Preclinical Model Of Brain Metastases Research Articles

DDDR-05. ERK INHIBITION INCREASES SENSITIVITY TO ANTI-PD-1 THERAPY IN A MOUSE MODEL OF BRAF MUTANT MELANOMA BRAIN METASTASIS

Abstract BACKGROUND Immune checkpoint inhibitors (ICI) have revolutionized the landscape of cancer treatment, including brain metastases. However, only a subset of patients with brain metastases respond to ICI. Activating mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, a key regulator of cell processes, are frequently found in brain metastases. LY3214996 (LY321) is a selective ERK 1/2 inhibitor that inhibits MAPK signaling and leads to regression of extracranial disease in preclinical models. Our objective was to evaluate whether with ERK inhibition (ERKi) can improve the efficacy of ICI in preclinical models of brain metastases. METHODS We tested sensitivity to LY321 in a BRAFV600E mutant, ICI resistant murine melanoma cell line (D4M) both in vitro and in vivo. To model brain metastases in vivo, we implanted D4M cells subcutaneously with subsequent intracranial implantation three days later. The treatment arms included LY321 and anti-PD-1 combination therapy, single agent therapies, and respective controls. We tested the efficacies of 2-week versus 4-week LY321 treatments, as well as 3-week treatment in an independent experiment. RESULTS D4M demonstrates in vitro sensitivity to LY321 (IC50 value of 4.75 µM). Our model demonstrated that LY321 in combination with anti-PD-1 improves both intracranial and extracranial disease control. Increased survival with combination therapy was observed with 3-week and 4-week LY321 administration. While individually, LY321 and anti-PD-1 produced better survival outcomes than control groups, combination therapy significantly outperformed both monotherapies. CONCLUSION Previous studies demonstrated that LY321 has minimal intracranial activity. However, we found that ERKi reduces intracranial tumor burden, with improved efficacy in combination with ICI in immunocompetent mice. Together, these data suggest that ERKi increases sensitivity to ICI therapy in pre-clinical brain metastasis models. Further investigation is needed to elucidate the mechanisms of this therapeutic approach, and to ultimately determine whether this combination will improve patient outcomes.

Abstract 3461: Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy

Abstract Background: Incidence of patients having brain metastasis of breast cancer has increased due to enhanced treatment for peripheral disease. Breast cancer brain metastasis is often fatal and occurs in 15-20% of patients diagnosed with stage IV breast cancer. Traditional chemotherapy delivery to the brain is limited by the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Low intensity focused ultrasound (LIFU) is a unique technique recently investigated to non-invasively disrupt these barriers and aid neuro-modulation. Herein, we demonstrate the use of a clinical LIFU device to modulate blood-tumor permeability and deliver combined chemotherapy in a novel preclinical model of brain metastases of breast cancer. Methods: Brain colonizing MDA-MB-231Br cells transfected with luciferase were injected into athymic Nu/Nu female mice by intracardiac injection into the left ventricle (per mouse 175,000 cells). Brain metastasis was allowed to develop, and tumor burden was monitored by bioluminescent imaging IVIS Spectrum CT (Perkin Elmer). Mice were randomized into sham or single treatment or combined treatment groups on Day 21 (Doxil nanoparticles 5.6 mg/kg or Paclitaxel 10mg/kg). Mice receiving LIFU were placed on a specially designed animal restraint with the skull partially immersed in degassed water. T1 weighted MRI was used to identify and localize sonication spots within the cortex, and mice were sonicated using 0.4uL/kg Definity© at 0.3 cavitation dose for 60sec. Treatment with LIFU was continued once weekly for three consecutive weeks. Survival and tumor burden was recorded until mice developed neurological symptoms. LIFU induced blood-brain barrier breakdown in healthy mice was also analyzed by in-situ brain perfusion with 14CSucrose, 3HGlucose, and 3HIvermectin to monitor BBB integrity and transporter function. Results: Mice receiving LIFU+paclitaxel+Doxil-NP showed significantly lower tumor burden (peak area 635.9) and higher median survival of 60 days as compared to vehicle (29d) and LIFU+vehicle (32d) groups. Analysis of BBB permeability in healthy mice demonstrated significantly higher passive permeability marker accumulation than non-sonicated contralateral hemisphere. Conclusion: Timing the combined chemotherapy with LIFU induced windows of maximal BBB opening resulted in slower tumor progression and increased survival in a preclinical model of breast cancer brain metastasis. Future directions aim to combine these results and evaluate underlying mechanisms of enhanced permeability post-LIFU. Citation Format: Tasneem A. Arsiwala, Kathryn E. Blethen, Samuel A. Sprowls, Ross A. Fladeland, Brooke Keilkowski, Morgan Glass, Trenton Pritt, Peng Wang, Ali Rezai, Paul R. Lockman. Focused ultrasound induced blood-tumor barrier permeability of combinatorial chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3461.

