Brain Tumor Vessels Research Articles

N-Acetyl-L-Cysteine (NAC) Blunts Axitinib-Related Adverse Effects in Preclinical Models of Glioblastoma.

Axitinib is a tyrosine kinase inhibitor characterized by a strong affinity for Vascular Endothelial Growth Factor Receptors (VEGFRs). It was approved in 2012 by Food and Drug Administration and European Medicines Agency as a second line treatment for advanced renal cell carcinoma and is currently under evaluation in clinical trial for the treatment of other cancers. Glioblastoma IDH-wild type (GBM) is a highly malignant brain tumor characterized by diffusely infiltrative growth pattern and by a prominent neo-angiogenesis. In GBM, axitinib has demonstrated a limited effectiveness as a monotherapy, while it was recently shown to significantly improve its efficacy in combination treatments. In preclinical models, axitinib has been reported to trigger cellular senescence both in tumor as well as in normal cells, through a mechanism involving intracellular reactive oxygen species (ROS) accumulation and activation of Ataxia Telangiectasia Mutated kinase (ATM). Limiting axitinib-dependent ROS increase by antioxidants prevents senescence specifically in normal cells, without affecting tumor cells. We used brain tumor xenografts obtained by engrafting Glioma Stem Cells (GSCs) into the brain of immunocompromised mice, to investigate the hypothesis that the antioxidant molecule N-Acetyl-L-Cysteine (NAC) might be used to reduce senescence-associated adverse effects of axitinib treatment without altering its anti-tumor activity. We demonstrate that the use of the antioxidant molecule N-Acetyl-Cysteine (NAC) in combination with axitinib stabilizes tumor microvessels in GBM tumor orthotopic xenografts, eventually resulting in vessel normalization, and protects liver vasculature from axitinib-dependent toxicity. Overall, we found that NAC co-treatment allows vessel normalization in brain tumor vessels and exerts a protective effect on liver vasculature, therefore minimizing axitinib-dependent toxicity.

Microbeam Radiation Therapy Opens a Several Days’ Vessel Permeability Window for Small Molecules in Brain Tumor Vessels

PurposeSynchrotron microbeam radiation therapy (MRT), based on an inhomogeneous geometric and microscopic irradiation pattern of the tissues with high dose and high dose rate X-rays, enhances the permeability of brain tumor vessels. This study attempts to determine time and size range of the permeability window induced by MRT in the blood brain (tumor) barrier. Methods and MaterialsRats-bearing 9L gliomas were exposed to MRT, either unidirectional (tumor dose 406 Gy), or bidirectional (crossfired) (2 × 203 Gy). We measured vessel permeability to molecules of 3 sizes (Gd-DOTA, Dotarem®, 0.56 kDa; gadolinium-labeled albumin, ∼74 kDa; gadolinium-labeled IgG, 160 kDa) by daily in vivo magnetic resonance imaging (MRI), from 1 day before to 10 days post-irradiation. ResultsAn equivalent tumor dose of bidirectional MRT delivered from two orthogonal directions increased tumor vessel permeability for the smallest molecule tested more effectively than unidirectional MRT. Bidirectional MRT also affected the permeability of normal contralateral vessels to a different extent than unidirectional MRT. Conversely, bidirectional MRT did not modify the permeability of normal or tumor vessels for both larger molecules (74 and 160 kDa). ConclusionsHigh-dose bidirectional (cross-fired) MRT induced a significant increase in tumor vessel permeability for small molecules between the first and the seventh day after irradiation, while permeability of vessels in normal brain tissue remained stable. Such a permeability window could facilitate an efficient and safe delivery of intravenous small molecules (≤0.56 kDa) to tumoral tissues. A permeability window was not achieved by molecules larger than gado-grafted albumin (74 kDa). Vascular permeability for molecules between these two sizes has not been determined.

