Kaci A. Bohn

Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer

Purpose: Brain metastases of breast cancer appear to be increasing in incidence, confer significant morbidity, and threaten to compromise gains made in systemic chemotherapy. The blood–tumor barrier (BTB) is compromised in many brain metastases; however, the extent to which this influences chemotherapeutic delivery and efficacy is unknown. Herein, we answer this question by measuring BTB passive integrity, chemotherapeutic drug uptake, and anticancer efficacy in vivo in two breast cancer models that metastasize preferentially to brain. Experimental Design:Experimental brain metastasis drug uptake and BTB permeability were simultaneously measured using novel fluorescent and phosphorescent imaging techniques in immune-compromised mice. Drug-induced apoptosis and vascular characteristics were assessed using immunofluorescent microscopy. Results: Analysis of over 2,000 brain metastases from two models (human 231-BR-Her2 and murine 4T1-BR5) showed partial BTB permeability compromise in greater than 89% of lesions, varying in magnitude within and between metastases. Brain metastasis uptake of 14C-paclitaxel and 14C-doxorubicin was generally greater than normal brain but less than 15% of that of other tissues or peripheral metastases, and only reached cytotoxic concentrations in a small subset (∼10%) of the most permeable metastases. Neither drug significantly decreased the experimental brain metastatic ability of 231-BR-Her2 tumor cells. BTB permeability was associated with vascular remodeling and correlated with overexpression of the pericyte protein desmin. Conclusions: This work shows that the BTB remains a significant impediment to standard chemotherapeutic delivery and efficacy in experimental brain metastases of breast cancer. New brain permeable drugs will be needed. Evidence is presented for vascular remodeling in BTB permeability alterations. Clin Cancer Res; 16(23); 5664–78. ©2010 AACR.

Vorinostat Inhibits Brain Metastatic Colonization in a Model of Triple-Negative Breast Cancer and Induces DNA Double-Strand Breaks

Purpose: As chemotherapy and molecular therapy improve the systemic survival of breast cancer patients, the incidence of brain metastases increases. Few therapeutic strategies exist for the treatment of brain metastases because the blood-brain barrier severely limits drug access. We report the pharmacokinetic, efficacy, and mechanism of action studies for the histone deactylase inhibitor vorinostat (suberoylanilide hydroxamic acid) in a preclinical model of brain metastasis of triple-negative breast cancer. Experimental Design: The 231-BR brain trophic subline of the MDA-MB-231 human breast cancer cell line was injected into immunocompromised mice for pharmacokinetic and metastasis studies. Pharmacodynamic studies compared histone acetylation, apoptosis, proliferation, and DNA damage in vitro and in vivo. Results: Following systemic administration, uptake of [14C]vorinostat was significant into normal rodent brain and accumulation was up to 3-fold higher in a proportion of metastases formed by 231-BR cells. Vorinostat prevented the development of 231-BR micrometastases by 28% (P = 0.017) and large metastases by 62% (P < 0.0001) compared with vehicle-treated mice when treatment was initiated on day 3 post-injection. The inhibitory activity of vorinostat as a single agent was linked to a novel function in vivo: induction of DNA double-strand breaks associated with the down-regulation of the DNA repair gene Rad52. Conclusions: We report the first preclinical data for the prevention of brain metastasis of triple-negative breast cancer. Vorinostat is brain permeable and can prevent the formation of brain metastases by 62%. Its mechanism of action involves the induction of DNA double-strand breaks, suggesting rational combinations with DNA active drugs or radiation. (Clin Cancer Res 2009;15(19):6148–57)

Paclitaxel–Hyaluronic NanoConjugates Prolong Overall Survival in a Preclinical Brain Metastases of Breast Cancer Model

Brain (central nervous system; CNS) metastases pose a life-threatening problem for women with advanced metastatic breast cancer. It has recently been shown that the vasculature within preclinical brain metastasis model markedly restricts paclitaxel delivery in approximately 90% of CNS lesions. Therefore to improve efficacy, we have developed an ultra-small hyaluronic acid (HA) paclitaxel nanoconjugate (∼5 kDa) that can passively diffuse across the leaky blood–tumor barrier and then be taken up into cancer cells (MDA–MB–231Br) via CD44 receptor–mediated endocytocis. Using CD44 receptor–mediated endocytosis as an uptake mechanism, HA-paclitaxel was able to bypass p-glycoprotein–mediated efflux on the surface of the cancer cells. In vitro cytoxicity of the conjugate and free paclitaxel were similar in that they (i) both caused cell-cycle arrest in the G2–M phase, (ii) showed similar degrees of apoptosis induction (cleaved caspase), and (iii) had similar IC50 values when compared with paclitaxel in MTT assay. A preclinical model of brain metastases of breast cancer using intracardiac injections of Luc-2 transfected MDA–MB–231Br cells was used to evaluate in vivo efficacy of the nanoconjugate. The animals administered with HA–paclitaxel nanoconjugate had significantly longer overall survival compared with the control and the paclitaxel-treated group (P < 0.05). This study suggests that the small molecular weight HA–paclitaxel nanoconjugates can improve standard chemotherapeutic drug efficacy in a preclinical model of brain metastases of breast cancer. Mol Cancer Ther; 12(11); 2389–99. ©2013 AACR.