Abstract 6370: Assessing the effect of blocking CCL2-CCR2 axis on breast cancer brain metastasis

Abstract Among the 300,000 newly diagnosed breast cancer patients every year, 5~20% of them will develop symptomatic brain metastasis, whose 1-year survival rate is lower than 20%. Our group previously reported that brain tumor microenvironment (TME)-mediated PTEN downregulation in brain metastatic tumor cells increases the secretion of the chemokine CCL2, which enhances the infiltration of CCR2+ bone marrow derived myeloid cells, eventually promoting brain metastasis outgrowth. However, the mechanism of CCL2-CCR2 axis promoting brain metastasis is not clear, and the efficacy of targeting either CCR2 or CCL2 remained to be assessed. Here we show that Ccr2 antagonism significantly blocked Ccr2high myeloid cells trafficking in the blood and infiltration into the brain TME. However, Ccr2 antagonism did not sufficiently impede breast cancer brain metastasis in multiple preclinical models. Nevertheless, knocking down CCL2 or targeting CCL2 exhibited significant inhibition of brain metastasis growth. Subsequent analysis of the brain TME through mass cytometry demonstrated dramatically decreased infiltration of both Ccr2high and Ccr2low myeloid cell populations in the CCL2 targeted group, implying that the Ccr2low myeloid cells are also involved in promoting brain metastasis growth. Our data highlights the differential responses between targeting CCR2 or CCL2 in multiple preclinical brain metastasis models, which direct us to explore the function of Ccr2low myeloid cells in regulating brain metastasis progression. Our findings also suggest that carefully choosing between CCR2 or CCL2 as a target to control brain metastasis is important in clinical practice. Citation Format: Yimin Duan, Yutao Qi, Lin Zhang, Xiangliang Yuan, Dihua Yu. Assessing the effect of blocking CCL2-CCR2 axis on breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6370.

RNAi screen identifies essential regulators of human brain metastasis-initiating cells.

Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein-protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-β ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.

Abstract P1-12-05: Etirinotecan pegol: Survival advantage over standard of care drugs in a model of brain metastases of breast cancer

Abstract BACKGROUND: Brain metastasis of breast cancer is a significant cause of death among women with disseminated breast cancer. Chemotherapy, radiation, and surgery for these metastases provide only minimal benefit with considerable toxicity. The long-acting topoisomerase I inhibitor etirinotecan pegol (EP) is a novel treatment for disseminated breast cancer. EP consists of irinotecan attached to a 20kDa branched polymer via a releasable linker. The large molecular weight of EP results in tumor accumulation, including brain metastases, providing increased and sustained exposure to cytotoxic SN38. In the Phase 3 BEACON study, EP showed a significant survival benefit (HR 0.51; 95% CI 0.30-0.86) in a prespecified subgroup of women with history of treated, stable brain metastases compared to treatment of physicians choice (TPC) consisting of one of seven approved chemotherapy drugs (median OS 10.0 vs 4.8 mo). Nektar has filed a conditional marketing authorization in Europe for EP in patients with advanced breast cancer and brain metastases. A Phase 3 trial in this patient population is being initiated to support a potential U.S. filing. To further support the biological rationale for EP in these patients, an experimental preclinical model of brain metastases of breast cancer was conducted to compare the efficacy of EP to TPC in BEACON and the planned subsequent study. METHODS: Female athymic nude mice were inoculated with 1.75x105 MDA-MB-231-BrLuc cells via intracardiac injection. Starting on day 21, gemcitabine (60 mg/kg) and eribulin (1.5 mg/kg) were dosed IP every 4 days; EP (50 mg/kg), irinotecan (50 mg/kg), paclitaxel (6 mg/kg), vinorelbine (10 mg/kg), docetaxel (10 mg/kg), and vehicle (saline or dextrose) were dosed weekly via tail vein. Efficacy was measured by tumor burden and survival. Tumor burden was determined based on twice weekly bioluminescence measurements. Animals were euthanized according to international animal care guidelines. RESULTS: Median survival for all control arm groups (docetaxel, vinorelbine, eribulin, gemcitabine, irinotecan, and paclitaxel) were 39, 43, 41, 48, 35, and 42 days, respectively, none of which were significantly different from vehicle control median survival of 40 days (p>0.05). No animals receiving these drugs survived until the end of the 90-day trial. Median survival for the EP treatment group was 86 days (p<0.05) with a 40% survival rate at 90 days, the trial endpoint. Tumor burden increased nearly 100-fold in all TPC and vehicle groups, whereas EP significantly inhibited tumor growth. Quantitative autoradiography showed that EP accumulated ~10-fold in brain metastases, while accumulation in non-tumor brain tissue was only ~2-fold compared to irinotecan. EP at 50 mg/kg achieved plasma trough concentrations comparable to those observed in patients receiving the recommended dose of 145 mg/m2 every 21 days. CONCLUSIONS: In this model of brain metastases, EP preferentially accumulates in brain metastases, significantly reduces tumor burden progression and significantly improves survival in brain metastases of breast cancer compared to the most active chemotherapeutic agents available for advanced breast cancer and those used in the BEACON trial and planned subsequent Phase 3 study. Citation Format: Shah N, Mohammad AS, Adkins CE, Dolan EL, Griffith J, Jagannathan R, Hoch U, Lockman PR. Etirinotecan pegol: Survival advantage over standard of care drugs in a model of brain metastases of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-12-05.