P10.21.A THE ABCB1/ABCG2 INHIBITOR ELACRIDAR IS A MORE POTENT PHARMACOENHANCER COMPARED TO TARIQUIDAR FOR TREATMENT OF INTRACRANIAL TUMORS WITH SMALL MOLECULE DRUGS

Abstract BACKGROUND The blood-brain barrier (BBB) is a major hurdle to successful pharmacotherapy of brain tumors. ABCB1 and ABCG2 are efflux transporters that are expressed in brain tumor vessels and reduce the uptake of many small molecule drugs. Elacridar and tariquidar are both dual ABCB1 and ABCG2 inhibitors. We aim to improve pharmacotherapy of brain cancer by concomitant use of potentially effective drugs with elacridar. Since tariquidar has mostly been used to assess the effects of ABC-transporters on brain uptake in human studies with PET tracers, we decided to compare both agents in our rodent models. MATERIAL AND METHODS We used Abcg2;Abcb1a/b double knockout (DKO), Abcb1a/b KO, Abcg2 KO and wild-type mice receiving a cocktail of 7 model drugs at a fixed low dose in combination with elacridar or tariquidar at a range of doses. Drugs were given by 3-h infusion. DKO mice are the reference for complete inhibition, while single KO mice allow interrogation of the remaining transporter in case of dual substrate drugs. Brain and plasma levels were measured by LC-MS/MS. RESULTS Complete inhibition of Abcb1 by elacridar was achieved when the plasma level is about 1.0 μM. However, a much higher plasma level (>4 μM) of tariquidar was needed. Inhibition of Abcg2 was more difficult. For erlotinib and palbociclib about 1.0 μM of elacridar was sufficient, but other more profound Abcg2 substrate drugs never reached the B/P ratios achieved in reference to DKO mice, even at 4 μM. Strikingly, tariquidar could not enhance the brain uptake of ABCG2-subtrate drugs in Abcb1a/b KO mice. The B/P ratio of elacridar in DKO mice was 6. Elacridar improved its own brain distribution in WT mice, achieving steady-state B/P ratios at plasma level above 1.4 μM. The B/P ratio of tariquidar in DKO mice was about 2, while in WT and Abcb1a/b KO mice was 0.3 and did not increase even at plasma concentration >8 μM. Only in Abcg2 KO mice, the B/P ratio increased to levels of DKO mice at tariquidar plasma levels of > 6 μM. CONCLUSION Elacridar is a much more potent inhibitor of Abcb1a/b and Abcg2 at the BBB. Elacridar can act as a pharmaco-enhancer of ABCB1 substrate drugs and weaker ABCG2 substrates. Tariquidar will enhance the brain penetration of ABCB1 substrates, but full inhibition is only achieved at levels above those reported in human subjects. The efflux of tariquidar by Abcg2 at the BBB severely compromises the usefulness of tariquidar as a pharmaco-enhancer for brain delivery.

289 (PB069) - The ABCB1/ABCG2 inhibitor elacridar is a more potent pharmacoenhancer compared to tariquidar for treatment of intracranial tumors with small molecule drugs

Background: The blood-brain barrier (BBB) is a major hurdle to successful pharmacotherapy of brain tumors. ABCB1 and ABCG2 are efflux transporters that are expressed in brain tumor vessels and reduce the uptake of many small molecule drugs. Elacridar and tariquidar are both dual ABCB1 and ABCG2 inhibitors. We aim to improve pharmacotherapy of brain cancer by concomitant use of potentially effective drugs with elacridar. Since tariquidar has mostly been used to assess the effects of ABC-transporters on brain uptake in human studies with PET tracers, we decided to compare both agents in our rodent models.

P11.31 EB1-dependent survival of glioblastoma tumor-bearing mice after treatment with the novel checkpoint activator BAL101553 is associated with inhibition of cancer stem-like cell-mediated tumor angiogenesis and astrocytic differentiation

