Tori B. Terrell-Hall

Characterization of passive permeability at the blood–tumor barrier in five preclinical models of brain metastases of breast cancer

The blood–brain barrier (BBB) is compromised in brain metastases, allowing for enhanced drug permeation into brain. The extent and heterogeneity of BBB permeability in metastatic lesions is important when considering the administration of chemotherapeutics. Since permeability characteristics have been described in limited experimental models of brain metastases, we sought to define these changes in five brain-tropic breast cancer cell lines: MDA-MB-231BR (triple negative), MDA-MB-231BR-HER2, JIMT-1-BR3, 4T1-BR5 (murine), and SUM190 (inflammatory HER2 expressing). Permeability was assessed using quantitative autoradiography and fluorescence microscopy by co-administration of the tracers 14C-aminoisobutyric acid (AIB) and Texas red conjugated dextran prior to euthanasia. Each experimental brain metastases model produced variably increased permeability to both tracers; additionally, the magnitude of heterogeneity was different among each model with the highest ranges observed in the SUM190 (up to 45-fold increase in AIB) and MDA-MB-231BR-HER2 (up to 33-fold in AIB) models while the lowest range was observed in the JIMT-1-BR3 (up to 5.5-fold in AIB) model. There was no strong correlation observed between lesion size and permeability in any of these preclinical models of brain metastases. Interestingly, the experimental models resulting in smaller mean metastases size resulted in shorter median survival while models producing larger lesions had longer median survival. These findings strengthen the evidence of heterogeneity in brain metastases of breast cancer by utilizing five unique experimental models and simultaneously emphasize the challenges of chemotherapeutic approaches to treat brain metastases.

Anti-cancer Antibody Trastuzumab-Melanotransferrin Conjugate (BT2111) for the Treatment of Metastatic HER2+ Breast Cancer Tumors in the Brain: an In-Vivo Study

PurposeThe ability of human melanotransferrin (hMTf) to carry a therapeutic concentration of trastuzumab (BTA) in the brain after conjugation (in the form of trastuzumab-melanotransferrin conjugate, BT2111 conjugate) was investigated by measuring the reduction of the number and size of metastatic human HER2+ breast cancer tumors in a preclinical model of brain metastases of breast cancer.MethodsHuman metastatic brain seeking breast cancer cells were injected in NuNu mice (n = 6–12 per group) which then developed experimental brain metastases. Drug uptake was analyzed in relation to metastasis size and blood-tumor barrier permeability. To investigate in-vivo activity against brain metastases, equimolar doses of the conjugate, and relevant controls (hMTf and BTA) in separate groups were administered biweekly after intracardiac injection of the metastatic cancer cells.ResultsThe trastuzumab-melanotransferrin conjugate (BT2111) reduced the number of preclinical human HER2+ breast cancer metastases in the brain by 68% compared to control groups. Tumors which remained after treatment were 46% smaller than the control groups. In contrast, BTA alone had no effect on reducing number of metastases, and was associated with only a minimal reduction in metastasis size.ConclusionsThe results suggest the novel trastuzumab-melanotransferrin conjugate (BT2111) may have utility in treating brain metastasis and validate hMTf as a potential vector for antibody transport across the Blood Brain Barrier (BBB).

A novel preclinical method to quantitatively evaluate early-stage metastatic events at the murine blood-brain barrier.

The observation that approximately 15% of women with disseminated breast cancer will develop symptomatic brain metastases combined with treatment guidelines discouraging single-agent chemotherapeutic strategies facilitates the desire for novel strategies aimed at outright brain metastasis prevention. Effective and robust preclinical methods to evaluate early-stage metastatic processes, brain metastases burden, and overall mean survival are lacking. Here, we develop a novel method to quantitate early metastatic events (arresting and extravasation) in addition to traditional end time-point parameters such as tumor burden and survival in an experimental mouse model of brain metastases of breast cancer. Using this method, a reduced number of viable brain-seeking metastatic cells (from 3,331 ± 263 cells/brain to 1,079 ± 495 cells/brain) were arrested in brain one week postinjection after TGFβ knockdown. Treatment with a TGFβ receptor inhibitor, galunisertib, reduced the number of arrested cells in brain to 808 ± 82 cells/brain. Furthermore, we observed a reduction in the percentage of extravasated cells (from 63% to 30%) compared with cells remaining intralumenal when TGFβ is knocked down or inhibited with galunisertib (40%). The observed reduction of extravasated metastatic cells in brain translated to smaller and fewer brain metastases and resulted in prolonged mean survival (from 36 days to 62 days). This method opens up potentially new avenues of metastases prevention research by providing critical data important to early brain metastasis of breast cancer events. Cancer Prev Res; 8(1); 68–76. ©2014 AACR.

