Efficient drug delivery to glioblastoma (GBM) is a major obstacle as the blood-brain barrier (BBB) and the blood-tumor barrier (BTB) prevent passage of the majority of chemotherapies into the brain. Here, we identified a transcriptional 12-gene signature associated with the BTB in GBM. We identified CDH5 as a core molecule in this set and confirmed its expression in GBM vasculature using transcriptomics and immunostaining of patient specimens. The indirubin-derivative, 6-bromoindirubin acetoxime (BIA), down-regulates CDH5 and other BTB signature genes, causing endothelial barrier disruption in vitro and in murine GBM xenograft models. Treatment with BIA increased intratumoral cisplatin accumulation and potentiated DNA damage by targeting DNA repair pathways. Last, using an injectable BIA nanoparticle formulation, PPRX-1701, we significantly improved cisplatin efficacy in murine GBM. Our work reveals potential targets of the BTB and the bifunctional properties of BIA as a BTB modulator and a potentiator of chemotherapy, supporting its further development.

The blood-brain barrier (BBB) is a highly specialized system that is critical for regulating transport between the blood and the central nervous system. In brain tumors, the vasculature system is compromised, and is referred to as the blood-tumor barrier (BTB). The ability to precisely model the unique physiological properties of the BTB is essential to decipher its role in tumor pathophysiology and for the rational design of efficacious therapeutics. Here, we introduce a robust and high-throughput in vitro 3D human BTB organoid model that recapitulates various key features of the BTB observed in vivo and in clinical GBM samples. The organoids are composed of patient-derived glioblastoma stem cells (GSCs), human brain endothelial cells (EC), astrocytes and pericytes, which are formed through self-assembly. Transcriptomic and functional analyses reveal that the GSCs in the BTB organoids exhibit enhanced level of stemness, mesenchymal signature, invasiveness and angiogenesis, and this is further confirmed in in vivo studies. We demonstrate the ability of the BTB organoids to model therapeutic delivery and drug efficacy on brain tumor cells. Collectively, our findings show that the BTB organoid model has broad utility as a clinically representative system for studying the BTB and evaluating brain tumor therapies. ### Competing Interest Statement The authors have declared no competing interest.

The development of gene therapies for the treatment of diseases of the central nervous system has been hindered by the limited availability of adeno-associated viruses (AAVs) that efficiently traverse the blood-brain barrier (BBB). Here, we report the rational design of AAV9 variants displaying cell-penetrating peptides on the viral capsid and the identification of two variants, AAV.CPP.16 and AAV.CPP.21, with improved transduction efficiencies of cells of the central nervous system on systemic delivery (6- to 249-fold across 4 mouse strains and 5-fold in cynomolgus macaques, with respect to the AAV9 parent vector). We also show that the neurotropism of AAV.CPP.16 is retained in young and adult macaques, that this variant displays enhanced transcytosis at the BBB as well as increased efficiency of cellular transduction relative to AAV9, and that it can be used to deliver antitumour payloads in a mouse model of glioblastoma. AAV capsids that can efficiently penetrate the BBB will facilitate the clinical translation of gene therapies aimed at the central nervous system.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, with a median survival of approximately 15 months. Treatment is limited by the blood-brain barrier (BBB) which restricts the passage of most drugs to the brain. We previously reported the design and synthesis of a BBB-penetrant macrocyclic cell-penetrating peptide conjugate (M13) covalently linked at the axial position of a Pt(IV) cisplatin prodrug. Here we show the Pt(IV)-M13 conjugate releases active cisplatin upon intracellular reduction and effects potent in vitro GBM cell killing. Pt(IV)-M13 significantly increased platinum uptake in an in vitro BBB spheroid model and intravenous administration of Pt(IV)-M13 in GBM tumor-bearing mice led to higher platinum levels in brain tissue and intratumorally compared with cisplatin. Pt(IV)-M13 administration was tolerated in naïve nude mice at higher dosage regimes than cisplatin and significantly extended survival above controls in a murine GBM xenograft model (median survival 33 days for Pt(IV)-M13 vs 24 days for Pt(IV) prodrug, 22.5 days for cisplatin and 22 days for control). Increased numbers of γH2AX nuclear foci, biomarkers of DNA damage, were observed in tumors of Pt(IV)-M13-treated mice, consistent with elevated platinum levels. The present work provides the first demonstration that systemic injection of a Pt(IV) complex conjugated to a brain-penetrant macrocyclic peptide can lead to increased platinum levels in the brain and extend survival in mouse GBM models, supporting further development of this approach and the utility of brain-penetrating macrocyclic peptide conjugates for delivering non-BBB penetrant drugs to the central nervous system.