Nicotine and cotinine increases the brain penetration of saquinavir in rat.

J. Neurochem. (2010) 115, 1495–1507.

P-glycoprotein mediated efflux limits substrate and drug uptake in a preclinical brain metastases of breast cancer model

The blood–brain barrier (BBB) is a specialized vascular interface that restricts the entry of many compounds into brain. This is accomplished through the sealing of vascular endothelial cells together with tight junction proteins to prevent paracellular diffusion. In addition, the BBB has a high degree of expression of numerous efflux transporters which actively extrude compounds back into blood. However, when a metastatic lesion develops in brain the vasculature is typically compromised with increases in passive permeability (blood-tumor barrier; BTB). What is not well documented is to what degree active efflux retains function at the BTB despite the changes observed in passive permeability. In addition, there have been previous reports documenting both increased and decreased expression of P-glycoprotein (P-gp) in lesion vasculature. Herein, we simultaneously administer a passive diffusion marker (14C-AIB) and a tracer subject to P-gp efflux (rhodamine 123) into a murine preclinical model of brain metastases of breast cancer. We observed that the metastatic lesions had similar expression (p > 0.05; n = 756–1214 vessels evaluated) at the BBB and the BTB. Moreover, tissue distribution of R123 was not significantly (p > 0.05) different between normal brain and the metastatic lesion. It is possible that the similar expression of P-gp on the BBB and the BTB contribute to this phenomenon. Additionally we observed P-gp expression at the metastatic cancer cells adjacent to the vasculature which may also contribute to reduced R123 uptake into the lesion. The data suggest that despite the disrupted integrity of the BTB, efflux mechanisms appear to be intact, and may be functionally comparable to the normal BBB. The BTB is a significant hurdle to delivering drugs to brain metastasis.

Quantitative Fluorescence Microscopy Measures Vascular Pore Size in Primary and Metastatic Brain Tumors.

Tumors residing in the central nervous system (CNS) compromise the blood-brain barrier (BBB) via increased vascular permeability, with the magnitude of changes dependent on the tumor type and location. Current studies determine penetrability of a cancer therapeutic by administering progressively larger molecules until cutoff is observed where little to no tumor accumulation occurs. However, decades-old experimental work and mathematical modeling document methods to calculate both the size of the vascular opening (pore) with solute permeability values. In this study, we updated this classic mathematical modeling approach with quantitative fluorescence microscopy in two preclinical tumor models, allowing simultaneous administration of multiple sized tracers to determine vascular permeability at a resolution of nearly one micron. We observed that three molecules ranging from 100 Da to 70 kDa permeated into a preclinical glioblastoma model at rates proportional to their diffusion in water. This suggests the solutes freely diffused from blood to glioma across vascular pores without steric restriction, which calculates to a pore size of >140 nm in diameter. In contrast, the calculated pore size of a brain metastasis of breast cancer was approximately 10-fold smaller than glioma vasculature. This difference explains why antibodies are effective against glioblastoma but generally fail in brain metastases of breast cancer. On the basis of our observations, we hypothesize that trastuzumab most likely fails in the treatment of brain metastases of breast cancer because of poor CNS penetration, while the similar sized antibody bevacizumab is effective in the same tumor type not because it penetrates the CNS degree better, but because it scavenges VEGF in the vascular compartment, which reduces edema and permeation. Cancer Res; 77(2); 238-46. ©2016 AACR.

Inhibition of VEGF and Angiopoietin-2 to Reduce Brain Metastases of Breast Cancer Burden

For metastases in the central nervous system, angiogenesis enhances metastatic potential and promotes progression. Primary factors which drive vessel growth are vascular endothelial growth factor (VEGF) and angiopoietin-2. Preclinical models show inhibition of either factor reduces metastases spread and inhibits growth. This work sets out to answer two questions in a preclinical mouse model. First, whether the combined inhibition of VEGF and angiopoietin-2, reduces passive permeability and limits drug uptake into brain metastases; and second, whether this inhibition reduces metastases burden in brain. We observed combinatorial inhibition of VEGF and angiopoietin-2, decreased (p < 0.05) angiogenesis and vascular branching in an aortic ring assay and decreased (p < 0.05) endothelial wound closure times. Using a brain metastases of breast cancer model (induced by intracardiac injections of brain seeking MDA-MB-231Br cells or 4T1Br cells), we observed, similar to VEGF, angiopoetin-2 expression correlates to increased angiogenesis (p < 0.05) and increased lesion permeability. To determine efficacy, animals were administered bevacizumab plus L1-10 (angiopoietin inhibitor) twice per week until neurological symptoms developed. Lesion permeability significantly decreased by ∼50% (p < 0.05) compared to untreated lesions, but remained ∼25% greater (p < 0.0%) than brain. In subsequent experiments, animals were administered similar regimens but sacrificed on day 32. The number of metastatic lesions developed was significantly (p < 0.001) reduced in the bevacizumab group (56%) and combination group (86%). Lesions’ size was reduced in bevacizumab treated lesions (∼67%) and bevacizumab and L1-10 treated lesions (∼78%) developing area < 0.5 mm2. In summary, combinatorial inhibition of VEGF and angiopoietin reduces lesion permeability and brain metastatic burden.

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 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