect on glioma.Blood-brain barrier(BBB)is a major limitation to the use of drugs in the brain.Some studies show that nearly 100% of macromolecular and 98% of micromolecular drugs cannot pass through the BBB.This limitation affects the development of effective drugs for many brain diseases.Studies show that the glioma vasculature permeability is heterogeneous,and there are barriers to drug therapy,such as edema and increased interstitial pressures.Furthermore,although the blood vessels in glioma often do not have a fully intact BBB and are somewhat permeable,the feeding capillaries reserve the characteristics of the BBB and form a blood-brain tumor barrier(BTB).BTB is similar to BBB,which is located between tumor cells and microvessels formed by highly specialized endothelial cells.Although the permeability of BTB is higher than that of BBB,the existence of a BTB obviously limits the delivery of drugs into glioma.Techniques that can help drugs pass across the BTB could enable the use of many anti-tumor agents in glioma therapy.Previous studies have demonstrated that microbubbles increase the possibility of acoustic cavitation and cavitation-related vascular damage effects,such as microvascular rupture and petechial hemorrhage,in which microbubbles served as cavitation nuclei to induce cavitation.Recently,some studies showed that the permeability of the BTB is increased by focused ultrasound and low-frequency therapeutic ultrasound in the presence of an ultrasound contrast agent.This technique non-invasively enhances the uptake of drugs or genes to the targeted glioma tissues.The above effect is reversible,allowing for a time window for drug delivery to the target site of the brain tumor.Therefore,in this study,we applied diagnostic ultrasound with microbubbles to increase the BTB permeability in order to enhance the delivery of bevacizumab into the central nervous system for glioma treatment.And the enhanced central nervous system bevacizumab penetration may confer benefit in overcoming the tumor vascular “normalization” encountered in glioma treatment.Objectives1.To explore the effect of microbubble-enhanced diagnostic ultrasound(MEUS)on the BTB permeability and the possible mechanism.2.To demonstrate that microbubble-enhanced diagnostic ultrasound can enhance the delivery of bevacizumab into the central nervous system for glioma treatment.3.To investigate the changes of MRI parameters in glioma after bevacizumab treatment and to explore possible pathological mechanism to these changes.Materials and Methods1.A study on the effect of microbubble-enhanced diagnostic ultrasound on the BTB permeability and the possible mechanismGlioma-bearing rats were randomized into three groups as follows: the microbubble-enhanced continued diagnostic ultrasound(MECUS)group;the microbubble-enhanced intermittent diagnostic ultrasound(MEIUS)group and the control group.The gliomas were insonicated through the skull with a diagnostic ultrasound and injected with microbubbles through the tail veins.Evans Blue(EB)and dynamic contrast-enhanced-MRI were used to test changes in the BTB permeability.Confocal laser scanning microscopy was used to observe the deposition of the EB in the tumor tissues.The distribution and expression of junctional adhesion molecule-A(JAM-A)and calcium-activated potassium channels(KCa channels)were detected by a Western blot,qRT-PCR and immunohistochemical staining.Hematoxylin and eosin(HE)and TUNEL staining were adopted to evaluate whether MEUS could cause damage to normal brain tissue.2.Microbubble-enhanced diagnostic ultrasound enhanced the delivery of bevacizumab into the central nervous system for glioma treatmentGlioma-bearing mice were randomized into four groups as follows: the microbubble-enhanced diagnostic ultrasound and bevacizumab group(MEUS+BEV group);the bevacizumab group(BEV group);the microbubble-enhanced diagnostic ultrasound group(MEUS group)and the control group.The gliomas were insonicated through the skull with a diagnostic ultrasound and injected with microbubbles through the tail veins.One hour later,bevacizumab was injected through the tail veins.Then enzyme linked immunosorbent assay(ELISA)were adopted to measure bevacizumab concentration in the tumor.CD34 staining was adopted to measure microvessel density(MVD)in the tumor after 3 days.Other forty-two nude mice intracranial gliomas were included.Bevacizumab(15 mg/kg)was injected via the tail vein of mice.Various MRI parameters(T2WI,apparent diffusion coefficient [ADC],T1w+contrast,dynamic contrast-enhanced-MRI [DCE-MRI])were acquired before and after treatment.The distribution and expression of Ki67,claudin-5,occludin and CD34 were detected by immunohistochemical staining.Tight junction changes of blood brain barrier were observed.Neovascularization pattern(intussusceptive microvascular growth(IMG))in tumor was observed after treatment.Spearman correlation analysis was used to compare the correlation between Ktrans value acquired from DCE-MRI and IMG,T1+contrast and tight junction proteins expression,ADC value and Ki67 positive cells.Results1.A study on the effect of microbubble-enhanced diagnostic ultrasound on the BTB permeability and the possible mechanismIn the MEUS groups,the EB extravasation(11.0 ± 2.2 ?g/g in MECUS group and 17.9 ± 2.3 ?g/g in MEIUS group)exhibited a significant increase compared with the control group(5.3 ± 0.9 ?g/g).The MEIUS group had more EB extravasation than the MECUS group.The Ktrans value in the MEUS groups was higher than that of the control group and correlated strongly with the EB extravasation in the tumor(R2=0.97).Western blot,q RT-PCR and immunohistochemical staining revealed that MEUS increased the KCa channels expression and reduced JAM-A expression in glioma.This change was more obvious in the MEIUS group than in the MECUS group.2.Microbubble-enhanced diagnostic ultrasound enhanced the delivery of bevacizumab into the central nervous system for glioma treatmentELISA showed that in the MEUS+BEV group,the bevacizumab concentration(88.8 ± 18 ?g/g)exhibited a significant increase compared with the BEV group(42.2 ± 15 ?g/g).And after 3 days,in MEUS+BEV group,MVD in tumor decreased more significantly compared with the BEV group.MRI results showed bevacizumab reduced tumor growth rate and suppressed tumor cell proliferation.Normalized ADC value was negatively correlated with the number of Ki67 positive cells in the tumor.After bevacizumab treatment,contrast enhancement on T1w+contrast and the Ktrans value showed a significant reduction in tumor.Transmission electron microscope showed tight junction was partially regained.Meanwhile,claudin-5 and occludin expression increased.In bevacizumab group,the number of intussusceptive microvascular growth in glioma was higher than that in control group.Ktrans value was negatively correlated with intussusceptive microvascular growth and normalized contrast enhancement value was negatively correlated with claudin-5 and occludin expression in the tumor.Conclusion1.MEUS increased the BTB permeability in a rat glioma model without causing damage to the normal brain tissue,at least,immediately after the procedure.The mechanism might involve affecting formation of tight junctions by reducing JAM-A expression and promoting pinocytosis by increasing KCa channels expression.Additionally,the Ktrans value could be a noninvasive biomarker to evaluate the BTB permeability in rat glioma after a MEUS treatment.These findings might provide some new guidance for glioma therapy.2.MEUS can enhance the delivery of bevacizumab into the central nervous system for glioma treatment.Bevacizumab reduced tumor growth rate and suppressed tumor cell proliferation.And bevacizumab effectively reduced the permeability of blood-brain tumor barrer in tumor.The mechanisms might involve neovascularization pattern change and reformation of tight junction.Additionally,ADC,Ktrans and T1 w +contrast might be noninvasive biomarkers to predict cell proliferation,neovascularization pattern and tight junction change in a U87 glioma model.