TMOD-23. DEVELOPMENT AND APPLICATION OF A NOVEL MODEL OF HUMAN LUNG-TO- BRAIN METASTASIS TO IDENTIFY GENETIC REGULATORS OF BRAIN METASTASIS INITIATING CELLS

Brain Metastases (BM) are the most common type of cerebral tumor in adult, occurring at a rate 10 times greater than that of primary brain cancers. The inherent abilities of a primary tumor cell capable of initiating a BM resembles that of a cancer stem cell (CSCs). We hypothesize that a subgroup of CSCs, termed brain metastasis-initiating cells (BMICs), are responsible for the initiation of BM and are identifiable by an exclusive subset of genes that regulate self-renewal and metastasis. Despite the prevalence and lethality of BM, there is no clinically relevant model that fully reflects metastasis in patients. We recently generated a novel human-mouse xenotransplantation model of BM that allows for interrogation of each phase of the metastatic process from lung to brain, through injection of human patient-derived BMICs into immunocompromised mice. Comprehensive interrogation of human BM using RNA interference screens with subsequent validation in our model identified SPOCK1 and TWIST2 as novel regulators of brain metastasis-initiating cell (BMIC) self-renewal and migration to the brain, respectively. A prospective cohort of primary lung cancer specimens showed SPOCK1 and TWIST2 over-expressed only in patients who ultimately developed BM. Protein-protein interaction network mapping identified novel BMIC regulatory genes with significant prognostic value in lung cancer patients. Specifically, the most significant connector between SPOCK1 and TWIST2, INHBA, a TGF-β ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1, defining a novel pathway between INHBA, SPOCK1 and TWIST2 and implicating the involvement of the TGF-β signaling pathway in BM development. Our development of a novel preclinical model of BM, through which we have identified several novel BMIC regulators, present potential therapeutic targets that could aid in blockage of the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.

Vinorelbine Delivery and Efficacy in the MDA-MB-231BR Preclinical Model of Brain Metastases of Breast Cancer.

To evaluate vinorelbine drug exposure and activity in brain metastases of the human MDA-MB-231BR breast cancer model using integrated imaging and analysis. Brain and systemic metastases were created by administration of cancer cells in female NuNu mice. After metastases developed, animals were administered vinorelbine at the maximal tolerated dose (12 mg/kg), and were evaluated thereafter for total and unbound drug pharmacokinetics, biomarker TUNEL staining, and barrier permeability to Texas red. Median brain metastasis drug exposure was 4-fold greater than normal brain, yet only ~8% of non-barrier systemic metastases, which suggests restricted brain exposure. Unbound vinorelbine tissue/plasma partition coefficient, Kp,uu, equaled ~1.0 in systemic metastases, but 0.03-0.22 in brain metastases, documenting restricted equilibration. In select sub-regions of highest drug-uptake brain metastases, Kp,uu approached 1.0, indicating complete focal barrier breakdown. Most vinorelbine-treated brain metastases exhibited little or no positive early apoptosis TUNEL staining in vivo. The in vivo unbound vinorelbine IC50 for TUNEL-positive staining (56 nM) was 4-fold higher than that measured in vitro (14 nM). Consistent with this finding, P-glycoprotein expression was observed to be substantially upregulated in brain metastasis cells in vivo. Vinorelbine exposure at maximum tolerated dose was less than one-tenth that in systemic metastases in >70% of brain metastases, and was associated with negligible biomarker effect. In small subregions of the highest uptake brain metastases, compromise of blood-tumor barrier appeared complete. The results suggest that restricted delivery accounts for 80% of the compromise in drug efficacy for vinorelbine against this model.

