Targeting Monoacylglycerol Lipase in Triple Negative Breast Cancer Reduced Tumor-Associated Inammation, Tumor Growth and Tumor Colonization in the Brain

While the prevalence of breast cancer metastasis in the brain is significantly higher in triple negative breast cancers (TNBCs), there is a lack of novel and/or improved therapies for these patients. Monoacylglycerol lipase (MAGL) is a hydrolase involved in lipid metabolism that catalyzes the degradation of 2-arachidonoylglycerol (2-AG) linked to generation of pro- and anti-inflammatory molecules. Here, we targeted MAGL in TNBCs, using the selective MAGL inhibitor AM9928 (hMAGL IC 50 = 9nM, with prolonged pharmacodynamic effects of 46 hours residence time). AM9928 blocked TNBC cell adhesion and transmigration across human brain microvascular endothelial cells (HBMECs) in 3D co-cultures. In addition, AM9928 inhibited the secretion of IL-6, IL-8, and VEGF-A from TNBC cells. TNBC-derived exosomes activated HBMECs resulting in secretion of elevated levels of IL-8 and VEGF, which were inhibited by AM9928. Using in vivo studies of syngeneic GFP-4T1-BrM5 mammary tumor cells, AM9928 inhibited tumor growth in the mammary fat pads and attenuated blood brain barrier (BBB) permeability changes, resulting in reduced TNBC colonization in brain. Together, these results support the potential clinical application of MAGL inhibitors as novel treatments for TNBC. series of highly potent carbamate MAGL inhibitors were synthesized and characterized at the Center for Drug Discovery, Northeastern University. In this study, we have investigated the inhibitor AM9928, which exhibited high potency against recombinant human MAGL with IC 50 value of 8.9nM and lacked any affinity for the cannabinoid receptors CB1 and CB2. 25-27 AM9928 demonstrated: (1) potency and selectivity for the target; (2) suitable physicochemical properties with low lipophilicity (ClogP 3-4); (3) good microsomal stability (>20 min) and plasma stability (>120 min); and (4) prolonged pharmacodynamic effects as determined by 1 H NMR spectroscopy, assessing the time required for MAGL reactivation (residence time) following the covalent inhibitory interaction between ligand and protein. investigated MAGL’s role in TNBC growth and tumor colonization in brain. MAGL is highly expressed in TNBCs, which secrete high levels of inflammatory cytokines and chemokines. We hypothesized that tumor growth in the mammary fat pads and tumor cell infiltration across the BBB is facilitated via inflammatory chemokines/cytokines secreted from TNBC cells, leading to activation of HBMECs and resulting in TNBC spreading and colonization in brain. Here, we have performed studies using several human and mouse cell lines: (1) a TNBC human cell line (MDA-MB-231 cells); (2) human and mouse brain-seeking TNBC cell lines (MDA-MB-BrM2 and GFP-4T1-BrM5, respectively; (3) human brain microvascular endothelial cells (HBMECs); and (4) the spontaneous breast metastasis mouse models (syngeneic). We found that TNBC adhesion to HBMECs and transmigration across HBMECs was inhibited by AM9928. AM9928 inhibited TNBC’s secretion of inflammatory cytokines such as IL-6 and IL-8, and the angiogenic factor VEGF-A. Notably, AM9928 inhibited i n vivo changes in BBB permeability and decreased TNBC colonization in brain. Taken together, these results demonstrate novel mechanisms by which MAGL mediates its effects on brain metastasis by activation of brain microvascular endothelium and modulating BBB permeability. These studies support the potential of clinical application of MAGL inhibitors as a novel treatment of TNBC tumor growth and TNBC-colonization in the brain. as compared to control were treated with vehicle control or with AM9928. Murine tumor cells in brain were detected by GFP immunostaining (GFP antibodies 1:50 dilutions; Abcam) and their respective controls were used under the same standardized conditions. Brain nuclei were counterstained with DAPI (blue). n = 10 mice/treatment; These are representative images of over 50 images from three independent experiments. Scale bar = 20 μ m. d. Quantitative analysis of tumor cells in the brain: Murine tumor cells in brain were detected by GFP immunostaining as described above. The tumor areas at day 28 were detected by immunostaining with GFP antibody: n = 10 mice/treatment; *p < 0.05, Mann– Whitney U฀test; **p < 0.005 as compared to vehicle control. Brain nuclei were counterstained with DAPI (blue). These are representative images of over 50 elds examined from three independent experiments. The magnication - x40; scale bar-10 μ m.