Liposomal Irinotecan Accumulates in Metastatic Lesions, Crosses the Blood-Tumor Barrier (BTB), and Prolongs Survival in an Experimental Model of Brain Metastases of Triple Negative Breast Cancer

PurposeThe blood-tumor barrier (BTB) limits irinotecan distribution in tumors of the central nervous system. However, given that the BTB has increased passive permeability we hypothesize that liposomal irinotecan would improve local exposure of irinotecan and its active metabolite SN-38 in brain metastases relative to conventional irinotecan due to enhanced-permeation and retention (EPR) effect.MethodsFemale nude mice were intracardially or intracranially implanted with human brain seeking breast cancer cells (brain metastases of breast cancer model). Mice were administered vehicle, non-liposomal irinotecan (50 mg/kg), liposomal irinotecan (10 mg/kg and 50 mg/kg) intravenously starting on day 21. Drug accumulation, tumor burden, and survival were evaluated.ResultsLiposomal irinotecan showed prolonged plasma drug exposure with mean residence time (MRT) of 17.7 ± 3.8 h for SN-38, whereas MRT was 3.67 ± 1.2 for non-liposomal irinotecan. Further, liposomal irinotecan accumulated in metastatic lesions and demonstrated prolonged exposure of SN-38 compared to non-liposomal irinotecan. Liposomal irinotecan achieved AUC values of 6883 ± 4149 ng-h/g for SN-38, whereas non-liposomal irinotecan showed significantly lower AUC values of 982 ± 256 ng-h/g for SN-38. Median survival for liposomal irinotecan was 50 days, increased from 37 days (p<0.05) for vehicle.ConclusionsLiposomal irinotecan accumulates in brain metastases, acts as depot for sustained release of irinotecan and SN-38, which results in prolonged survival in preclinical model of breast cancer brain metastasis.

Trastuzumab distribution in an in-vivo and in-vitro model of brain metastases of breast cancer