Background: Glioblastoma (GBM) is the most common and deadliest malignant primary brain tumor, contributing significant morbidity and mortality among patients. As current standard-of-care demonstrates limited success, the development of new efficacious GBM therapeutics is urgently needed. Major challenges in advancing GBM chemotherapy include poor bioavailability, lack of tumor selectivity leading to undesired side effects, poor permeability across the blood–brain barrier (BBB), and extensive intratumoral heterogeneity. Methods: We have previously identified a small, soluble peptide (BTP-7) that is able to cross the BBB and target the human GBM extracellular matrix (ECM). Here, we covalently attached BTP-7 to an insoluble anti-cancer drug, camptothecin (CPT). Results: We demonstrate that conjugation of BTP-7 to CPT improves drug solubility in aqueous solution, retains drug efficacy against patient-derived GBM stem cells (GSC), enhances BBB permeability, and enables therapeutic targeting to intracranial GBM, leading to higher toxicity in GBM cells compared to normal brain tissues, and ultimately prolongs survival in mice bearing intracranial patient-derived GBM xenograft. Conclusion: BTP-7 is a new modality that opens the door to possibilities for GBM-targeted therapeutic approaches.

INTRODUCTION. Glioblastoma is the most aggressive primary malignant brain tumor and has a median survival of approximately 15 months. Treatment is hindered by the blood-brain barrier (BBB) that restricts entry of the vast majority of cancer therapeutics to the brain. We have designed a peptide-drug conjugate, (Pt(IV)-M13), comprised of M13, a perfluoroaryl-stapled cell-penetrating peptide, covalently linked to Pt(IV) via an amide bond. Upon intracellular reduction the Pt(IV) prodrug releases active cisplatin eliciting DNA damage and cell death. To investigate the properties of Pt(IV)-M13 in detail, we determined in vitro cell killing, platinum brain accumulation, biodistribution, and efficacy in mouse models. METHODS. Pt(IV)-M13 tumor cytotoxicity was assessed in vitro using standard approaches. In vivo platinum levels were measured in the brain by ICP-MS, and BBB penetration assessed through a multicellular 3D in vitro BBB model, and biodistribution to peripheral organs was determined. Toxicity from bi-weekly injections of the Pt(IV)-M13 platinum conjugate was monitored from 5mg/kg to 30mg/kg. Animal survival was determined in a glioblastoma xenograft model. DNA damage was quantified by γH2AX staining. RESULTS. Pt(IV)-M13 possesses in vitro tumor cell killing effects similar to cisplatin and Pt(IV) alone. Pt(IV)-M13 showed increased BBB penetration when compared to cisplatin in our in vitro BBB spheroid model (>20-fold), in brain tissue (7-fold) and glioblastoma tumor-bearing mice (8-fold). Five hours after intravenous injection, biodistribution of platinum delivered by Pt(IV)-M13 was higher than cisplatin in the spleen, but lower in the heart. Bi-weekly injections of Pt(IV)-M13 were well-tolerated in nude mice to at least 15mg/kg, three times the reported cisplatin MTD. Finally, Pt(IV)-M13 treatment increased survival in a murine glioblastoma xenograft model (median survival= 33 days for Pt(IV)-M13 vs. 22.5 days for cisplatin and 24 days Pt(IV) alone. Elevated γH2AX foci were observed in Pt(IV)-M13 treated mice providing direct support for the improved delivery of cisplatin to brain tumors using this approach. CONCLUSION. Pt(IV)-M13 leads to enhanced BBB penetration and cisplatin accumulation in glioblastoma models, with consequent animal survival benefit. Overall, our data support further investigation and development of Pt(IV)-M13 for glioblastoma treatment. Future studies will include additional glioblastoma models, and further optimization of this approach. Citation Format: Jorge-Luis Jimenez-Macias, Yen-Chun Lee, Tomer Finkelberg, Gilles Berger, Michal Nowicki, Choi-Fong Cho, Bogdan Fedeles, Andrei Loas, Brad Penetelute, Sean E. Lawler. Pt(IV)-M13, a blood-brain-barrier penetrant macrocyclic peptide-platinum(IV) conjugate, leads to enhanced drug uptake and efficacy in murine glioblastoma models [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 5329.

