Brain Metastasis Formation Research Articles

Metabolic advantages and vulnerabilities in brain metastases.

Metabolic adaptations permit tumor cells to metastasize to and thrive in the brain. Brain metastases continue to present clinical challenges due to rising incidence and resistance to current treatments. Therefore, elucidating altered metabolic pathways in brain metastases may provide new therapeutic targets for the treatment of aggressive disease. Due to the high demand for glucose in the brain, increased glycolytic activity is favored for energy production. Primary tumors that undergo Warburg-like metabolic reprogramming become suited to growth in the brain microenvironment. Indeed, elevated metabolism is a predictor of metastasis in many cancer subtypes. Specifically, metabolic alterations are seen in primary tumors that are associated with the formation of brain metastases, namely breast cancer, lung cancer, and melanoma. Because of this selective pressure, inhibitors of key metabolic factors may reduce tumor cell viability, thus exploiting metabolic pathways for cancer therapeutics. This review summarizes the metabolic advantages and vulnerabilities of brain metastases.

A Non-random Spatial Distribution of Brain Metastases

We sought to quantitatively describe the spatial distribution of brain metastases (BM) in a single institution experience treating metastatic disease to the brain, with the alternative hypothesis that BM spatial frequency does not follow a stochastic distribution. It has been demonstrated that metastatic disease infrequently occurs in the hippocampus, leading to a low risk of failure within the region when using hippocampal-sparing whole brain radiotherapy protocols. Probabilistic mapping of BM to other regions of the brain, however, has not been previously quantified. We retrospectively analyzed patients with newly diagnosed and recurrent brain metastases from 2006-2016. Each lesion was manually defined on MRI with T1 gadolinium-enhanced imaging. Standard brain atlases dividing the brain into 52 anatomical regions were used to identify the frequency of metastasis in each region. This was compared to the expected probability of occurrence, which was assumed to be a random distribution based on region volume. The quantified BM maps were analyzed across different primary cancers and histologic subtypes. Results indicate a non-random distribution of brain metastases that agree with prior reports of a low risk of developing disease in the hippocampi. Analyses revealed additional regions – the frontal pole, lateral occipital cortex, supramaginal gyrus, and temporal pole – had a lower incidence of BM than predicted. We also demonstrate an increase in risk of involvement for several regions, notably the precentral gyrus and paracingulate gyrus relative to what would be expected based on brain volume (p<0.01, corrected for multiple comparisons). Clinically, a more granular understanding of BM distribution may permit sparing of additional brain regions with partial brain radiotherapy. This analysis forms the framework for a deterministic model to approximate the non-stochastic process of BM formation. Further analyses correlating prognostic factors such as age, histology, previous therapeutic interventions and additional imaging data (e.g. cerebral blood flow mapping) with our current findings are necessary to further develop this model.

Claudin-5 regulates blood-brain barrier permeability by modifying brain microvascular endothelial cell proliferation, migration, and adhesion to prevent lung cancer metastasis.

To investigate the roles of Claudin-5 (CLDN5) in regulating the permeability of the blood-brain barrier (BBB) during lung cancer brain metastasis. By silencing and overexpressing the CLDN5 gene in human brain vascular endothelial (hCMEC/D3) cells, we demonstrated the attenuation of cell migration ability and CLDN5's significant positive role in cell proliferation in CLDN5-overexpressing hCMEC/D3 cells and observed the opposite result in the CLDN5 knockdown group. The reinforced CLDN5 expression reduced the paracellular permeability of hCMEC/D3 cells and decreased the invasion of lung adenocarcinoma A549 cells. Overall, 1685 genes were found to be differentially expressed between the CLDN5-overexpressing cells and the control cells using the Affymetrix Human Transcriptome Array 2.0 (HTA 2.0), and the function of these genes was determined by Gene Ontology and pathway analyses. The possible biological functions of the 1685 genes include cell proliferation, adhesion molecules, and the Jak-STAT, PI3K-Akt, Wnt, and Notch signaling pathways. The identified sets of mRNAs that were specific to CLDN5-overexpressing hCMEC/D3 cells were verified by a qRT-PCR experiment. CLDN5 regulates the permeability of BBB by regulating the proliferation, migration, and permeability of hCMEC/D3 cells, especially through the cell adhesion molecule signaling pathway, to enhance the function of the tight junctions, which was involved in reducing the formation of lung cancer brain metastasis.