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

The 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. Human 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. The 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. The 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).

Abstract A55: A novel mediator of brain metastasis from breast cancer

Abstract Purpose: Brain metastasis poses a major treatment challenge and remains an unmet clinical need. Finding novel therapies to prevent and treat brain metastases requires an understanding of the biology and molecular basis of the process, which currently is constrained by a dearth of experimental models and specific therapeutic targets. The purpose of this study was to identify molecular mediators of brain metastasis from breast cancer using novel preclinical brain metastasis models we developed recently. Methods: GFP-labeled cells were injected via tail vein into SCID/beige mice and metastatic colonization to the brain and lung was evaluated by fluorescent stereomicroscope and histology 8-weeks after injection. miRNA microarray was performed with miRNA 3.0 Array. Stable knockdown and overexpression of miR-141 was achieved with lentiviral vectors. miR-141 serum levels in patients with metastatic breast cancer was measured using quantitative PCR and was associated with outcome data. Results: We developed novel brain metastasis models in which tail-vein injection of parental triple-negative and a HER2-overexpressing inflammatory breast cancer lines led to a high rate of brain metastases (67%) in SCID/Beige mice (SUM149, 6 of 9 mice; MDA-MB-IBC3, 10 of 15 mice). Sub-lines derived from brain metastases (BrMS) or lung metastases (LuMS) tissues were morphologically and molecularly distinct. The BrMS showed epithelial morphology and overexpressed epithelial markers and miR-141 while the LuMS showed mesenchymal morphology and overexpressed mesenchymal markers. Knockdown of miR-141 significantly inhibited metastatic colonization to the brain compared to parental controls in both cell lines (miR-141 knockdown vs. control: SUM149, 0 of 8 mice vs. 6 of 9 mice, p=0.009; MDA-MB-IBC3, 2 of 14 mice vs. 10 of 15 mice, p=0.007). Further, ectopic expression of miR-141 in non-expressing MDA-MB-231 significantly enhanced brain metastatic colonization via tail-vein injection (5 of 9 mice vs. 0 of 10 mice, P=0.02). On multivariate analyses high serum level of miR-141 was an independent predictor of progression free survival [HR 4.8 (95%CI, 2.6-8.7), P<0.001] and overall survival [HR 7.0 (95%CI 3.5-15.1), P<0.001] in patients with metastatic breast cancer. Importantly, it is associated with shorter brain metastasis free survival (P=0.03). Conclusion: We have generated novel brain metastasis models that can be utilized to develop therapies. Further, we demonstrated miR-141 as a key regulator of brain metastasis and provided clinical evidence supporting the prognostic relevance of miR-141 in patients with brain metastasis. Our work could lead to novel targets for the prevention and treatment of brain metastasis that could improve patient survival in addition to fundamentally advancing the field of brain metastasis research. We propose that miR-141 should be examined as a biomarker and potential target to prevent or treat brain metastases. Citation Format: Bisrat G. Debeb, Lara Lacerda, Simone Anfossi, Parmeswaran Diagaradjane, Richard Larson, Khoi Chu, Lei Huo, Daniel Smith, Li Li, Cristina Ivan, Pamela Allen, Xiang Zhang, George Calin, Savitri Krishanmurthy, Thomas Buchholz, Naoto Ueno, James Reuben, Wendy A. Woodward. A novel mediator of brain metastasis from breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A55.