Abstract INTRODUCTION Glioblastoma (GBM), an aggressive brain tumor with a poor prognosis, presents an average of 2% of patients surviving beyond 2 years after diagnosis. Therapies to effectively manage glioblastoma are hindered due to the presence of the blood-brain barrier (BBB). Previously, a cell-penetrating peptide, M13, was conjugated to a Pt(IV) cisplatin prodrug, via amide bond formation. The conjugated Pt(IV) releases active cisplatin upon intracellular reduction. Herein, we investigated the BBB-penetrance and biodistribution of M13 conjugated to Pt(IV), as well as its effectiveness against GBM in mouse models. METHODS M13 platinum-conjugate tumor cell killing capacity was assessed by luminescent cell viability assays in vitro. By using Inductively-Coupled Plasma Mass-Spectrometry for platinum detection, BBB penetration and bio-distribution studies were performed in a three-dimensional BBB spheroid in vitro model and in vivo in mouse brain, intracranial tumor, and peripheral organs. Dose-regime studies involved observations of symptomatology and weight variations after bi-weekly injections of platinum compounds at 2mg/kg and 5mg/kg. RESULTS The Pt(IV)-M13 conjugate possesses tumor cell killing effects similar to cisplatin when tested in GBM cell lines in vitro. Platinum increased by using Pt(IV)-M13 when compared to cisplatin in our in vitro BBB-spheroid model (20-fold, p-value=0.0033), in brain tissue (10-fold, p< 0.0001) and GBM tumor-bearing mice models (7.5-fold, p< 0.0001). Bio-distribution of platinum delivered by Pt(IV)-M13 in spleen, heart and blood was significantly different to cisplatin 5hrs. after intravenous injection (p< 0.001). Bi-weekly dose regimes of Pt(IV)-M13 are tolerable in nude mice without toxicity at a similar concentration to reported tolerable cisplatin doses at 5 mg/kg. Finally, Pt(IV)-M13 significantly increased survival in a murine glioblastoma xenograft model compared with controls (median 24 days vs. 29 days, p-value=0.0071). CONCLUSION Overall, our data support the further development of BBB-crossing peptide-drug conjugates for GBM treatment.

Glioblastoma (GBM) is the most common and deadliest form of brain tumor and remains amongst the most difficult cancers to treat. Brevican (Bcan), a central nervous system (CNS)‐specific extracellular matrix protein, is upregulated in high‐grade glioma cells, including GBM. A Bcan isoform lacking most glycosylation, dg‐Bcan, is found only in GBM tissues. Here, dg‐Bcan is explored as a molecular target for GBM. In this study, a d ‐peptide library is screened to identify a small 8‐amino acid dg‐ B can‐ T argeting P eptide (BTP) candidate, called BTP‐7 that binds dg‐Bcan with high affinity and specificity. BTP‐7 is preferentially internalized by dg‐Bcan‐expressing patient‐derived GBM cells. To demonstrate GBM targeting, BTP‐7 is radiolabeled with 18 F, a radioisotope of fluorine, and increased radiotracer accumulation is found in intracranial GBM established in mice using positron emission tomography (PET) imaging. dg‐Bcan is an attractive molecular target for GBM, and BTP‐7 represents a promising lead candidate for further development into novel imaging agents and targeted therapeutics.