Mutations targeting the coagulation pathway are enriched in brain metastases

Brain metastases (BMs) are the most common malignancy of the central nervous system. Recently it has been demonstrated that plasminogen activator inhibitor serpins promote brain metastatic colonization, suggesting that mutations in serpins or other members of the coagulation cascade can provide critical advantages during BM formation. We performed whole-exome sequencing on matched samples of breast cancer and BMs and found mutations in the coagulation pathway genes in 5 out of 10 BM samples. We then investigated the mutational status of 33 genes belonging to the coagulation cascade in a panel of 29 BMs and we identified 56 Single Nucleotide Variants (SNVs). The frequency of gene mutations of the pathway was significantly higher in BMs than in primary tumours, and SERPINI1 was the most frequently mutated gene in BMs. These findings provide direction in the development of new strategies for the treatment of BMs.

Abstract LB-215: Astrocytes promote brain metastasis of triple-negative great cancer through TGF-β2/ANGPTL4 axis

Abstract Breast cancer has a high propensity for brain metastasis (BM), especially in triple negative (ER-/PR-/Her2-) breast cancer (TNBC) subsets. To date, the underlying mechanism for BM is poorly understood, and no targeted therapies have been developed. Metastatic outcome depends on the interactions of metastatic cells with a specific organ microenvironment. Astrocyte is the most abundant cell type in the brain microenvironment. Previous observations suggest a role for astrocytes in tumor BM, but the underlying mechanism is unknown. Our goal is to determine how astrocytes promote TNBC cell BM and to develop novel therapeutic strategies to treat this deadly disease To investigate the effect of astrocyte on BM formation of TNBC cells, we passaged TNBC cells in astrocyte-cultured medium (ACM), and compared these cells to DMEM passaged cells (control) for their genetic and functional difference. Next, we determined the role of a significant ACM up-regulated gene, angiopoietin-like 4 (Angptl4), in BM formation in a mouse model. Furthermore, we investigated the mechanisms for how TNBC cells and astrocytes interaction to up-regulate the ANGPTL4 expression, and how ANGPTL4 contributes to BM formation. Finally, we analyzed the ANGPTL4 expression in brain metastatic tumors and plasma from TNBC patients. We found that 1) ACM-conditioned MDA-MB-231 cell facilitated BM formation and up-regulated ANGPTL4 expression. In addition to 231 cells, other TNBC cells (MDA-MB-468 and HCC1937) showed similar up-regulation of ANGPTL4 expression; 2) knockdown of Angptl4 by shRNA reduced ACM-induced BM formation and mortality; 3) astrocytes produced transforming growth factor-beta2 (TGF-β2), which was enhanced by tumor cell-derived IL-1β and TNF-α. The TGF-β2 through Smad signaling in part is responsible for the up-regulation of ANGPTL4 in TNBCs; 4) passaging TNBC cells in ACM led to a large amount of cells died, but cells survived in ACM revealed cancer stem cell like phenotype with CD44+/CD24-, α-SMA high and ID1 low. They started to gradually grow when extended to passage them in ACM. However, knockdown of ANGPTL4 resulted to a contrary consequence; and 5) the higher ANGPTL4 level was observed in brain mets compared to primary tumors, and the higher ANGPTL4 level in plasma was also identified in TNBC patients with BM compared to without BM. Collectively, our results suggest that TNBC cells interact with astrocytes to promote astrocytes secrete TGF-β2, which in turn up-regulates ANGPTL4 expression in TNBC cells. This up-regulation of ANGPTL4 attributes to the survival and activation of a small population of breast cancer stem like cells from TNBC to facilitate BM formation. This work presents a novel mechanism of how astrocytes and tumor cells communicate in a paracrine and autocrine manner to facilitate BM of TNBC. Our work suggests that ANGPTL4 is a potential therapeutic molecule that can be targeted for the treatment of BM of TNBCs. Note: This abstract was not presented at the meeting. Citation Format: Ling Wang, Zhimin Hou, Emily Gronseth, Janet Rader, David Harder, Ramani Ramchandran. Astrocytes promote brain metastasis of triple-negative great cancer through TGF-β2/ANGPTL4 axis [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 LB-215. doi:10.1158/1538-7445.AM2017-LB-215

Abstract 1090: Preclinical evaluation of combination therapy with dasatinib and sorafenib for the treatment of brain metastases