Abstract BACKGROUND Glioblastoma (GBM) patients have limited treatment options. Cancer stem-like cells (CSLCs) contribute to GBM invasiveness, representing promising targets. BAL101553 is a novel small molecule tubulin-binding agent, promoting tumor cell death through spindle assembly checkpoint activation, which is currently in Phase 1/2a in advanced solid tumor patients including GBM. We have shown that overexpression of microtubule + end-binding 1-protein (EB1) correlates with GBM progression and poor survival. This study aimed to evaluate the effect of short-term IV and long-term daily oral BAL101553 treatment on mice orthotopically grafted with GBM CSLCs (GBM6) according to EB1 expression-level, and to decipher its mechanism of action on CSLCs. MATERIAL AND METHODS The A2B5+ GBM6 cell line was established from CSLCs isolated from a GBM patient. GBM6 cells display a mesenchymal phenotype with a high tumorigenicity and infiltrative pattern of migration. As EB1 is overexpressed in GBM6, we investigated drug activity on GBM6 according to EB1 expression, by using EB1-expressing cells (GBM6-GFP-sh0) and EB1-downregulated (GBM-GFP-shEB1) cells. BAL27862 is the active moiety of the prodrug BAL101553, which was developed to improve solubility. Two sets of animal experiments were performed with 4 groups randomized for GBM6-GFP-sh0- and GBM6-GFP-shEB1-bearing mice, receiving either BAL101553 (25 mg/kg) or vehicle intravenously on day 30, 33, and 36 after glioma implantation, or BAL101553 (30 mg/kg) or vehicle orally from day 35–135 post-grafting (5 days dosing/week). Survival and tumor invasion were analyzed. In vitro, BAL27862 activity was tested by using a cytotoxicity assay, and endothelial cell capillary-like tube formation assay. RESULTS Both treatment schedules provided an EB1-expression level-dependent survival benefit; with daily oral treatment this was almost one year longer than observed in vehicle-treated mice. Moreover, the anti-proliferative and anti-invasive effects of BAL101553 were more potent in mice bearing EB1-expressing tumors as compared to EB1-downregulated tumors. This was associated with inhibition of stem cell properties. Formation of vascular structures by the fluorescent GBM6-GFP-sh0 cells was observed in the brains of grafted mice. Drug treatment counteracted the formation of brain tumor vessels by inhibiting GBM6 trans-differentiation into tumor-derived endothelial cells, as well as VEGF secretion; thus, preventing normal endothelial cell migration activated by CSLCs. CONCLUSION BAL101553/BAL27862 triggers astrocytic differentiation and counteracts tumor angiogenesis by acting on CSLCs; thus, potentially adding to the direct anti-tumor cell effect. A high level of EB1 expression in CSLCs potentiates the drug’s effects, suggesting the potential of EB1 expression as a response-predictive biomarker for BAL101553 in GBM.

Abstract LB-255: Acquired and intrinsic resistance to vemurafenib in BRAFv600e-driven melanoma brain metastases

Abstract Purpose: BRAFV600-mutated melanoma brain metastases (MBMs) are generally responsive to BRAF and MEK inhibitors, albeit that responses are generally less durable than of extracranial metastases. We have studied the impact of the drug efflux proteins P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) at the blood-brain barrier (BBB) on the efficacy of vemurafenib against BRAF-mutated A375 MBMs. Experimental design: We have implanted BRAF-mutated A375 tumor cells in the brains of wildtype and Abcb1a/b;Abcg2-/- mice. We characterized the BBB in the tumors, treated the mice with oral vemurafenib, analyzed drug levels in plasma and brain lesions and determined the efficacy in brain metastases and subcutaneous lesions. Results: Although A375 MBMs disrupt the integrity of the BBB, as shown by contrast-enhanced MRI, vemurafenib achieves greater antitumor efficacy in Abcb1a/b;Abcg2-/- mice compared to wild-type mice. P-gp and BCRP in brain tumor vessels limit vemurafenib penetration into A375 MBMs. Vemurafenib efficacy in both strains was similar against subcutaneous A375 tumors. Intriguingly, although initially responsive, A375 MBMs rapidly developed therapy resistance, even in Abcb1a/b;Abcg2-/- mice, and this was unrelated to pharmacokinetic or target inhibition issues. Rather, MBMs likely resorted to non-canonical growth signaling, as target inhibition of canonical MAPK and PI3K pathway signaling components was maintained in resistant intracranial A375 tumors. Conclusions: In line with clinical data, BRAFV600E-positive MBMs are less responsive to vemurafenib. This is partly due to protection by the BBB, but also because they rapidly acquire further resistance by resorting to non-canonical growth signaling. Citation Format: Mark C. de Gooijer, Ping Zhang, Levi C. Buil, Stephan Freriks, Gang Li, Jos H. Beijnen, Olaf Van Tellingen. Acquired and intrinsic resistance to vemurafenib in BRAFv600e-driven melanoma brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-255.