Effective delivery to the brain limits the development of novel glioblastoma therapies. Here, we introduce conjugation between platinum(IV) prodrugs of cisplatin and perfluoroaryl peptide macrocycles to increase brain uptake. We demonstrate that one such conjugate shows efficacy against glioma stem-like cells. We investigate the pharmacokinetics of this conjugate in mice and show that the amount of platinum in the brain after treatment with the conjugate is 15-fold greater than with cisplatin after 5 h.

Abstract High-grade gliomas are deadly cancers, and current standard-of-care has demonstrated limited success. The ability to specifically target glioma cells allows for the development of safer and more efficacious brain cancer therapy strategies. Brevican, a CNS-specific extracellular matrix protein is upregulated in glioma cells and its expression correlates with tumor progression. Particularly, a brevican isoform lacking glycosylation, B/bΔg is a unique glioma marker and not expressed in non-cancerous tissues. Therefore, B/bΔg represents a valuable target for anti-cancer strategies. Here, we describe the utilization of state-of-the-art platforms to screen a one-bead-one-compound combinatorial peptide library to discover a novel “B/bΔg-Targeting Peptides”, called BTP-7 that can bind B/bΔg with high affinity and specificity. BTP-7 displayed 260 nanomolar affinity for recombinant B/bΔg protein. Binding to a specific site on B/bΔg was confirmed in a competitive binding assay using BTP-7 functionalized with a UV-crosslinker and BTP-7 had little association with the fully glycosylated isoform of brevican (control). Scrambling of the BTP-7 sequence led to complete abrogation of B/bΔg binding. Furthermore, BTP-7 is preferentially taken up by B/bΔg-expressing glioma cells compared with non-expressing cells. We also discovered that BTP-7 can cross the blood-brain barrier in both the in vitro BBB organoid model and in mice. BTP-7 displayed 10x greater binding to intracranial GBM-6 tumors than control peptides, and 4x higher tumor uptake than in normal brain tissues. Conjugation of BTP-7 to camptothecin (an anti-tumor drug) via a cleavable linker led to increased DNA damage in intracranial GBM-6 tumors and prolonged survival in tumor-bearing mice. Our results show the potential of BTP-7 for the development of next-generation targeted therapeutics that could greatly benefit the outcome of patients with advanced brain cancer.

High-grade gliomas are deadly cancers, and current standard-of-care has demonstrated limited success. The ability to specifically target glioma cells can allow for the development of safer and more efficacious brain cancer therapy strategies. Brevican, a CNS-specific extracellular matrix protein is upregulated in glioma cells and its expression correlates with tumor progression. Particularly, a brevican isoform lacking glycosylation, B/bΔg is a unique glioma marker and not expressed in non-cancerous tissues. Therefore, B/bΔg represents a valuable target for anti-cancer strategies. Here, we describe the utilization of state-of-the-art platforms to screen a one-bead-one-compound combinatorial peptide library to discover a novel "B/bΔg-Targeting Peptides", called BTP-7 that can bind B/bΔg with high affinity and specificity. BTP-7 displayed 260 nanomolar affinity for recombinant B/bΔg protein, and had little association with the fully glycosylated isoform of brevican (control). Scrambling of the BTP-7 sequence led to complete abrogation of B/bΔg binding. Furthermore, BTP-7 is preferentially taken up by B/bΔg-expressing glioma cells compared with non-expressing cells. We also discovered that BTP-7 can cross the blood-brain barrier using both the in vitro BBB organoid model and in mice. BTP-7 displayed 10x greater binding to intracranial GBM-6 tumors than control peptides, and 4x higher tumor uptake than in normal brain tissues. Conjugation of BTP-7 to camptothecin (an anti-tumor drug) via a cleavable linker led to increased DNA damage in intracranial GBM-6 tumors and prolonged survival in tumor-bearing mice. Our results show the potential of BTP-7 for the development of next-generation targeted therapeutics that could greatly benefit the outcome of patients with advanced brain cancer.