Abstract Brain metastases, the most common type of intracranial tumors in adults, carry a dismal prognosis with a median survival of less than 18 months regardless of primary status or treatment given. As part of an ongoing project, this study is concerned with the preclinical evaluation of targeted combination therapy for the treatment of brain metastases. The cell line used in this study was the H1915 (ATCC® CRL-5904TM) human lung cancer cell line, which was originally derived from a brain metastasis from primary lung adenocarcinoma. The cell line was stably transfected with a plasmid that allows expression of firefly luciferase (i.e., H1915-luc) so that the tumor growth in a mouse orthotopic model of brain metastases can be assessed in real time using the Xenogen in vivo bioluminescence imaging (BLI). Our previous study has demonstrated that the synergistic effect of combined dasatinib and sorafenib treatment was cell-line dependent. In this study, the cytotoxicity of dasatinib and sorafenib, when used alone or in combination at the fixed molar ratios of 3:1, 1:1 and 1:3 (dasatinib:sorafenib), was further examined in H1915-luc cells using the MTT assay. The results demonstrated that simultaneous and continuous exposure of H1915-luc cells to dasatinib and sorafenib at the fixed concentration ratio of 1:1 and 1:3 for 72 h showed synergism (the combination index value between 0.7 and 0.8) at effect levels of 40 and 80% inhibition of cell viability. For the in vivo study, an orthotopic mouse model of brain metastases was established by injecting H1915-luc cells into the right general carotid artery of individual athymic nude mice followed by permanent ligation. The preliminary study showed that following the intracarotid injection of H1915-luc cells, brain metastases were formed and readily to be detected by BLI. The obtained whole brain samples were fixed in OCT and stored at -80oC. The brain sections are to be subjected to the immunofluorescence double staining of CD31 and firefly luciferase to assess the potential effect of the dasatinib-sorafenib combination on microscopic brain metastasis formation and progression and vessel co-option. Given the fact that brain metastases usually have a poor response to chemotherapeutic agents in part due to the presence of blood-brain barrier, a pharmacokinetic study is underway to characterize the distribution of sorafenib and dasatinib in normal brain tissues and brain metastases using the serial blood sampling and brain microdialysis technique. Overall, results from the in vitro cytotoxicity study demonstrated the synergistic effect of dasatinib and sorafenib on H1915-luc human lung cancer cells, providing a rationale for the in vivo evaluation the potential of this combination for the treatment of brain metastases from lung carcinomas. Citation Format: Qingyu Stephanie Zhou, Xiaofang Guo. Preclinical evaluation of combination therapy with dasatinib and sorafenib for the treatment of brain metastases [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 1090. doi:10.1158/1538-7445.AM2017-1090

OS03.1 The role of the immune system in facilitating the formation of brain metastasis of breast cancer

OS03.1 The role of the immune system in facilitating the formation of brain metastasis of breast cancer

Role of HYAL1 expression in primary breast cancer in the formation of brain metastases.

The incidence of brain metastases in breast cancer patients has increased in the last years. However, the knowledge about tumor cell invasion in the brain is still very limited. Based on our recent study on cDNA microarray data of breast cancer patients, we hypothesized that two enzymes involved in the hyaluronan metabolism, namely, hyaluronan synthase 2 (HAS2) and hyaluronidase 1 (HYAL1) are associated with brain metastases formation. Protein expression levels of hyaluronan, HAS2, and HYAL1 were analyzed in primary breast cancer, and metastatic tissue samples from different localizations (brain, bone, skin, liver, and lung) were included in four different cohorts by immunohistochemistry. Correlations of expression levels with clinical and pathological parameters were performed within the individual cohorts. Higher HYAL1 expression was detected among primary tumors from patients with subsequent brain metastases compared with those without brain metastases (p=0.011). Interestingly, brain metastatic tissue showed a significantly reduced HYAL1 expression compared with the corresponding primary tumor (p=0.003). HYAL1 expression in brain metastases was also significantly lower than in skin, liver, and lung metastases. Further, hyaluronan staining in brain metastases was mainly located on the surface of the tumor cells, whereas in all other metastatic sites hyaluronan was only detected in the extracellular matrix. We could not show an association of HAS2 with the formation of brain metastases. In conclusion, our results suggest that the enzyme HYAL1 plays a role in tumor dissemination and brain-specific colonization, rather than in subsequent metastatic out-growth.