Permeability of Brain Tumor Vessels Induced by Uniform or Spatially Microfractionated Synchrotron Radiation Therapies

Tocompare the blood-brain barrier permeability changes induced by synchrotron microbeam radiation therapy (MRT, which relies on spatial fractionation of the incident x-ray beam into parallel micron-wide beams) with changes induced by a spatially uniform synchrotron x-ray radiation therapy. Male rats bearing malignant intracranial F98 gliomas were randomized into 3 groups: untreated, exposed to MRT (peak and valley dose: 241 and 10.5Gy, respectively), or exposed to broad beam irradiation (BB) delivered at comparable doses (ie, equivalent to MRT valley dose); both applied by 2 arrays, intersecting orthogonally the tumor region. Vessel permeability was monitored invivo by magnetic resonance imaging 1day before (T-1) and 1, 2, 7, and 14days after treatment start. To determine whether physiologic parameters influence vascular permeability, we evaluated vessel integrity in the tumor area with different values for cerebral blood flow, blood volume, edema, and tissue oxygenation. Microbeam radiation therapy does not modify the vascular permeability of normal brain tissue. Microbeam radiation therapy-induced increase of tumor vascular permeability was detectable from T2 with a maximum at T7 after exposure, whereas BB enhanced vessel permeability only at T7. At this stage MRT was more efficient at increasing tumor vessel permeability (BB vs untreated: +19.1%; P=.0467; MRT vs untreated: +44.8%; P<.0001), and its effects lasted until T14 (MRT vs BB, +22.6%; P=.0199). We also showed that MRT was more efficient at targeting highly oxygenated (high blood volume and flow) and more proliferative parts of the tumor than BB. Microbeam radiation therapy-induced increased tumor vascular permeability is: (1) significantly greater; (2) earlier and more prolonged than that induced by BB irradiation, especially in highly proliferative tumor areas; and (3) targets all tumor areas discriminated by physiologic characteristics, including those not damaged by homogeneous irradiation.

Disruption of Vascular Barriers in Tumors and in the Brain

While most brain tumors do not have an intact blood-brain barrier (BBB), vascular barriers in the tumor and the surrounding intact brain tissue are major challenges for the use of chemotherapy for CNS malignancies. The permeability of brain tumor vessels can be highly heterogeneous, and factors such as high interstitial pressures can prevent agents from reaching an effective concentration in all tumor regions. Furthermore, tumors such as glioma are highly infiltrative, and these infiltrating tumor cells in the brain tissue surrounding the vascular portion of the tumor are protected by the BBB.

Evaluation of brain tumor vessels specific contrast agents for glioblastoma imaging.

A mouse model of glioblastoma multiforme was used to determine the accumulation of a targeted contrast agent in tumor vessels. The contrast agent, consisting of superparamagnetic iron oxide coated with dextran, was functionalized with an anti-insulin-like-growth-factor binding protein 7 (anti-IGFBP7) single domain antibody. The near infrared marker, Cy5.5, was also attached for an in vivo fluorescence study. A 9.4T magnetic resonance imaging (MRI) system was used for in vivo studies on days 10 and 11 following tumor inoculation. T(2) relaxation time was used to measure the accumulation of the contrast agent in the tumor. Changes in tumor to brain contrast because of active targeting were compared with a nontargeted contrast agent. Effective targeting was confirmed with near infrared measurements and fluorescent microscopic analysis. The results showed that there was a statistically significant (P < .01) difference in normalized T(2) between healthy brain and tumor tissue 10 min, 1 h, and 2 h point postinjection of the anti-IGFBP7 single domain antibody targeted and nontargeted iron oxide nanoparticles. A statistical difference remained in animals treated with targeted nanoparticles 24 h postinjection only. The MRI, near infrared imaging, and fluorescent microscopy studies showed corresponding spatial and temporal changes. We concluded that the developed anti-IGFBP7-iron oxide single domain antibody-targeted MRI contrast agent selectively binds to abnormal vessels within a glioblastoma. T(2)-weighted MRI and near infrared imaging are able to detect the targeting effects in brain tumors.