Background Targeted therapies for malignant brain cancer that are currently available have little clinical activity, highlighting an urgent need for the development of novel precision medicines. Brevican (Bcan), a central nervous system (CNS)-specific extracellular matrix protein is upregulated in glioma cells. A brevican isoform lacking glycosylation, dg-Bcan, is a unique glioma marker and thus represents a valuable target for anti-cancer therapy. In this study, we aimed to find a versatile dg-Bcan specific ligand to facilitate glioma targeting. Methods We screened a D-peptide library to identify dg- B can- T argeting P eptide (BTP) candidates, which were characterized extensively through binding kinetic analyses, cell uptake tests and animal studies. Results The top candidate, BTP-7 binds dg-Bcan with high affinity and specificity, is preferentially internalized by Bcan-expressing glioma cells and can cross the blood-brain barrier in vitro and in mice. Functionalization of camptothecin with BTP-7 led to increased drug delivery to intracranial glioblastoma and cytotoxicity in tumor tissues, as well as prolonged survival in tumor-bearing mice. Conclusion dg-Bcan is an attractive therapeutic target for high-grade gliomas, and BTP-7 represents a promising lead candidate for further development into novel targeted therapeutics. Key points BTP-7 is a high affinity peptide ligand for the dg-Bcan protein and Bcan-expressing cells. BTP-7 targets human intracranial GBM xenografts in mice. Functionalization of a toxic anti-cancer drug with BTP-7 enables targeted delivery of the therapeutic to intracranial GBM in mice Importance of the Study Targeted therapies for malignant brain cancer that are currently available have little clinical activity, highlighting an urgent need for the development of novel precision medicines that can selectively recognize and kill high-grade glioma tissues. A protein called dg-Bcan is an ideal target because it is present only in the extracellular matrix of high-grade glioma cells and is absent from normal brain tissues. Here, we describe the discovery of a novel dg- B can- T argeting P eptide, called BTP-7 that can bind specifically to high-grade glioma cells/tissues, and thus serve as a promising drug delivery vehicle.