Direct mapping of melanoma cell - endothelial cell interactions.

The most life-threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood-brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high-resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi-physiological conditions. Hereby, we present our latest results by using this melanoma-decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single-cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic-related processes.

Abstract 1571: PTEN is an important mediator of tumor and glia cell crosstalk in breast cancer brain metastasis

Abstract With the improvement of therapeutic options for the treatment of breast cancer, the development of brain metastases has become a major limitation to life expectancy in many patients. Loss of Phosphatase and tensin homolog (PTEN) is correlated with occurrence of breast cancer brain metastases, but its functional role in metastases formation remains unclear. Here, we first assessed the clinical role of PTEN expression in brain metastases, by performing immunohistochemical analyses on a TMA consisting of 132 breast cancer brain metastases samples. Loss of PTEN protein expression was found in 48.6% of brain metastases, and significantly associated with the triple-negative breast cancer subtype (67.5%, p = 0.01). Loss of PTEN was furthermore significantly associated with shorter overall survival (p = 0.048). We then investigated the biological role of PTEN in breast cancer brain metastasis formation by overexpressing PTEN in the triple-negative brain metastatic breast cancer cell cline MDA-MB-231 BR. Overexpression of PTEN was shown to reduce the AKT pathway activation especially by reducing the Akt1 kinase activity. Coculture experiments with glia cells (astrocytes and microglia) showed that astrocytes can inhibit cell proliferation of the tumor cells in a PTEN dependent manner. Both astrocytes and microglia promote migration of MDA-MB-231 BR cells in a PTEN-dependent manner in vitro. Using an organotypic brain slice model as a representation of the brain tumor microenvironment, the PTEN-dependent invasion of tumor cells could be confirmed. Furthermore, PTEN overexpression reduced the astrocyte activation. Coculture experiments with glia cells showed PTEN-dependent, differential cytokine expression. We could identify an endocrine and paracrine regulation between tumor cells and astrocytes, where GM-CSF, CSF2RA, EGF and PTEN play an important role. In conclusion, loss of PTEN is associated with poor prognosis and is an important mediator of a “vicious circle” mediated by the cross talk between tumor cells and glia cells. Citation Format: Harriet Wikman, Ina Hohensee, Han-Ning Chuang, Astrid Grottke, Stefan Werner, Alexander Schulte, Jakob Matschke, Markus Glatzel, Stefan Horn, Manfred Jücker, Tobias Pukrop, Klaus Pantel. PTEN is an important mediator of tumor and glia cell crosstalk in breast cancer brain metastasis. [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 1571.

Cancer Cell–Astrocyte Gap Junctions Promote Brain Metastasis Formation

Abstract Carcinoma–astrocyte gap junctions drive growth and chemoresistance of brain metastases.

Breast Cancer Exosomes Breach the Blood-Brain Barrier.

Breast Cancer Exosomes Breach the Blood-Brain Barrier.

Abstract P6-17-06: Relevance of the hyaluronidase-1 (HYAL1) in brain metastasis formation of breast cancer patients

Abstract The incidence of brain metastases in breast cancer (BMBC) patients has increased in the last years and represents the major life-limiting problem for metastatic breast cancer (BC) patients. However, the knowledge about tumor cell invasion in the brain is still very limited and new markers for brain metastasis incidence are urgently needed in order to early detect high risk patients. In a recent study based on cDNA microarray data of primary breast tumors, we could show that mRNA expression of certain glycosylation enzymes significantly correlates with an organ-specific metastatic spread. Interestingly, hyaluronan synthase 2 (HAS2) and hyaluronidase-1 (HYAL1), both genes involved in hyaluronan (HA) metabolism, showed an independent prognostic value and a significant correlation with brain metastasis formation. In order to corroborate the role of these enzymes at protein level, we examined the expression of HYAL1 and HAS2 on a tissue microarray including 200 primary BC samples. Here, the prognostic impact of HAS2 could not be validated, whereas for HYAL1 shorter disease free survival (DFS) was observed for patients with high HYAL1-expression levels. This trend could be additionally verified in a second cohort of 107 BC samples, using western blot analysis. Moreover, significantly higher HYAL1 expression was detected among primary tumors with subsequent brain metastases compared with those without brain metastases using immunohistochemistry (IHC). No impact of HYAL1 expression on disease progression of BCBM patients could be observed after analysis of 87 brain metastasis samples. Here, quantification of tumor-associated HA revealed a significant positive correlation with triple negative tumors and a trend towards shorter progression-free survival. Taken together, our data suggest a role of the enzyme HYAL1 for progression and especially for the development of brain metastases in breast cancer patients. Citation Format: Oliveira-Ferrer L, Anna M, Wickman H, Matschke J, Schumacher U, Milde-Langosch K, Müller V, Witzel I. Relevance of the hyaluronidase-1 (HYAL1) in brain metastasis formation of breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-17-06.