Background Drug and antibody delivery to brain metastases has been highly debated in the literature. The blood-tumor barrier (BTB) is more permeable than the blood-brain barrier (BBB), and has shown to have highly functioning efflux transporters and barrier properties, which limits delivery of targeted therapies. Methods We characterized the permeability of 125I-trastuzumab in an in-vivo, and fluorescent trastuzumab-Rhodamine123 (t-Rho123) in a novel microfluidic in-vitro, BBB and BTB brain metastases of breast cancer model. In-vivo: Human MDA-MB-231-HER2+ metastatic breast cancer cells were grown and maintained under static conditions. Cells were harvested at 80% confluency and prepped for intra-cardiac injection into 20 homozygous female Nu/Nu mice. In-vitro: In a microfluidic device (SynVivo), human umbilical vein endothelial cells were grown and maintained under shear stress conditions in the outer compartment and co-cultured with CTX-TNA2 rat brain astrocytes (BBB) or Met-1 metastatic HER2+ murine breast cancer cells (BTB), which were maintained in the central compartment under static conditions. Results Tissue distribution of 125I-trastuzumab revealed only ~3% of injected dose reached normal brain, with ~5% of injected dose reaching brain tumors. No clear correlation was observed between size of metastases and the amount of 125I-trastuzumab localized in-vivo. This heterogeneity was paralleled in-vitro, where the distribution of t-Rho123 from the outer chamber to the central chamber of the microfluidic device was qualitatively and quantitatively analyzed over time. The rate of t-Rho123 linear uptake in the BBB (0.27 ± 0.33 × 104) and BTB (1.29 ± 0.93 × 104) showed to be significantly greater than 0 (p < 0.05). The BTB devices showed significant heterogenetic tendencies, as seen in in-vivo. Conclusions This study is one of the first studies to measure antibody movement across the blood-brain and blood-tumor barriers, and demonstrates that, though in small and most likely not efficacious quantities, trastuzumab does cross the blood-brain and blood-tumor barriers.BACKGROUND Drug and antibody delivery to brain metastases has been highly debated in the literature. The blood-tumor barrier (BTB) is more permeable than the blood-brain barrier (BBB), and has shown to have highly functioning efflux transporters and barrier properties, which limits delivery of targeted therapies. METHODS We characterized the permeability of 125I-trastuzumab in an in-vivo, and fluorescent trastuzumab-Rhodamine123 (t-Rho123) in a novel microfluidic in-vitro, BBB and BTB brain metastases of breast cancer model. In-vivo: Human MDA-MB-231-HER2+ metastatic breast cancer cells were grown and maintained under static conditions. Cells were harvested at 80% confluency and prepped for intra-cardiac injection into 20 homozygous female Nu/Nu mice. In-vitro: In a microfluidic device (SynVivo), human umbilical vein endothelial cells were grown and maintained under shear stress conditions in the outer compartment and co-cultured with CTX-TNA2 rat brain astrocytes (BBB) or Met-1 metastatic HER2+ murine breast cancer cells (BTB), which were maintained in the central compartment under static conditions. RESULTS Tissue distribution of 125I-trastuzumab revealed only ~3% of injected dose reached normal brain, with ~5% of injected dose reaching brain tumors. No clear correlation was observed between size of metastases and the amount of 125I-trastuzumab localized in-vivo. This heterogeneity was paralleled in-vitro, where the distribution of t-Rho123 from the outer chamber to the central chamber of the microfluidic device was qualitatively and quantitatively analyzed over time. The rate of t-Rho123 linear uptake in the BBB (0.27 ± 0.33 X 104) and BTB (1.29 ± 0.93 X 104) showed to be significantly greater than 0 (p < 0.05). The BTB devices showed significant heterogenetic tendencies, as seen in in-vivo. CONCLUSIONS This study is one of the first studies to measure antibody movement across the blood-brain and blood-tumor barriers, and demonstrates that, though in small and most likely not efficacious quantities, trastuzumab does cross the blood-brain and blood-tumor barriers.

Semi-automated rapid quantification of brain vessel density utilizing fluorescent microscopy

BACKGROUND Measurement of vascular density has significant value in characterizing healthy and diseased tissue, particularly in brain where vascular density varies among regions. Further, an understanding of brain vessel size helps distinguish between capillaries and larger vessels like arterioles and venules. Unfortunately, few cutting edge methodologies are available to laboratories to rapidly quantify vessel density. NEW METHOD We developed a rapid microscopic method, which quantifies the numbers and diameters of blood vessels in brain. Utilizing this method we characterized vascular density of five brain regions in both mice and rats, in two tumor models, using three tracers. RESULTS We observed the number of sections/mm(2) in various brain regions: genu of corpus callosum 161±7, hippocampus 266±18, superior colliculus 300±24, frontal cortex 391±55, and inferior colliculus 692±18 (n=5 animals). Regional brain data were not significantly different between species (p>0.05) or when using different tracers (70kDa and 2000kDa Texas Red; p>0.05). Vascular density decreased (62-79%) in preclinical brain metastases but increased (62%) a rat glioma model. COMPARISON WITH EXISTING METHODS Our values were similar (p>0.05) to published literature. We applied this method to brain-tumors and observed brain metastases of breast cancer to have a ∼2.5-fold reduction (p>0.05) in vessels/mm(2) compared to normal cortical regions. In contrast, vascular density in a glioma model was significantly higher (sections/mm(2) 736±84; p<0.05). CONCLUSIONS In summary, we present a vascular density counting method that is rapid, sensitive, and uses fluorescence microscopy without antibodies.