2 Breast cancer is a common cause of brain metastases occurring in at least 10-16% of patients and reaching 25-35% of TNBC patients. 1-5 Unfortunately, patients who are diagnosed with brain metastases often have poor prognosis with short overall survival times. 6 Triple negative breast cancer (TNBC) more frequently affects younger patients and have higher prevalence in African-American and Hispanic women. 6,7 TNBC tumors are larger in size and more biologically aggressive with lymph node involvement. TNBC patients often have a higher rate of distant recurrence and a poorer prognosis than patients with other breast cancer subtypes. 8. Breast cancer metastases in brain (BCM/B) show significant morphological and genomic heterogeneity. [1][2][3][4][5][6][9][10][11][12][13] Patients with BCM/B have high mortality resulted from the brain lesions and are resistant to chemotherapy treatments. [9][10] Metastatic breast tumor cells transmigrate across the blood-brain barrier (BBB) and form colonies of tumor cells in brain. 9,10,15 Under normal conditions, the BBB is a highly selective barrier due to existence of tight junctions (TJs) between adjacent brain microvascular endothelial cells (BMECs). However, in the process of invasion of tumor cells to the brain, inflammatory cytokines and chemokines secreted by these infiltrating tumor cells disrupt the BBB integrity. Although the BBB prevents the delivery of most therapeutic drugs into the brain, the circulating metastatic tumor cells invade the damaged BBB to form colonies in the brain. 9-10. Several genes were shown to be involved in the development of brain metastases which include cyclooxygenase COX-2, EGFR ligand HBEGF and α-2,6-sialyltransferase ST6GALNAC5 9,16 , all involved in facilitating cancer cell passage through the BBB. We have previously reported the roles of the proinflammatory peptide P in impairing the BBB integrity and the angiogenic factor Angiopoietin-2 in mediating activation of brain microvascular endothelial cells and BBB impairment, resulting in infiltration of TNBCs into the brain. 17,18. Further, loss of E-cadherin was found in breast cancer metastasis and its expression inversely correlates with tumor stage, pathologic stage, and prognosis of cancers of epithelial origin. 19 The tumor microenvironment (TME) is important in cancer progression. [10][11][12] TME is characterized by chronic inflammation which stimulates tumorigenesis, especially in inflammatory breast cancer, where metastasis arises at the initial stage. TME is comprised of cells surrounding the tumor (such as macrophages, fibroblasts, and endothelial cells) and the extracellular matrix (ECM). TME in tumor niches differs from the healthy tissue microenvironments in cell type composition and phenotype. [10][11][12] TME promotes tumor development by complex signaling molecules that include soluble secreted molecules such as cytokines, chemokines, growth factors, proinflammatory enzymes, exosomes and matrix remodeling proteinases. [10][11][12][13][14] Interestingly, extracellular vesicles, a heterogeneous group of cell-derived membranous structures comprising of exosomes and microvesicles, were shown recently to breach intact BBB via Transcytosis. 21 Although tumor-derived exosomes are being recognized 3 as essential mediators of intercellular communication between cancer and immune cells, it is not known whether exosomes derived from breast cancer cells can activate directly brain endothelial cells.
Cancer cells endogenously synthesize 95% of free fatty acids (FFAs) de novo 15 and incorporate and remodel exogenous palmitate into structural and oncogenic glycerophospholipids, sphingolipids and ether lipids. 15 Fatty acid synthesis (FAS) pathway confers a survival advantage to cancer cells and especially to tumor cells that are resistant to chemotherapy. 15 Fatty acid-binding protein 5 (FABP5) promoted lipolysis droplets, de-novo fatty acid synthesis, and coordinated lipid signaling that promoted prostate cancer metastasis. 20 Increased FFA levels in tumors lead to enhanced tumor aggressiveness by increasing lipid synthesis mediated by lipolytic pathways. 15,[22][23][24] Thus, targeting lipid metabolism may prevent the gain of survival advantage and improve treatment response in cancer cells.