Breast cancer brain metastases: biology and new clinical perspectives.

Because of improvements in the treatment of patients with metastatic breast cancer, the development of brain metastases (BM) has become a major limitation of life expectancy and quality of life for many breast cancer patients. The improvement of management strategies for BM is thus an important clinical challenge, especially among high-risk patients such as human epidermal growth factor receptor 2-positive and triple-negative patients. However, the formation of BM as a multistep process is thus far poorly understood. To grow in the brain, single tumor cells must pass through the tight blood–brain barrier (BBB). The BBB represents an obstacle for circulating tumor cells entering the brain, but it also plays a protective role against immune cell and toxic agents once metastatic cells have colonized the cerebral compartment. Furthermore, animal studies have shown that, after passing the BBB, the tumor cells not only require close contact with endothelial cells but also interact closely with many different brain residential cells. Thus, in addition to a genetic predisposition of the tumor cells, cellular adaptation processes within the new microenvironment may also determine the ability of a tumor cell to metastasize. In this review, we summarize the biology of breast cancer that has spread into the brain and discuss the implications for current and potential future treatment strategies.

Blood-brain Barrier Remodeling during Brain Metastasis Formation.

Our understanding of the process of metastatic progression has improved markedly over the past decades, yet metastasis remains the most enigmatic component of cancer pathogenesis. This lack of knowledge has serious health-related implications, since metastasis is responsible for 90% of all cancer-related mortalities. The brain is considered a sanctuary site for metastatic tumor growth, where the blood-brain barrier (BBB) and other components of the brain microenvironment, provide protection to the tumor cells from immune surveillance, chemotherapeutics and other potentially harmful substances. The interactions between tumor cells and the brain microenvironment, principally brain vascular endothelium, are the critical determinants in their progression toward metastasis, dormancy, or clearance. This review discusses current knowledge of the biology of metastatic progression, with a particular focus on the tumor cell migration and colonization in the brain.

BMET-31DEVELOPMENT AND APPLICATION OF A NOVEL MODEL OF HUMAN LUNG-TO-BRAIN METASTASIS TO IDENTIFY UNIQUE METASTATIC GENE SIGNATURES

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). Previous work conducted in our lab identified a population of CSCs within lung-derived brain metastases. 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 GFP-expressing BMICs into immunocompromised mice via three routes: 1) intracranial (IC), 2) intrathoracic (IT) and 3) intravascular/intracardiac (IV). GFP+ cells were harvested from the lungs and/or brains from each injection route, and RNA was submitted for microarray analysis to identify a unique metastatic and tissue-specific gene signature. In order to identify genes involved in self-renewal and tumor initiation, we also performed RNA interference screens in vitro and in vivo on BMIC lines against 150 genes implicated in BM formation. Further in vitro validation of select hit genes identified SPOCK1 and TWIST2 as essential regulators of cell proliferation, sphere formation, and migration. Future work will validate the role of these genes in our established in vivo metastasis models. We also aim to examine their expression in primary lung FFPE samples to determine if they are predictive biomarkers of lung-to-brain metastasis in prospective cohorts of newly diagnosed lung cancer patients, and to determine their potential as therapeutic targets.