Abstract 3693: Quantitative fluorescent microscopy to measure permeability in brain adjacent to tumor

Abstract Introduction: Chemotherapeutics typically distribute poorly into healthy brain due to the restrictive characteristics of the blood-brain barrier (BBB). Brain tissue immediately surrounding tumors (brain adjacent to tumor, BAT) is an important target for chemotherapy as tumorigenic cells proliferate and invade this region during tumor progression. Drug permeability in BAT is not well defined and discrepancies exist among studies estimating drug accumulation in this region. This study seeks to address these discrepancies by evaluating BAT permeability using quantitative fluorescent microscopy (QFM) in a preclinical model of brain metastases of breast cancer. Methods: 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 were allowed to grow for 4-6 weeks. After neurological symptoms developed, a fluorescence marker (Texas Red 625Da) was injected intravenously and allowed to circulate. Blood and brain concentrations of Texas Red were determined by QFM and regional brain uptake was determined. Lastly, we mapped fluorescent tracer concentration at 8 μm increments extending 300 μm inwardly and outwardly from the edge of selected metastases. Results: The permeability of Texas Red in MDA-MB-231Br brain metastases are heterogeneous, ranging nearly ∼10-fold between metastases for Texas Red 625Da. Quantitatively, most metastases (∼75%) exhibit modest increases in permeability (∼2 to 5-fold, p<0.05, n = 133), while ∼15% of metastases have greater than a 5-fold increase in permeability (p0.05, n = 15). Texas Red concentrations in BAT regions are elevated (p<0.05) relative to healthy brain and approximate healthy brain levels ∼100μm beyond tumor margins. The estimated accumulation of fluorescent tracer in BAT region reaches ∼30-40% of average concentrations observed in the core region of metastases. Conclusions: The BAT of experimental metastases of breast cancer exhibit elevated permeability relative to healthy brain. The increased accumulation of tracer in the BAT may result from either an increase in permeability at the local BAT region or from diffusion and flow of the tracer from the tumor core region into the BAT, or a combination thereof. However, this model suggests that this region accumulates approximately ∼50-60% less tracer than the respective tumor. Clinically, these data suggest that chemotherapeutic uptake in the BAT, where tumor cell replication and invasion occurs, remains limited and likely contributes to poor chemotherapeutic efficacy. Citation Format: Chris E. Adkins, Rajendar K. Mittapalli, Paul Lockman. Quantitative fluorescent microscopy to measure permeability in brain adjacent to tumor. [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 3693. doi:10.1158/1538-7445.AM2015-3693

Abstract 3087: MicroRNA 141: A novel regulator of brain metastasis from breast cancer

Abstract Purpose: Brain metastasis poses a major treatment challenge and remains an unmet clinical need. Finding novel therapies to prevent and treat brain metastases requires an understanding of the biology and molecular basis of the process, which currently is constrained by a dearth of experimental models and specific therapeutic targets. The purpose of this study was to identify molecular mediators of brain metastasis from breast cancer using novel preclinical brain metastasis models we developed recently. Methods: GFP-labeled cells were injected via tail vein into SCID/beige mice and metastatic colonization to the brain and lung was evaluated by fluorescent stereomicroscope and histology 8-weeks after injection. miRNA microarray was performed with miRNA 3.0 Array. Stable knockdown and overexpression of miR-141 was achieved with lentiviral vectors. miR-141 serum levels in patients with metastatic breast cancer was measured using quantitative PCR and was associated with outcome data. Results: We developed novel brain metastasis models in which tail-vein injection of parental triple-negative and a HER2-overexpressing inflammatory breast cancer lines led to a high rate of brain metastases (67%) in SCID/Beige mice (SUM149, 6 of 9 mice; MDA-MB-IBC3, 10 of 15 mice). Sub-lines derived from brain metastasis (BrMS) or lung metastasis (LuMS) tissues were morphologically and molecularly distinct. The BrMS showed epithelial morphology and overexpressed epithelial markers and miR-141 while the LuMS showed mesenchymal morphology and overexpressed mesenchymal markers. Knockdown of miR-141 significantly inhibited metastatic colonization to the brain in both cell lines (miR-141 knockdown vs. control: SUM149, 0 of 8 mice vs. 6 of 9 mice, p = 0.009; MDA-MB-IBC3, 2 of 14 mice vs. 10 of 15 mice, p = 0.007). Ectopic expression of miR-141 in non-expressing MDA-MB-231 significantly enhanced brain metastatic colonization via tail-vein injection (5 of 9 mice vs. 0 of 10 mice, P = 0.02). On multivariate analyses high serum level of miR-141 was an independent predictor of progression free survival [HR 4.8 (95%CI, 2.6-8.7), P<0.001] and overall survival [HR 7.0 (95%CI 3.5-15.1), P<0.001], and was associated with shorter brain metastasis free survival (P = 0.03). Conclusion: We have generated novel brain metastasis models, demonstrated miR-141 as a key regulator of brain metastasis and provided clinical evidence supporting the prognostic relevance of miR-141 in patients with brain metastasis. We propose that miR-141 should be examined as a biomarker and potential target for the prevention and treatment of brain metastases from breast cancer. Citation Format: Bisrat G. Debeb, Lara Lacerda, Simone Anfossi, Parmesh Diagaradjane, Khoi Chu, Lei Huo, Caimiao Wei, Richard Larson, Adam Wolfe, Wei Xu, Li Li, Daniel Smith, Cristina Ivan, Pamela Allen, Savitri Krishnamurthy, George Calin, Xiang Zhang, Thomas Buchholz, Naoto Ueno, James Reuben, Wendy Woodward. MicroRNA 141: A novel regulator of brain metastasis from 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 3087. doi:10.1158/1538-7445.AM2015-3087