MAGL is a serine hydrolase that regulates a fatty acid network that promotes cancer pathogenesis by enriching pro-tumorigenic signaling molecules. MAGL is a key hydrolytic enzyme in the FFA tumor network reported in colorectal cancer, neuroblastoma, nasopharyngeal carcinoma, and other cancers. [21][22][23][24] MAGL primarily hydrolyzes endocannabinoid 2-AG and other monoacylglycerides. In addition, MAGL indirectly controls the levels of free fatty acids derived by their hydrolysis and other lipids derived by metabolism of fatty acids with pro-inflammatory or pro-tumorigenic effects. 24 Recently, 2-AG was shown to be the source for arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. 45,51,53 Accumulated studies showed that MAGL inhibition impaired cell migration, invasion and tumorigenicity in some types of cancers. 20,[22][23][24] Further, MAGL promoted progression of hepatocellular carcinoma via NF-kB mediated epithelial-mesenchymal transition. 52 Taken together, although MAGL was shown to be involved in some cancers and inflammatory diseases, its specific roles in TNBC tumor progression and the brain metastasis process is still unknown.
A new series of highly potent carbamate MAGL inhibitors were synthesized and characterized at the Center for Drug Discovery, Northeastern University. In this study, we have investigated the inhibitor AM9928, which exhibited high potency against recombinant human MAGL with IC50 value of 8.9nM and lacked any affinity for the cannabinoid receptors CB1 and CB2. [25][26][27] AM9928 demonstrated: (1) potency and selectivity for the target; (2) suitable physicochemical properties with low lipophilicity (ClogP 3-4); (3) good microsomal stability (>20 min) and plasma stability (>120 min); and (4) prolonged pharmacodynamic effects as determined by 1 H NMR spectroscopy, assessing the time required for MAGL reactivation (residence time) following the covalent inhibitory interaction between ligand and protein.
Since MAGL is important for lipid metabolism and lipid metabolism plays various roles in tumorigenesis, we investigated MAGL's role in TNBC growth and tumor colonization in brain. MAGL is highly expressed in TNBCs, which secrete high levels of inflammatory cytokines and chemokines. We hypothesized that tumor growth in the mammary fat pads and tumor cell infiltration across the BBB is facilitated via inflammatory chemokines/cytokines secreted from TNBC cells, leading to activation of HBMECs and resulting in TNBC spreading and colonization in brain. Here, we have performed studies using several human and mouse cell lines: (1) a TNBC human cell line (MDA-MB-231 cells); (2) human and mouse brain-seeking TNBC cell lines (MDA-MB-BrM2 and GFP-4T1-BrM5, respectively; (3) human brain microvascular endothelial cells (HBMECs); and (4) the spontaneous breast metastasis mouse models (syngeneic). We found that TNBC adhesion to HBMECs and transmigration across HBMECs was inhibited by AM9928. AM9928 inhibited TNBC's secretion of inflammatory cytokines such as IL-6 and IL-8, and the angiogenic factor VEGF-A. Notably, AM9928 inhibited in vivo changes in BBB permeability and decreased TNBC colonization in brain. Taken together, these results demonstrate novel mechanisms by which MAGL mediates its effects on brain metastasis by activation of brain microvascular endothelium and modulating BBB permeability. These studies support the potential of clinical application of MAGL inhibitors as a novel treatment of TNBC tumor growth and TNBC-colonization in the brain. form metastatic brain tumors [16][17][18] and generate multifocal lesions in the cerebrum, the cerebellum, and the brainstem, with features typical of brain metastasis in cancer patients.

5
MAGL inhibitors: MAGL inhibitor AM9928 was synthesized and characterized at the Center for Drug Discovery, Northeastern University. AM9928 inhibits human MAGL (hMAGL) with an IC50 value of 8.9 nM . [25][26][27] lacked any affinity for the cannabinoid receptors CB1 and CB2. 25 Isolation of Exosomes: The culture supernatants of MDA-MB-231 and MDA-MB-BrM2 cells untreated or treated with AM9928 for 24hours, at approximately 65% confluence and were harvested after 16 hours conditioning in serum-free media. Cells and debris were cleared from the supernatants by centrifugation (500 g, 10 min) followed by filtration using 0.22-micron filters (Millipore Inc.). Exosomes were prepared from cell-free supernatants using the Exosome Isolation Kit (Kit# EIK-01, Creative Biolabs, NY). The quantitative and qualitative analysis of exosomes were performed on double sandwich enzyme-linked immunoassay using the Total Exosome Capture & Quantification Kit (Kit#EQK-04Creative Biolabs).
In vivo effects of MAGL on BBB integrity and TNBC colonization in brain: Female (6wks) BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were housed at an AAALAC-accredited facility at Beth Israel Deaconess Medical Center, Boston. The mice were handled in accordance with the animal care policy of Harvard Medical School. Mice were euthanized humanely by CO2 inhalation in the end of the experiments following treatment and tumor samples were harvested for further study as described below.