BMET-34DRUG REPURPOSING DISCOVERS BETA-SITOSTEROL AS AN EFFECTIVE THERAPEUTIC AGENT AGAINST MELANOMA BRAIN METASTASES IN VIVO

Despite major therapeutic advances in the management of patients with metastatic melanoma, brain metastases remain an intractable problem. It has proven difficult to identify and target single genes or pathways critical to brain metastasis formation. Here, we explore a more global and preventive treatment approach to melanoma brain metastases within an established xenograft model of human melanoma. Mice were injected intracardially and followed for six weeks with whole-body BLI and brain MRI. Tumor-bearing brains, adrenals, ovaries, and femurs were harvested and enzymatically dissociated before three tumor cell samples from each organ were flow-sorted and subjected to RNA sequencing. We were able to define a 108-gene brain metastasis gene signature, and queried the Connectivity Map database for candidate drugs, i.e. drugs that induce an opposite gene expression profile in various cancer cell lines. We found the cholesterol analogue beta-sitosterol to effectively inhibit brain metastases and improve survival, both in established and preventive therapeutic settings. Beta-sitosterol extensively suppressed the MAPK pathway, and effectively reduced mitochondrial respiration through Complex I inhibition. This novel mechanistic synergy may open new avenues of systemic therapy against metastatic melanoma, as increased mitochondrial respiration is a key mediator of resistance to MAPK-targeted drugs.

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

Abstract 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&amp;lt; 0.01) in branching compared to control at 48 and 72 hours (211 ± 18.2 and 303 ± 47.9 branches) in wells treated with the combination of Ang-2 (100 ng/mL) and VEGF (10 ng/mL). Upon addition of bevacizumab (20 μg/mL), a significant decrease (p&amp;lt; 0.05) in branching was seen at 24 hours (54 ± 10 branches) and 48 hours (161 ± 9.8) but not 72 hours. Similarly, L1-10 (6.5 μg/mL) administration reduced endothelial branching significantly (p&amp;lt; 0.05) at all time points. We observed that nearly all lesions had evidence of hypoxia, VEGF, and Ang-2. We observed the number of metastatic lesions which developed using bevacizumab and L1-10 (10 ± 4) was significantly less (p&amp;lt; 0.001) than bevacizumab treatment (31.6 ± 6) or untreated controls (72 ± 10). The percentage of large lesions (7.7 ± 5) in the combinatorial group was significantly less than bevacizumab treatment (14.6 ± 4; p&amp;lt;0.05) and significantly less (p&amp;lt; 0.05) than un-treated controls (34.6 ± 8). 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

The inflammatory microenvironment in brain metastases: potential treatment target?

Brain metastases (BM) are frequent in cancer patients and are associated with high morbidity and mortality. The incidence of BM is increasing and only limited treatment strategies are available. Immunomodulatory agents are emerging as effective cancer therapeutics and a deeper understanding of the inflammatory microenvironment in BM might reveal new treatment possibilities. Although the brain has long been considered an 'immune-privileged' organ with limited capacity for inflammatory response, BM do contain tumor infiltrating lymphocytes (TILs). Moreover, dense infiltration with TIL showed an association with favorable patients' overall survival times. Microglia/macrophages were shown to be involved in the pivotal steps of BM formation by providing a "pre-metastatic niche" attracting BM initiating cells and to promote growth and survival of tumor cells in the CNS microenvironment. The anti-cytotoxic T lymphocyte antigen 4 (CTLA4) immune checkpoint inhibitor ipilimumab showed activity against melanoma BM in a clinical trial, thus providing proof of concept for effective therapeutic immunomodulation in patients with CNS metastases. Collectively, the available data show that BM harbors an active inflammatory microenvironment that may be exploited as treatment target.

Exercise maintains blood–brain barrier integrity during early stages of brain metastasis formation

Tumor cell extravasation into the brain requires passage through the blood–brain barrier, which is a highly protected microvascular environment fortified with tight junction (TJ) proteins. TJ integrity can be regulated under physiological and pathophysiological conditions. There is evidence that exercise can modulate oxidation status within the brain microvasculature and protect against tumor cell extravasation and metastasis formation. In order to study these events, mature male mice were given access to voluntary exercise on a running wheel (exercise) or access to a locked wheel (sedentary) for five weeks. The average running distance was 9.0 ± 0.2 km/day. Highly metastatic tumor cells (murine Lewis lung carcinoma) were then infused into the brain microvasculature through the internal carotid artery. Analyses were performed at early stage (48 h) and late stage (3 weeks) post tumor cell infusion. Immunohistochemical analysis revealed fewer isolated tumor cells extravasating into the brain at both 48 h and 3 weeks post surgery in exercised mice. Occludin protein levels were reduced in the sedentary tumor group, but maintained in the exercised tumor group at 48 h post tumor cell infusion. These results indicate that voluntary exercise may participate in modulating blood–brain barrier integrity thereby protecting the brain during metastatic progression.