Abstract 2653: Anti-cancer antibody trastuzumab-melanotransferrin conjugate (BT2111) for the treatment of metastatic HER2+ breast cancer tumors in the brain: An in vivo study

Abstract Background: Approximately 20% of patients with Her2+ metastatic breast cancer may be diagnosed with brain metastases. Trastuzumab, has been used successfully to treat peripheral Her2+ breast cancers, but has shown limited efficacy in brain metastases. Human melanotransferrin analog (MTf) is a vector to transport drugs across the blood brain barrier. In the current work, we investigated brain and brain metastases uptake of a MTf-trastuzumab conjugate (BT2111), and determined if the conjugate had efficacy in reducing the number and size of metastatic lesions in a preclinical model of brain metastases of breast cancer. Methods: Human metastatic brain seeking breast cancer cells (MDA-MB-231-Her2) were injected intracardially in 8 week old female NuNu mice (n=6-12 per group), allowed to circulate, and eventually develop experimental brain metastases. To examine drug uptake, radiolabeled conjugate (BT2111), trastuzumab and MTf was injected intravenously, and allowed to circulate for 30 min, 2 and 8 hours. Serial blood samples were drawn during circulation and terminal brain and blood samples were obtained. Drug uptake (Kin) was analyzed in relation to metastasis size and blood-tumor barrier permeability. To investigate in-vivo activity against brain metastases, we administered equimolar doses of the conjugate, and relevant controls in separate groups, biweekly starting 21 days after intracardiac injection of the metastatic cancer cells. Efficacy was assessed by determining both the number and size of metastases in brain at defined terminal endpoints. Results: Uptake of trastuzumab in “normal” brain (brain distant to tumor; BDT) was 0.17 + 0.004 x 10-6 mL/s/g and nearly 10 fold higher on average in metastatic lesions (1.1 + 0.04 x 10-6 mL/s/g). MTf uptake into BDT (18.4 + 0.5 x 10-6 mL/s/g) and brain metastases (53.0 + 3.1 x 10-6 mL/s/g) was significantly higher (p<0.01) than that of the antibody alone. BT2111 was nearly 30 fold higher (p<0.01) than that of the antibody alone in BDT (3.91 + 0.3 x 10-6 mL/s/g) and 10 fold higher (p<0.01) in metastases (10.7 + 0.43 x 10-6 mL/s/g). BT2111 reduced the number of preclinical human HER2+ breast cancer metastases in the brain of control animals (85 ± 6.3) by 68% (27.6 ± 3.9; P<0.001). Further, lesions in control animals were 54% larger (1.7 ± 0.7 µm2) than those in the BT2111 treated animals (0.78 ± 0.6 µm2; P<0.001). In contrast, trastuzumab alone had no effect on reducing the number of metastases (74 ± 5.0, P>0.05) and was associated with only a minimal (21%) reduction in metastasis size (1.34 ± 0.04 µm2). Conclusions: These results provide evidence that the MTf-trastuzumab conjugate, BT2111, has efficacy in treating brain metastasis in preclinical models and validates the role MTf has as a vector for the transport of antibodies across the BBB. Citation Format: Mohamed Nounou, Chris Adkins, Tori R. Terrell, Afroz Mohamed, Tim Vitalis, Reinhard Gabathuler, Paul R. Lockman. Anti-cancer antibody trastuzumab-melanotransferrin conjugate (BT2111) for the treatment of metastatic HER2+ breast cancer tumors in the brain: An in vivo study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2653. doi:10.1158/1538-7445.AM2014-2653

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.