We have selected AM9928 for our in vivo studies based on its prolonged target engagement. [25][26][27] We expected that the prolong inhibitory effect of AM9928 would be translatable to a longer pharmacodynamic effect when compared to other MAGL inhibitors such as AM4301.To that end, we studied the effects of AM9928 on BMEC-TJs and tumor colonization in the brain using the spontaneous breast cancer metastasis mouse model (syngeneic) of mammary tumor cells. We used GFP-4T1-BrM5 cells which migrate to the brain [16][17][18] and form breast metastasis in the brains of Balb/c mice. We administered GFP-4T1-BrM5 tumor cells (5x10 4 cells) into the mammary fat pads of Balb/c mice. More than 70% of these mice developed mammary tumors within 3 weeks, while brain microtumors were developed in about 5 weeks. Here, following administration of GFP-4T1-BrM5 cells, mice were injected with AM9928 (10 mg/kg, i.v.) or the vehicle control twice a week for 3 weeks (10 mice/group/treatment). Tumor sizes in the mammary fat pads were measured using calipers, and the volume was calculated, using the formula: V = 0.52 × (length) × (width) 2 . The BBB integrity at the end of the experiments was analyzed by Evan blue test in control groups and in mice treated with AM9928 as compared to vehicle control.
Briefly, Evans's blue (EB) (Sigma Chemical Co. St. Louis, MO. USA.) dye (25% in 0.9% NaCl solution) was intravenously injected at dose of 25 mg/kg under anesthesia. One hour after the injection, animals were sacrificed.
Brains were weighed, clipped, and individually placed within formamide p.a. (2mL/brain). The content of dye extracted from each brain was determined by spectrophotometer (Photometer 4010, Boehringer) at 620nm and compared to standard graph created through the recording of optical densities from serial dilutions of EB in 0.9% NaCl solution. For in vivo imaging, spectral fluorescence images were obtained using a Maestro-based imaging system (CRI Inc., Woburn, MA, USA). Five sets of side-by-side whole-brain images of animals with tumors from the GFP-4T1BrM5 cells treated with vehicle control or with AM9928 were obtained. In the end of the experiment, mice were sacrificed, and brain and mammary fat pad tissues were collected for further analysis. Based on these observations we selected AM9928 for our studies.
The toxicity effects and time kinetics of treatment with AM9928: First, the toxicity of AM9928 on TNBC cells were analyzed. TNBC cells include the highly invasive MDA-MB-231 and the brain-seeking variant MDA-MB-BrM2 cells. The following doses were examined: 250, 500 or 1000nM, for durations of 72 hours. No significant toxicity of AM9928 (Figure 2a) was observed on the tested cells.
AM998 inhibited adhesion and transmigration of breast cancer cells through human brain microvascular endothelial cells (HBMECs): Next, in vitro adhesion assay was performed using co-cultures of the brain-seeking Tumor growth in mammary fat pads was significantly reduced in mice treated with AM9928 as compared to mice treated with vehicle control (Figure 4b). Quantitation of GFP-4T1-BrM5 tumor cell colonization in brain was determined by ex vivo imaging of the brain and by immunohistochemistry using GFP antibody. The expression of GFP positive tumor cells was significantly lower in the mice group treated with AM9928, while the presence of GFP-4T1-BrM5 tumor cells in brain was significantly higher in the untreated GFP-4T1BrM5 cells treated mice or the vehicle control treated mice (Figure 4c, 4d).
Increased in BBB permeability was found in mice administered with tumor cells or with vehicle treated mice, while mice treated with AM9928 had lower BBB permeability changes as compared to control mice at day 28 ( Figure 5a). We then analyzed BMEC-tight junction proteins' expression of ZO-1 and Claudin-5 in vivo in normal brain, as compared to TJs in tumor brains from syngeneic GFP-4T1-BrM5 mammary tumor cell model. ZO-1 is the main scaffolding protein and claudin-5 is known as the TJ sealer protein of the BBB. As shown in Figure 5b, both expression of ZO-1 and claudin-5 were abundant in the normal brain vasculature. Following GFP-4T1-BrM5 cells administration to the mammary fat pads, the GFP-tagged tumor cells migrated across the BBB after about 4 weeks. The GFP tagged tumor cells were detected by immunostaining with either Monoclonal Pan-Cytokeratin (Pan-CK) antibodies or GFP antibodies. No staining of normal brain sections with Pan-ck antibodies was observed (data not shown). These tumor cells were usually located around the brain capillaries and in-close proximity to BMECs (BMECS were detected with CD31 antibodies) (figure 5c).
