The Blood-Brain Barrier at Sites of Metastasis
The Blood-Brain Barrier at Sites of Metastasis
Background Effective chemotherapeutics for primary systemic tumors have limited access to brain metastases because of the blood–brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilizing the BBB at sites of cerebral metastases.
Methods BALB/c mice were injected intracardially to induce brain metastases. After metastasis induction, either tumor necrosis factor (TNF) or lymphotoxin (LT) was administered intravenously, and 2 to 24 hours later gadolinium- diethylenetriaminepentaacetic acid, horseradish peroxidase, or radiolabeled trastuzumab (In-BnDTPA-Tz) was injected intravenously. BBB permeability was assessed in vivo using gadolinium-enhanced T1-weighted magnetic resonance imaging and confirmed histochemically. Brain uptake of In-BnDTPA-Tz was determined using in vivo single photon emission computed tomography/computed tomography. Endothelial expression of TNF receptors was determined immunohistochemically in both mouse and human brain tissue containing metastases. Group differences were analyzed with one-way analysis of variance followed by post hoc tests, Wilcoxon signed rank test, and Kruskal–Wallis with Dunn's multiple comparison test. All statistical tests were two-sided.
Results Localized expression of TNF receptor 1 (TNFR1) was evident on the vascular endothelium associated with brain metastases. Administration of TNF or LT permeabilized the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect at 6 hours. Metastasis-specific uptake ratio of In-BnDTPA-Tz was also demonstrated after systemic TNF administration vs control (0.147±0.066 vs 0.001±0.001). Human brain metastases displayed a similar TNF receptor profile compared with the mouse model, with predominantly vascular TNFR1 expression.
Conclusions These findings describe a new approach to selectively permeabilize the BBB at sites of brain metastases to aid in detection of micrometastases and facilitate tumor-specific access of chemotherapeutic agents. We hypothesize that this permeabilization works primarily though TNFR1 activation and has the potential for clinical translation.
Brain metastases pose a substantial challenge for chemotherapeutic treatment because of the impermeable nature of the blood–brain barrier (BBB), which limits access of drugs and thus prevents accumulation of clinically effective doses at tumor sites. Substances with good penetration of the BBB have limited activity against breast cancer, one of the most common cancers metastasizing to the brain, whereas the most active therapeutics for breast cancer (including doxorubicin and trastuzumab) appear not to reach the central nervous system (CNS) because of their hydrophilic nature. At the same time, the impermeable BBB also prevents early diagnosis of small brain metastases by the current clinical gold-standard method of gadolinium-enhanced magnetic resonance imaging (MRI). This diagnostic approach enables detection of large cerebral metastases and primary brain tumors but only when gross structural abnormalities have developed. Moreover, although BBB compromise may allow limited access of drugs to the tumor in later stages, this BBB permeability is frequently inhomogenous and generally poor around the tumor margin. Thus, the late stage of BBB disruption and inhomogeneity across the tumor mean treatment is largely ineffective. Smaller metastases possessing an intact BBB evade both detection and treatment and will inevitably develop into symptomatic tumors.
A number of approaches to transiently circumvent the BBB have been investigated for the delivery of chemotherapeutics to brain tumors [for a review see ]. Bradykinin B2 receptor activation by cereport (RPM-7) was the first pharmacological treatment to be shown to transiently modify the BBB in a receptor-mediated manner and to increase drug transport into rat and human gliomas. This approach, however, did not improve the efficacy of carboplatin in a phase II trial in glioma patients at the dose used because of the dose-limiting side effect of hypotension. The efficacy of RMP-7 in brain metastases has not been investigated. Alternatively, intravenous infusion of the hyperosmotic agent mannitol has been shown to globally induce endothelial cell shrinkage and tight junction separation and has been proposed as a means of transiently providing access to cerebral tumors. This approach has been performed in humans and has been shown to cause BBB disruption. However, the lack of specificity for tumor sites is a serious confounder with regard to healthy brain tissue, whereas the short working window limits therapeutic efficacy. Alternatively, ultrasound-mediated focused BBB disruption is a promising technique but relies on prior knowledge of metastatic sites. Thus, further work in this area is critical if brain metastases are to be detected and treated effectively.