Highlights of This Issue

Despite the breakdown of the blood-brain barrier in brain metastases of breast cancer, traditional chemotherapy does not typically accumulate at sufficient concentrations to induce cytotoxicity. Here, Mittapalli and colleagues have designed a nanoconjugate of hyaluronic acid and paclitaxel that is small enough to passively get through the leaky tumor vasculature and to enter into the tumor interstitial space. Once there, the hyaluronic polymers promote CD44 receptor-mediated internalization of the nanoconjugate. Once the nanoconjugate is taken up into the cell, paclitaxel remains cytotoxic. This nanoconjugate system reduced lesion burden in the brain and improved overall survival in a preclinical model of brain metastases of breast cancer.Members of conventional drug classes targeting the MET pathway have faced multiple challenges, including specificity, selectivity, partial agonism, and HGF-dependent activity. Olwill and colleagues discovered and characterized for the first time a novel MET inhibitor (PRS-110) based on a new class of biopharmaceuticals called Anticalins. The monovalent binding nature of Anticalins makes them exquisitely suited for the antagonism of receptor tyrosine kinases, unlike bivalent mAbs, which can lead to receptor activation. The manuscript demonstrates the drug-like properties of this novel inhibitor, which supports its evaluation in a future clinical trial.Synthetic cannabinoids (CB), such as WIN 55,212-2, inhibit the growth of solid tumors and mechanisms can be CB receptor-dependent or -independent. Sreevalson and Safe showed that WIN 55,212-2 inhibited colon cancer cell growth and survival, and an important underlying pathway for these responses was due to downregulation of the specificity protein (Sp) transcription factors Sp1, Sp3, Sp4, and Sp-regulated genes. The mechanism was CB receptor-independent and due to induction of protein phosphatase 2A-mediated disruption of miR-27a-ZBTB10 (an Sp repressor). The design of future applications of WIN 55,212-2 for individual or combined chemotherapies can now be guided by these novel mechanistic insights.MicroRNA-141 (miR-141) was shown to play an important role in tumorigenesis. However, the exact role of miR-141 in pancreatic cancer has not yet been elucidated. In the present study, Zhao and colleagues discovered that downregulation of miR-141 in pancreatic cancer was correlated with poor overall survival and MAP4K4 was the direct target of miR-141. Restoration of miR-141 increased the chemosensitivity in vitro. Moreover, overexpression of miR-141 or knockdown of MAP4K4 inhibited the tumor growth of mice xenografts in vivo. All these findings suggest that miR-141 targets MAP4K4 and acts as a tumor suppressor in pancreatic cancer cells.

Abstract 1559: Evaluation of MR imaging biomarkers of the vascular and infiltrative phenotype in intracranial MDA-MB-231 tumors.

Abstract Tumors growing in the brain often invade into the parenchyma and co-opt the existing vasculature leaving the blood brain barrier (BBB) intact; extravasation of the Gd-DTPA contrast agent used in MRI is therefore precluded. Anti-angiogenic agents can restore the compromised BBB of neovascular regions resulting in ‘pseudoresponse’ in human glioblastoma (GBM), and preclinical brain metastasis models. Non-invasive imaging strategies are required to accurately interrogate brain specific growth patterns. Preclinical models that faithfully emulate infiltrative gliomas are limited. Intracranial implantation of MDA-MB-231 breast cancer cells results in tumors exhibiting substantial local invasion, principally occurring along existing blood vessels. Multiparametric MRI was used to identifying sensitive biomarkers of infiltrative tumor growth in this model. Alteration in the vascular phenotype was assessed in response to pan-VEGFR inhibitor cediranib or cross reactive human/mouse anti-VEGF-A antibody B20-4.1.1. Intracranial MDA-MB-231 tumors were heterogeneous; areas of high apparent diffusion coefficient (ADC, mm2s−1), a biomarker of cellularity, correlated with the most hyperintense regions on T2-weighted images and areas of histologically confirmed edema. Regions of high Gd-DTPA extravasation were distinct from the areas of high ADC, but correlated with regions of high fractional blood volume (fBV) detected using intravascular USPIO particles. Infiltrative regions identified on H&amp;E images corresponded to areas of limited Gd-DTPA leakage and low fBV. Cediranib (3 x 6mg/kg) caused significant tumor growth delay after 48h compared to controls (cediranib 160 ± 9% pre-treatment volume, vehicle 210 ± 14%, p&amp;lt;0.05, unpaired t-test). A 50% reduction in Ktrans (min−1), a compound biomarker of permeability/perfusion, was observed following cediranib treatment (p&amp;lt;0.05, paired t-test). Perfused tumor area, assessed using Hoechst 33342, was lower in treated tumors (cediranib 5.4 ± 0.6%, vehicle 9.1 ± 1.5%, p&amp;lt;0.05), confirming reduced vascular function. Cediranib also led to a 9% reduction in tumor ADC (p&amp;lt;0.05), possibly due to resolution of edema, as observed in GBM patients. A single 10mg/kg dose of B20-4.1.1 resulted in significant tumor growth delay over 48h (B20-4.1.1 120 ± 18%, control antibody 230 ± 8%) and a 58% reduction in Ktrans (p&amp;lt;0.05), but no change in ADC was observed. BBB integrity following treatment is being evaluated to inform on the contributions of its restoration, and altered vascular function, to the observed changes in vascular imaging biomarkers. Intracranial MDA-MB-231 tumors provide a useful model for the development of imaging biomarkers of the infiltrative and vascular phenotype of tumors in the brain, which are essential for the assessment of the novel invasive orthotopic glioma models currently being developed, and their response to therapeutics. Citation Format: Jessica KR Boult, Alexa Jury, Sergey Popov, Lara Perryman, Gary Box, Chris Jones, Suzanne A. Eccles, Simon P. Robinson. Evaluation of MR imaging biomarkers of the vascular and infiltrative phenotype in intracranial MDA-MB-231 tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1559. doi:10.1158/1538-7445.AM2013-1559