Both ZO-1 and claudin-5 structures were damaged in the brain tumor vasculature (Figure 6a) as compared to the control brain (Figure 5b). In some images, both ZO-1 and claudin-5 structures withing BMECs were seen to colocalized with tumor cells, indicating the proximity of BMECs to the tumor cells in vivo. Interestingly, following treatment with AM9928, the expression of both ZO-1 and claudin-5 was less impaired as compared to the mice treated with vehicle control (Figure 6a). The number of CD31 + vessels expressing TJ proteins, ZO-1, and claudin-5, was performed and images were analyzed using Adobe Photoshop CS2. The expression of ZO-1, as determined by average sum of intensity was 582,096 and following treatment with AM9928 the average sum 9 of intensity was 610,920 ( Figure 6b). The average sum of intensity of Claudin-5 expression in untreated group was 518,599 and following treatment with AM9928 the average sum of intensity was 681,457 ( Figure 6c). Although these differences in TJ numbers were not statistically significant between the untreated mice and the treated AM9928 mice, both ZO-1 and claudin-5 were observed as less damaged structures in the AM9928 treated mice, as compared to the vehicle treated mice.
In summary, the number of mice positive with mammary tumors and brain tumors was significantly higher in the vehicle treated group as compared to AM9928 treated mice (Table 1). Importantly, the number of alive mice at day 28 was higher in the AM9928 treated mice as compared to the vehicle control group (Table 1). Thus, AM9928 significantly inhibited changes in the BBB permeability and reduced TNBC colonization in brain.

Discussion
During tumor progression, cells undergo reprogramming of metabolic pathways that regulate glycolysis and the production of lipids. 24,[38][39][40] Since MAGL is important in assigning the lipid stores in the direction of protumorigenic signaling lipids in cancer cells 41,42 , we studied the mechanisms by which MAGL contributes to promoting TNBC metastases. The energy supply of lipids comes from de novo synthesis rather than circulating lipids during tumor development, which are modulated and regulated by MAGL. 38 A. The TME cells are the source of proinflammatory cytokines, 10,15 which are secreted by both autocrine and paracrine manner though activation of STAT3. 44 The strong inhibition of IL-6 by AM9928 indicates the potential of AM9928 to target inflammation associated with TNBC. Therefore, MAGL may regulate lipid quality and/or quantity to promote aggressiveness such as migration and inflammation in breast cancer cells.
VEGF-A/VEGF-R signaling functions as an important survival pathway in breast cancer cells. [48][49][50] VEGF-A promoted tumor cell self-renewal through VEGF-A/VEGF-R/STAT3 signaling resulting in activation of Myc, Sox2 and STAT3. [48][49][50] High VEGF-A levels strongly correlated with both STAT3 and Myc expression as well as with tumor metastatic potential. Interestingly, we found significant inhibition of VEGF-A expression by AM9928 (Figure 3c), strongly suggesting the effectiveness of targeting MAGL in inhibition of angiogenesis. The permeability of the BBB plays a crucial role in brain metastasis and is implicated in the initial stages of cancer cell extravasation. In the presence of invading tumor cells, the local BBB is altered and exhibits heterogeneous permeability. [16][17][18] The changes in permeability of the BBB-BMEC-TJs are a dynamic process dependent on the interaction between TNBCs and BMECs. Our results showed that TNBC transmigration across the BBB resulted in increased BBB permeability (Figures 5). Since AM9928 inhibited tumor growth in the mammary fat pads ( Figure 4) and prevented changes in BBB integrity in vivo, resulting in reduced tumor cell colonization in brain, we conclude that MAGL plays a role in TNBC tumor growth and TNBC transmigration across the BBB, in addition to its roles in neurological and neurodegenerative diseases. 51,53 Taken together, we suggest that targeting MAGL may provide a new approach to reducing expression of proinflammatory factors as well as inhibiting tumor growth and tumor cell colonization in brain.