Preclinical studies aimed at increasing drug delivery to systemic tumors have demonstrated the ability of an intravenous bolus dose of recombinant human tumor necrosis factor (TNF), a proinflammatory cytokine, to disrupt endothelial tight junctions in the tumor vasculature through the RhoA/Rho kinase pathway. This approach was shown to enhance the permeability of tumor vasculature and to facilitate virus particle delivery to a solid subcutaneous xenograft EL4 lymphoma model in mice. TNF has two endogenous receptors (TNFR1 and TNFR2), which mediate endothelial cytoskeletal reorganization and destabilization of interendothelial adhesion complexes. Although their activation is generally associated with pathophysiological processes, the effect of TNF receptor activation in controlled low-dose administration may be beneficial. However, the normal adult brain microvasculature, unlike peripheral blood vessels, is known to be resistant to the permeabilizing effects of cytokines. This resistance can be modified by a number of factors, and in previous work we have shown that microinjection of TNF into the rat brain can cause focal, but delayed, disruption of the BBB in association with a focal inflammatory response. Our recent work has shown that the early phases of metastasis development in the brain are associated with a strong inflammatory response and activation of the cerebral endothelium.
Based on the above findings, we hypothesized that cerebral metastases may provide a unique environment for TNF receptor activation on the associated vasculature and that this might yield a target for specific and local opening of the tumor-associated BBB. The primary aims of this study, therefore, were to determine whether TNFR1 and TNFR2 are expressed on metastasis-associated vasculature, and to determine whether intravenous administration of TNF, or its endogenous analogue lymphotoxin (LT), can permeabilize the BBB specifically at sites of cerebral metastasis throughout the brain to an extent that allows entry of 1) diagnostic imaging agents and 2) a clinically relevant anticancer drug.
Abstract and Introduction
Abstract
Background Effective chemotherapeutics for primary systemic tumors have limited access to brain metastases because of the blood–brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilizing the BBB at sites of cerebral metastases.
Methods BALB/c mice were injected intracardially to induce brain metastases. After metastasis induction, either tumor necrosis factor (TNF) or lymphotoxin (LT) was administered intravenously, and 2 to 24 hours later gadolinium- diethylenetriaminepentaacetic acid, horseradish peroxidase, or radiolabeled trastuzumab (In-BnDTPA-Tz) was injected intravenously. BBB permeability was assessed in vivo using gadolinium-enhanced T1-weighted magnetic resonance imaging and confirmed histochemically. Brain uptake of In-BnDTPA-Tz was determined using in vivo single photon emission computed tomography/computed tomography. Endothelial expression of TNF receptors was determined immunohistochemically in both mouse and human brain tissue containing metastases. Group differences were analyzed with one-way analysis of variance followed by post hoc tests, Wilcoxon signed rank test, and Kruskal–Wallis with Dunn's multiple comparison test. All statistical tests were two-sided.
Results Localized expression of TNF receptor 1 (TNFR1) was evident on the vascular endothelium associated with brain metastases. Administration of TNF or LT permeabilized the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect at 6 hours. Metastasis-specific uptake ratio of In-BnDTPA-Tz was also demonstrated after systemic TNF administration vs control (0.147±0.066 vs 0.001±0.001). Human brain metastases displayed a similar TNF receptor profile compared with the mouse model, with predominantly vascular TNFR1 expression.
Conclusions These findings describe a new approach to selectively permeabilize the BBB at sites of brain metastases to aid in detection of micrometastases and facilitate tumor-specific access of chemotherapeutic agents. We hypothesize that this permeabilization works primarily though TNFR1 activation and has the potential for clinical translation.
