Neal Shah

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.

Combination of Eribulin and Aurora A Inhibitor MLN8237 Prevents Metastatic Colonization and Induces Cytotoxic Autophagy in Breast Cancer

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule-targeting drug eribulin on different stages of metastasis in triple-negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process, including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells, providing foundation for the combination therapy. Mechanistically, AURKA inhibition induces cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. These preclinical data provide a new understanding of the mechanisms by which MLN8237 mediates its antimetastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early-stage solid tumors. Mol Cancer Ther; 15(8); 1809–22. ©2016 AACR.

Investigational chemotherapy and novel pharmacokinetic mechanisms for the treatment of breast cancer brain metastases

&NA; In women, breast cancer is the most common cancer diagnosis and second most common cause of cancer death. More than half of breast cancer patients will develop metastases to the bone, liver, lung, or brain. Breast cancer brain metastases (BCBM) confers a poor prognosis, as current therapeutic options of surgery, radiation, and chemotherapy rarely significantly extend life and are considered palliative. Within the realm of chemotherapy, the last decade has seen an explosion of novel chemotherapeutics involving targeting agents and unique dosage forms. We provide a historical overview of BCBM chemotherapy, review the mechanisms of new agents such as poly‐ADP ribose polymerase inhibitors, cyclin‐dependent kinase 4/6 inhibitors, phosphatidyl inositol 3‐kinaseinhibitors, estrogen pathway antagonists for hormone‐receptor positive BCBM; tyrosine kinase inhibitors, antibodies, and conjugates for HER2+ BCBM; repurposed cytotoxic chemotherapy for triple negative BCBM; and the utilization of these new agents and formulations in ongoing clinical trials. The mechanisms of novel dosage formulations such as nanoparticles, liposomes, pegylation, the concepts of enhanced permeation and retention, and drugs utilizing these concepts involved in clinical trials are also discussed. These new treatments provide a promising outlook in the treatment of BCBM. Graphical abstract Figure. No caption available.

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.

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 1832: Bioluminescent pharmacokinetics of luciferin in preclinical brain metastases of breast cancer models

Background: Approximately 20% of breast cancer patients with disseminated disease will develop brain metastases. Preclinical models of brain metastases of breast cancer rely on ex-vivo histology to evaluate drug efficacy. However, bioluminescence imaging allows more precise quantification of tumor burden and progression through counting photons per second without requiring animal sacrifice. This methodology has not readily been applied to hematogenously derived brain metastases models given the heterogeneity of tumor burden and growth. Herein we demonstrate repeatable methodology to quantify brain metastases of breast cancer progression which is verified with histology. Methods: Approximately 1.75 x 10 5 JIMT-1 (n=3) and 231-BrLuc (n=4) brain seeking subclones were injected intracardially to produce metastatic brain lesions. Mice were given 150 mg/kg of luciferin IP and luminescence was captured every 2-minutes for 60 minutes starting 24 hours after cell implantation. Radiance (photons per second per square centimeter per steridian) was plotted to observe peak luminescence. Imaging was repeated twice weekly to evaluate progression until euthanasia. Results: Following intracardiac injection, both brain seeking metastatic models produced a maximum luminescence signal between 14-20 minutes after luciferin injection. For all subsequent imaging, 5 minute imaging between 15-20 minutes after luciferin injection was used. Longitudinally, both tumor cell lines produce bioluminescence 24 hours after cell injection, which is used to ensure tumor implantation and randomization. The BLI signal decreases to undetectable limits in both models between 3-7 days after cell implantation. Re-emergence of signal occurs on day 14 for the JIMT-1 line and day 21 for 231-Br line. JIMT-1 bioluminescence on day 14 begins at ~10 5 photons/sec/cm 2 /sr and then over time signal increases with an ~slope of 5.6x10 5 until reaching a maximum of 10 7 units on day 28. 231-Br bioluminescence on day 21 begins at ~10 4 photons/sec/cm 2 /sr and then over time signal increases with an ~ slope of 1.1x10 6 until reaching a maximum of ~10 7 on day 53. Conclusion: To accurately measure bioluminescence as a surrogate for tumor burden in hematogenously implanted metastases, the optimal circulation time is approximately 15 minutes with a 5 minute imaging period. Bioluminescence of the 231Br line initially starts at a lower magnitude and later in time than the JIMT-1 line, but slopes of both intracardiac models are similar and result in similar bioluminescent curves. Citation Format: Neal Shah, Chris E. Adkins, Afroz S. Mohammad, Paul R. Lockman. Bioluminescent pharmacokinetics of luciferin in preclinical brain metastases of breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1832. doi:10.1158/1538-7445.AM2017-1832