The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This study was supported by departmental grants to Hava Karsenty Avraham and Shalom Avraham.     In vivo effects of AM9928 on TNBC tumor growth and brain metastases: a. Schematic presentation of time injection of AM9928 and GFP/4T1BrM5 cells: AM9928 (at 10 mg/kg) or vehicle control were injected by I.V. twice a week for 3 weeks. b. Tumor formation in mammary fat pads following treatment with AM9928: Murine tumor formation in mammary fat pads were measured at day 28: n = 10 mice/treatment; *p < 0.05, Mann-Whitney Utest; **p < 0.005 as compared to vehicle control. c. Detection of GFP-positive cells in brain tumors: GFP-4T1BrM5 tumor cells were administered to the mammary fat pads as compared to control mice and were treated with vehicle control or with AM9928. Murine tumor cells in brain were detected by GFP immunostaining (GFP antibodies 1:50 dilutions; Abcam) and their respective controls were used under the same standardized conditions. Brain nuclei were counterstained with DAPI (blue). n = 10 mice/treatment; These are representative images of over 50 images from three independent experiments. Scale bar = 20μm. d. Quantitative analysis of tumor cells in the brain: Murine tumor cells in brain were detected by GFP immunostaining as described above. The tumor areas at day 28 were detected by immunostaining with GFP antibody: n = 10 mice/treatment; *p < 0.05, Mann-Whitney Utest; **p < 0.005 as compared to vehicle control.

Figure 5
In vivo effect of AM9928 on transmigration of GFP/4T1BrM5 cells across the BBB and on BMEC-Tight Junctions expression a: In vivo analysis of the BBB permeability: Following injection of Evans blue, a statistically signi cant increase of the dye was found in the hippocampal region in mice administered with 4T1-BrM5 cells and treated with vehicle control, but not in mice treated with AM9928. The level of dye in the control WT mice is shown also for comparison between the groups. This is a representative experiment of three separate experiments. The error bars indicate standard deviations. *p < 0.05 as compared to WT mice control. b: Immunodetection of tight junctions in normal brain CD31+ microvasculature (BMECs). The normal brain microvasculature was immunoassayed with CD31+ antibody (1:500 dilution) as a marker for BMECs, using FITC-conjugated secondary antibody. Brain sections were immunoassayed for ZO-1(1:500 dilution) and claudin-5 (1:500 dilution) using uorescence Texas Red secondary antibody to detect ZO-1 and claudin-5 expression in normal mouse brain sections. Arrows show the intact ZO-1 and claudin-5 structures. Brain nuclei were counterstained with DAPI (blue).
These are representative images of over 50 elds examined in three independent experiments. Scale bar = 20μm. c: In vivo effects of GFP-4T1BrM5 tumor cells on the BBB integrity and on tight junctions' structures: Upper Panel: The tumor cells were detected by pan-cytokeratin (Pan-CK) antibody (1:500 dilution) as primary antibody and uorescence Texas red was used as a secondary antibody to detect the tumor cells in brain sections of mice administered with GFP-4T1BrM5 cells. The brain microvasculature was immunoassayed with CD31+ antibody (1:500 dilution) to detect BMECs. The arrows indicate the tumor cells or the CD31 BMECs as indicated. These are representative images of over 50 elds examined in three independent experiments. Magni cation -x40; scale bar-10 μm. Middle and Lower Panels: Immunodetection of tight junctions in brain tumors: Expression of claudin-5 (middle panel) and ZO-1 (lower panel) in tumor brain sections were performed by immunostaining using FITC-conjugated secondary antibody as indicated. Detection of GFP-4T1BrM5 cells in vivo was performed by immunostaining with Pan-CK antibody. Co-immunostaining of the tumor cells with claudin-5 or with ZO-1 are shown in the merged gures. Brain nuclei were counterstained with DAPI (blue). These are representative images of over 50 elds examined from three independent experiments. The magni cation -x40; scale bar-10 μm. a.Analysis of ZO-1 and Claudin-5 expression in the BBB in mice treated with AM9928: Fluorescence Texas Red was used as a secondary antibody to detect ZO-1 and claudin-5 expression in mouse brain tumor sections. ZO-1 and claudin-5 structures are shown by arrows. Brain nuclei were counterstained with DAPI (blue). These are representative images of over 50 elds examined from three independent experiments. The magni cation -x40; scale bar-20 μm. b-c: Quantitative analysis of claudin-5 and ZO-1 proteins expression in the brain in mice treated with AM9928: The expression level was quantitated and compared with 4T1-BrM5 injected mice and mice treated with vehicle control. Changes in BMEC-TJ proteins in brain section samples after AM9928 treatment as compared to BMEC-TJ proteins from control mice is shown. Data are presented as the mean ± S.D. of two experiments. Number of mice per group per treatment, n = 10. Scale bar = 20μm