Abstract 2080: Demonstration of casual relationship between blood-tumor barrier permeability changes and chemotherapeutic uptake and effect in brain micrometastases of breast cancer

Background: 10-16% of women with advanced breast cancer will develop symptomatic brain metastases, the survival rate of which is less than 2 years. When metastasis develop within the brain, the BBB anatomy changes due to increased angiogenesis with increase in permeability of BBB in the tumor region (> 1mm diameter). We have also demonstrated in our previous study that, once the metastases are established in the brain (>1 mm in diameter) chemotherapy fails to induce cytotoxicity in most of them. The efficacy of a chemotherapeutic agent in the metastatic brain tumors significantly correlates to its uptake in the micro metastases ( Methods: Female nude mice were intracardially injected with different human brain seeking breast cancer cell and allowed it to metastasize. Metastases were allowed to develop until neurologic symptoms appeared and animals were injected with IV bolus dose of permeability markers 14C-AIB (104 Da) and Texas Red dextran (3kDa) 10 minutes prior to euthanasia. After euthanasia rain sample were harvested, sectioned and analyzed by autoradiography and fluorescent microscopy. Results: Passive permeability changes in 4T1 metastatic lesions was 3.171 + 1.52 (SD) for 14C-AIB, and 2.529 + 0.84 (SD) for the passive diffusion marker 3kDa Texas Red Dextran(TRD). MDA-MB-231BR HER2 metastatic lesions has 14C-AIB permeability 4.936 + 3.6 (SD) and for TRD 1.301 + 0.49 (SD). For MDA-MB-231Br cell line permeability changes with 14C-AIB was found to be 3.001 ± 0.1514 (SD) and the BTB permeability to TRD ranged was found to be 1.159 ± 0.3137 (SD). In JIMT-1Br, 14C-AIB permeability was found to be 2.212 + 0.72 (SD) and TRD was 1.502 + 0.344 (SD). Sum190 micrometastases showed maximum 14C-AIB permeability in the range of 1 to 28.64 fold (Mean was 10.11 ± 5.68; SD) and TRD was found to be in the range of 1 to 2.10 (Mean was 1.287 ± 0.293; SD). Conclusions: We have demonstrated that there are differences in passive permeability between each cell line and there are differences in overall survival and the size of lesions at the time neurological symptoms develop. The increased permeability of the vasculature of CNS micrometastases is critically important for both diagnosis and treatment. Citation Format: Afroz Shareef Mohammad, Chris E. Adkins, Emma L. Dolan, Tori B. Terrell-Hall, Paul R. Lockman. Demonstration of casual relationship between blood-tumor barrier permeability changes and chemotherapeutic uptake and effect in brain micrometastases of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2080.

Abstract 2616: Inhibition of TGFß to prevent brain metastases of breast cancer

Introduction: Because systemic chemotherapy fails to effectively treat brain metastases, the need for novel strategies designed to prevent brain metastasis is urgent. Recently, transforming growth factor beta (TGFs) has been shown to influence the development of brain metastases of breast cancer and may offer a therapeutic target to prevent metastatic disease in brain. This study seeks to evaluate the efficacy of TGFs inhibition, using losartan, at preventing metastases in an experimental model of brain metastases of breast cancer. Methods: Human breast cancer cells (MDA-MB-231Br-Luc) were inoculated into female NuNu mice after TGFs knockdown or treatment with inhibitor; after 1-week mice were sacrificed, their brains removed and sliced, and metastatic cells counted and localized to determine seeding. Survival studies were then initiated with daily low (10mg/kg) or high (25mg/kg) doses of losartan, or SB431542 (5mg/kg) prior to the injection of one of two human breast cancer cell lines (MDA-MB-231Br-Luc or JIMT-1-Br3-Luc) transfected with luciferase. Treatments resumed daily until neurological symptoms developed. Animals were sacrificed, brains removed, and sliced to evaluate the number of metastases and their respective sizes. Results: The number of metastatic cells seeding brain was reduced after knockdown of TGFs (from 3,331 ± 263 cells to 1,079 ± 495 cells; p 0.05) in median survival was observed for the JIMT-1-Br3-Luc losartan (30 days, low; 26 days, high) or inhibitor (29 days) treatment groups. Conclusions: Inhibition of TGFs reduced the total number of metastatic MDA-MB-231Br cells seeding brain and simultaneously reduced the percentage of cells that had extravasated the vessel. While losartan did not increase survival for animals inoculated with JIMT-1-Br3-Luc cells, the MDA-MB-231Br model exhibited enhanced survival with losartan (15% survival rate; high dose), inhibitor (20% survival rate) treatment, and after TGFs knockdown (38% survival rate). The data indicate that TGFs offers a potential therapeutic target to reduce the risk of brain metastases of breast cancer. Citation Format: Chris E. Adkins, Afroz S. Mohammad, Emma Dolan, Jessica Griffith, Tori Terrell-Hall, Paul R. Lockman. Inhibition of TGFs to prevent brain metastases of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2616.