Small unilamellar vesicles: a platform technology for molecular imaging of brain tumors

Molecular imaging enables the non-invasive investigation of cellular and molecularprocesses. Although there are challenges to overcome, the development of targeted contrastagents to increase the sensitivity of molecular imaging techniques is essential for theirclinical translation. In this study, spontaneously forming, small unilamellar vesicles (sULVs)(30 nm diameter) were used as a platform to build a bimodal (i.e., optical and magneticresonance imaging (MRI)) targeted contrast agent for the molecular imaging of braintumors. sULVs were loaded with a gadolinium (Gd) chelated lipid (Gd-DPTA-BOA),functionalized with targeting antibodies (anti-EGFR monoclonal and anti-IGFBP7 singledomain), and incorporated a near infrared dye (Cy5.5). The resultant sULVs werecharacterized in vitro using small angle neutron scattering (SANS), phantom MRI anddynamic light scattering (DLS). Antibody targeted and nontargeted Gd loadedsULVs labeled with Cy5.5 were assessed in vivo in a brain tumor model in miceusing time domain optical imaging and MRI. The results demonstrated that aspontaneously forming, nanosized ULVs loaded with a high payload of Gd canselectively target and image, using MR and optical imaging, brain tumor vessels whenfunctionalized with anti-IGFBP7 single domain antibodies. The unique featuresof these targeted sULVs make them promising molecular MRI contrast agents.

Antiangiogenesis and Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Targeting as Part of a Combined-Modality Approach to the Treatment of Cancer

Antiangiogenesis and Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Targeting as Part of a Combined-Modality Approach to the Treatment of Cancer

Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiationRole of oxygenation, angiopoietin-1, and matrix metalloproteinases

The recent landmark Phase III clinical trial with a VEGF-specific antibody suggests that antiangiogenic therapy must be combined with cytotoxic therapy for the treatment of solid tumors. However, there are no guidelines for optimal scheduling of these therapies. Here we show that VEGFR2 blockade creates a “normalization window”—a period during which combined radiation therapy gives the best outcome. This window is characterized by an increase in tumor oxygenation, which is known to enhance radiation response. During the normalization window, but not before or after it, VEGFR2 blockade increases pericyte coverage of brain tumor vessels via upregulation of Ang1 and degrades their pathologically thick basement membrane via MMP activation.

Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: Role of oxygenation, angiopoietin-1, and matrix metalloproteinases

The recent landmark Phase III clinical trial with a VEGF-specific antibody suggests that antiangiogenic therapy must be combined with cytotoxic therapy for the treatment of solid tumors. However, there are no guidelines for optimal scheduling of these therapies. Here we show that VEGFR2 blockade creates a “normalization window”—a period during which combined radiation therapy gives the best outcome. This window is characterized by an increase in tumor oxygenation, which is known to enhance radiation response. During the normalization window, but not before or after it, VEGFR2 blockade increases pericyte coverage of brain tumor vessels via upregulation of Ang1 and degrades their pathologically thick basement membrane via MMP activation.

Multidrug resistance-associated protein MRP1 expression in human gliomas: chemosensitization to vincristine and etoposide by indomethacin in human glioma cell lines overexpressing MRP1.

The 190 kDa multidrug resistance protein MRP1 is likely to be involved in the multidrug resistance phenotype of human gliomas. MRP1 expression was evaluated in surgical tumor samples from 17 patients with gliomas. In addition, the impact of the MRP's inhibitor, indomethacin, on the chemosensitivity to etoposide (VP16) and vincristine (VCR) of two glioblastoma cell lines expressing MRP1 (GL15 and 8MG) was investigated. When evaluated in tumor samples, MRP1 expression was observed in all of them with more than 90% of stained tumor cells in 14/15 high-grade gliomas. MRP1 was also strongly expressed at the membrane of the vascular endothelial cells in the same 14 tumor samples, suggesting that the permeability to anticancer drugs could be also limited across brain tumor vessels. At concentrations comprised between 5 and 50 microM, indomethacin significantly increased the cytotoxic effect of etoposide in both cell lines but it was more efficient in increasing the cytotoxicity of VCR on GL15 cells, as compared with 8MG cells. These results suggest that the association of indomethacin to VCR or etoposide could be of interest in the clinical management of gliomas.