Introduction
Brain metastases pose a substantial challenge for chemotherapeutic treatment because of the impermeable nature of the blood–brain barrier (BBB), which limits access of drugs and thus prevents accumulation of clinically effective doses at tumor sites. Substances with good penetration of the BBB have limited activity against breast cancer, one of the most common cancers metastasizing to the brain, whereas the most active therapeutics for breast cancer (including doxorubicin and trastuzumab) appear not to reach the central nervous system (CNS) because of their hydrophilic nature. At the same time, the impermeable BBB also prevents early diagnosis of small brain metastases by the current clinical gold-standard method of gadolinium-enhanced magnetic resonance imaging (MRI). This diagnostic approach enables detection of large cerebral metastases and primary brain tumors but only when gross structural abnormalities have developed. Moreover, although BBB compromise may allow limited access of drugs to the tumor in later stages, this BBB permeability is frequently inhomogenous and generally poor around the tumor margin. Thus, the late stage of BBB disruption and inhomogeneity across the tumor mean treatment is largely ineffective. Smaller metastases possessing an intact BBB evade both detection and treatment and will inevitably develop into symptomatic tumors.
A number of approaches to transiently circumvent the BBB have been investigated for the delivery of chemotherapeutics to brain tumors [for a review see ]. Bradykinin B2 receptor activation by cereport (RPM-7) was the first pharmacological treatment to be shown to transiently modify the BBB in a receptor-mediated manner and to increase drug transport into rat and human gliomas. This approach, however, did not improve the efficacy of carboplatin in a phase II trial in glioma patients at the dose used because of the dose-limiting side effect of hypotension. The efficacy of RMP-7 in brain metastases has not been investigated. Alternatively, intravenous infusion of the hyperosmotic agent mannitol has been shown to globally induce endothelial cell shrinkage and tight junction separation and has been proposed as a means of transiently providing access to cerebral tumors. This approach has been performed in humans and has been shown to cause BBB disruption. However, the lack of specificity for tumor sites is a serious confounder with regard to healthy brain tissue, whereas the short working window limits therapeutic efficacy. Alternatively, ultrasound-mediated focused BBB disruption is a promising technique but relies on prior knowledge of metastatic sites. Thus, further work in this area is critical if brain metastases are to be detected and treated effectively.
Preclinical studies aimed at increasing drug delivery to systemic tumors have demonstrated the ability of an intravenous bolus dose of recombinant human tumor necrosis factor (TNF), a proinflammatory cytokine, to disrupt endothelial tight junctions in the tumor vasculature through the RhoA/Rho kinase pathway. This approach was shown to enhance the permeability of tumor vasculature and to facilitate virus particle delivery to a solid subcutaneous xenograft EL4 lymphoma model in mice. TNF has two endogenous receptors (TNFR1 and TNFR2), which mediate endothelial cytoskeletal reorganization and destabilization of interendothelial adhesion complexes. Although their activation is generally associated with pathophysiological processes, the effect of TNF receptor activation in controlled low-dose administration may be beneficial. However, the normal adult brain microvasculature, unlike peripheral blood vessels, is known to be resistant to the permeabilizing effects of cytokines. This resistance can be modified by a number of factors, and in previous work we have shown that microinjection of TNF into the rat brain can cause focal, but delayed, disruption of the BBB in association with a focal inflammatory response. Our recent work has shown that the early phases of metastasis development in the brain are associated with a strong inflammatory response and activation of the cerebral endothelium.
Based on the above findings, we hypothesized that cerebral metastases may provide a unique environment for TNF receptor activation on the associated vasculature and that this might yield a target for specific and local opening of the tumor-associated BBB. The primary aims of this study, therefore, were to determine whether TNFR1 and TNFR2 are expressed on metastasis-associated vasculature, and to determine whether intravenous administration of TNF, or its endogenous analogue lymphotoxin (LT), can permeabilize the BBB specifically at sites of cerebral metastasis throughout the brain to an extent that allows entry of 1) diagnostic imaging agents and 2) a clinically relevant anticancer drug.
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