Abstract 1388: Inhibition of VEGF and angiopoietin-2 to reduce brain metastases of breast cancer burden

Introduction: In cancer, vascular destabilization and angiogenesis enhance tumor growth, metastatic potential, and correlate with poor patient outcome. A primary driver of angiogenesis is vascular endothelial growth factor (VEGF), secreted by tumor cells in response to decreased vessel density and hypoxia; and is highly expressed in breast cancer. A secondary driver, angiopoietin-2 (Ang-2), is activated by hypoxia, and induces vessel destabilization upon Tie2 receptor binding. VEGF and Ang-2 have shown to independently and synergistically induce angiogenesis. Preclinical brain metastases models of breast cancer have shown administering bevacizumab results in reduced vascular permeability, and reduces overall metastatic burden in brain. Based upon these observations, we hypothesize that inhibition of both VEGF and angiopoetin-2 will further reduce metastatic burden in brain. Methods: To determine if inhibition of VEGF and Ang-2 results in decreased metastatic burden, a human breast cancer (MDA-MB-231Br) cell line transfected to stably express eGFP was injected into the peripheral circulation via the left cardiac ventricle and CNS metastases grew for 4-6 weeks. As metastases seeded the brain, treatments were administered beginning on day 10. Bevacizumab (10 mg/kg, twice weekly) and L1-10 (Ang-2 inhibitor, 4 mg/kg twice weekly) were administered until day 32, when animals were euthanized and metastatic burden in brain was determined. Results: To confirm that VEGF and Ang-2 synergistically induced angiogenesis in vitro with bevacizumab and L1-10, we used aortic ring and wound healing assays. We observed significant increase (p Conclusion: In summary, we demonstrate an integral role of Ang-2 and VEGF in angiogenesis and brain metastases progression. Simultaneous inhibition of these two angiogenic factors may potentially reduce the formation of experimental brain metastases. Further work is required to determine if simultaneous inhibition of VEGF and Ang-2 may be effective to reduce brain metastasis formation. Citation Format: Kaci A. Bohn, Emily R. Sechrest, Chris E. Adkins, Rajendar K. Mittapalli, Mohamed I. Nounou, Tori B. Terrell-Hall, Afroz S. Mohammad, Paul R. Lockman. Inhibition of VEGF and angiopoietin-2 to reduce brain metastases of breast cancer burden. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2015-1388

Abstract 5207: Vascular remodeling is associated with increased permeability of experimental brain metastases of breast cancer

Approximately 1 in 8 women will develop breast cancer. Of these women, a subset develop metastatic breast cancer, of which 10-16% develop brain metastases. Treatment options are limited. The blood-brain barrier (BBB) serves as a physical barrier, limiting drug entry into the brain, as well as the active efflux of drugs by P-Glycoprotein, Breast Cancer Resistance Protein, and/or Multidrug Resistance-associated Protein located on the capillary endothelial cell membranes composing the BBB. Active remodeling occurs when cancer cells extravasate through the BBB and begin to metastasize forming fully colonized tumors, creating the Blood-Tumor Barrier (BTB). In order for active remodeling to occur, induction of angiogenesis must be initiated, resulting in formation of new blood vessels. These new blood vessels undergo active remodeling, and are associated with increased permeability when compared to intact BBB. By measuring vascular density, our goals were to understand permeability as it relates to vascular density, determine if increases in lesion permeability facilitate paclitaxel uptake into the lesion, and evaluate permeability as it relates to vascular remodeling. In this study, we utilize two experimental models (murine: 4T1-BR5; and human: MDA-MB-231-BR-Her2) to analyze permeability, drug uptake and vascular characteristics. In an analysis of over 2,000 metastatic lesions, we observed an increased permeability (a range over 30 fold) of nearly all lesions, with paclitaxel concentrations elevated (p Citation Format: Kaci A. Bohn, Tori B. Terrell-Hall, Chris E. Adkins, Rajendar K. Mittapalli, Mohamed I. Nounou, Afroz S. Mohammad, Paul R. Lockman. Vascular remodeling is associated with increased permeability of experimental brain metastases of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5207. doi:10.1158/1538-7445.AM2015-5207