Correlation of intraluminal thrombosis in brain tumor vessels with postoperative thrombotic complications: a preliminary report.

Thrombotic complications (deep vein thrombosis and/or pulmonary embolization [DVT/PE]) occur in 18 to 50% of patients harboring brain tumors who undergo neurosurgical procedures. Such patients are at risk for DVT/PE because of immobility, paresis, hypovolemia, and lengthy surgery. The present study was undertaken to see whether tumor patients at highest risk for DVT/PE could be identified so that augmentation of prophylactic measures might be used to reduce the incidence of thrombotic complications. The authors conducted a retrospective analysis of 488 patients enrolled in their brain tumor registries between 1988 and 1995, identifying 57 patients (12%) with recorded symptomatic DVT, PE, or both postoperatively. In 24 of these 57 cases histological specimens were retrievable for review, allowing an in-depth analysis. Forty-five patients were lost to follow-up review, and the remaining 386 patients had no record of systemic thrombosis. Slides of pathological specimens were retrievable in 50 cases in which there was no DVT/PE. From these 50 cases, 25 were selected at random to represent the control group by a blinded observer. Seventeen (71%) of the 24 brain tumor specimens obtained in patients with DVT/PE stained positively for intraluminal thrombosis (ILT) after hematoxylin and eosin had been applied. The odds ratio associated with the presence of ILT was 17.8, with a confidence interval ranging from 4 to 79.3. No evidence of ILT was found in 22 patients (88%) within the control group (p < 0.0001, Fisher's exact test). Other factors that may predispose patients with brain tumors to DVT/PE-limb paresis, extent of tumor removal, and duration of the surgery-were also analyzed and found not to be statistically significant. Therefore, these factors were not the basis for differences seen between the study and control groups. These preliminary observations suggest that the presence of ILT within malignant glioma or glioblastoma tumor vessels may represent a marker of tumor-induced hypercoagulability.

Altered response to histamine in brain tumor vessels: the selective increase of regional cerebral blood flow in transplanted rat brain tumor

The authors studied the effect of intracarotid administration of histamine on the regional cerebral blood flow (rCBF) in transplanted rat C6 glioma by the hydrogen clearance method. Histamine infusion at doses of 1 and 10 micrograms/kg/min produced an increase of rCBF in the tumor (24.6% +/- 16.4%, p < 0.002, and 37.6% +/- 18.2%, p < 0.0001, respectively) and also in brain surrounding the tumor (26.8% +/- 16.2%, p < 0.002, and 34.9% +/- 9.2%, p < 0.0001, respectively) without any significant changes in the ipsilateral hemisphere. Intravenous administration of pyrilamine (H1 antagonist) and cimetidine (H2 antagonist) reduced blood flow responses to histamine; cimetidine was a more effective blocking agent than pyrilamine. Intracarotid infusion of histamine (1 and 10 micrograms/kg/min) with intravenous injection of Evans blue dye disclosed the selective extravasation of dye in the tumor and the brain surrounding the tumor. These results indicated that brain tumor vessels could respond to histamine differently than normal brain capillaries. The mechanism of selective response to histamine could be explained either by increased permeability or by altered characteristics of histamine receptors in the tumor vessels.

ONTOGENIC STUDIES ON THE RAT BRAIN CAPILLARIES IN RELATION TO THE HUMAN BRAIN TUMOR VESSELS.

One of the major morphological disparities of brain capillaries between newborn or young and adult rats might be reflected in the aspect of development of the basement membrane. The basement membrane in young animals is clearly evident to be poorly developed up to 15 days of age, and then better developed to possess some similarity to that in adult animals. In addition, it also should be emphasized that slightly distended extracellular space is evident in the nervous tissue of newborn animals.