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.

Abstract 2373: Heterogeneity of permeability in a preclinical model of brain metastases of breast cancer does not correlate with vascular density, HIF-1α expression, or tumor survival

Abstract Introduction: High vascular density of brain metastases of breast cancer has been correlated with both poor patient prognosis and with potential chemotherapeutic success. Inversely a low vascular density implies there would be decreased distribution of anti-cancer chemotherapeutics, and over time reduced efficacy of tumor kill. Given that there is heterogeneity in response to chemotherapeutics in human brain metastases and metastases are different than primary tumors; we used a novel brain metastases of breast cancer model to characterize vascular density, and corresponding changes in tumor permeability, hypoxia and survival. Methods: Brain seeking metastatic tumor lines MDA-MB-231-Br and 4T1-Br5 were injected via the left cardiac ventricle in separate experiments and brain metastases developed for ∼28 days and ∼15 days respectively. After development, both 14C-AIB and indocyanine green (ICG) were injected and allowed to circulate for 10 and 1 min respectively. Near infrared imaging was used to identify ICG filled vessels to quantify vascular density of lesions compared to normal brain. To determine permeability, concentrations of 14C-AIB in blood and brain tissue were calculated using autoradiography. Immunofluorescent analysis was performed using anti-HIF-1α and anti Ki67 antibodies and co-stained with DAPI. Results: A significant amount of metastatic lesions were observed to have developed in each model. Lesions varied in size from 0.028 to 2.3mm2. Vascular density was significantly decreased (MDA 176 ± 96 and 4T1 201 ± 76) compared to control brain (429 ± 25) and to an RG2 implanted glioma used as a positive control (736 ± 168). Based on accumulation of 14C-AIB, metastases (range: 22 nCi/g to 1682 nCi/g) were considered to be either permeable (3 fold or greater uptake compared to adjacent control tissue) or non-permeable. There was no strong correlation between lesion size and permeability (r2=0.23) or vascular density (r2 = 0.06). HIF-1α was expressed in both permeable and non-permeable lesions. Lastly, no significant difference was seen in the percentage of Ki67 positive cells in the MDA (permeable 41 ± 2.9, n=7; and non-permeable 40.8 ± 2.7, n=14) and 4T1 model (permeable 52.0 ± 4.6, n=6; and non-permeable 45.0 ± 2.8, n=6). Discussion: This data provides a correlation of permeability to vascular density, HIF-1α and Ki67 in a brain metastases model. While, both brain model systems exhibited a high degree of heterogeneous permeability, in neither model did permeability strongly correlate with vascular density, lesion size, HIF-1α expression and positivity for Ki67. Based upon the data, this experimental model may reflect the heterogeneity seen in human brain metastases of breast cancer and suggests there are other biological factors besides vascular density which correlate with drug distribution and or tumor/survival growth. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2373.