brain cancer: brain cancerThe brain is arguably the most important organ in the body. Unlike all other blood vessels in the body, the brain vasculature system has evolved a highly specialized protective system, called the blood-brain barrier (BBB) to selectively transport essential biomolecules needed to support the functions of the central nervous system (CNS) while preventing the entry of most other foreign agents that might be potentially harmful to the brain. Unfortunately, the presence of the BBB also poses a significant challenge for therapeutic delivery to treat diseases within the CNS; and indeed, this problem is highly relevant to brain cancer therapy. Despite increasing development and approval of multiple new therapies, the survival rate of patients with malignant brain tumors (such as glioblastoma) has not improved (Drug Resist Updat 2015;19:1-12, Am Health Drug Benefits 2014:7;140-149). Patients still face abysmal prognosis even after surgical excision and aggressive post-operative chemo-radiotherapy (N Engl J Med 2005;352:987-996) and, to date, brain cancer is the leading cause of death in children and adolescents (CA Cancer J Clin 2016;66:7-30). Healthy BBB forms a protective “wall” along the brain capillaries and is made up of predominantly brain capillary endothelial cells that are surrounded by closely associating pericytes and astrocytic endfeet (Figure) (Nat Rev Neurosci 2006;7:41-53). Paracellular flux at the BBB is restricted by the presence of tight junctions that connect adjacent endothelial cells (Trends Neurosci 2001;24:719-725). Essential nutrients required for normal brain function (such as glucose and amino acids) are shuttled into the brain through specific transporters, while other large molecules or complexes (such as insulin and transferrin) are actively transported from the blood into the brain through receptor-mediated transcytosis (Trends Neurosci 2001;24:719-725). Additionally, efflux transporters (such as P-glycoprotein) on the surface of endothelial cells play an important role in actively pumping potentially toxic foreign agents back into the bloodstream (Adv Drug Deliv Rev 1999;36:179-194). Solid tumors recruit new blood vessels through angiogenesis for a continuous supply of oxygen, nutrients, and necessary growth factors for rapid propagation. Angiogenic blood vessels have defective architecture and exhibit enhanced vascular permeability or “leakiness.” This phenomenon, which is universal in all solid tumors, is known as the enhanced permeability and retention (EPR) effect, and facilitates delivery of chemotherapeutics to tumor tissues (Adv Drug Deliv Rev 2011;63:136-151). In contrast to the BBB, abnormal and dysfunctional blood vessels in brain tumors form the blood-tumor barrier (BTB). Blood vessels in brain tumors are heterogenous, consisting of capillary populations that are: continuous and non-fenestrated (such as those of normal brain); continuous but fenestrated (exhibit enhanced permeability): and non-continuous (with interendothelial gaps) (Neuro-Oncol 2000;2:45-59). Figure:: Structure of the Blood-Brain Barrier vs. Blood-Tumor BarrierA) Schematic showing blood vessels with 1) intact blood-brain barrier (BBB); and 2) blood-tumor barrier (BTB). B) Cross section of a vessel with intact BBB (left), showing the organization of brain capillary endothelial cells, pericytes, and astrocytes. The tight junctions in-between endothelial cells prevent paracellular transport from the circulation into the brain. The presence of tumor cells (right) causes major disruption to the organization and function of the BBB, resulting in the formation of the BTB, where fenestrations between endothelial cells leading to vessel “leakiness,” as well as disrupted basement membrane are observed.In areas where fenestration or endothelial discontinuity is observed, drug delivery level in the tumor is substantially higher. This phenomenon is mostly found at or near the tumor core, but even then, due to the heterogeneity of the BTB, the distribution of drug permeability in the tumor is highly variable (Clin Cancer Res 2010;16:5664-5678). Invasive cells at the leading edge of the brain tumor border continue to thrive under the protection of the BBB (Figure) (Drug Metab Dispos 2013;41:33-39). Failure to deliver therapeutics to the invasive cancer cells behind a functional BBB is one of the major causes for disease recurrence, even after primary tumor debulking by surgical means. Additionally, even though most tumors exhibit some increased BTB permeability, a high percentage of these lesions do not respond to cytotoxic drugs (such as paclitaxel and doxorubicin), highlighting the critical role of active efflux pumps in both the BTB and brain tumor cells in conferring chemoresistance (Cancer Res 2005;65:11419-11428). Therapeutic Delivery to Tumors Osmotic shrinkage using hypertonic solutions (such as mannitol) have been used to increase BBB permeability for enhancing drug delivery to the brain in the clinic. Various other therapeutic strategies are also employed to increase drug delivery to brain tumors, including injecting the chemotherapy agents directly into the CNS through intrathecal, intranasal, or intraventricular administration. Direct drug administration into the brain interstitial system using biodegradable wafers (such as carmustine) and catheter-based convection-enhanced delivery (CED) have also been applied in the clinics. Although they have been successful in improving patient survival, clinicians remain hesitant with the utility of these approaches due to morbidity associated with perioperative complications (Curr Pharm Des 2016;22:1177-1193, J Neurooncol 2012;107:373-378). BBB disruption by radiation (Oncol Rep 2001;9:683-688) or imaging-guided focused ultrasound using intravenously administered microbubbles (Proc Natl Acad Sci USA 2006;103:11719-11723), where activation of the microbubbles in the vessels by an acoustic field produced by ultrasound treatment temporarily disrupts the tight junctions that connect the endothelial cells of the BBB have also been explored (J Acoust Soc Am 2011;130:3059-3067). While there are safety concerns relating to toxicity and long-term effects due to radiation, the use of focused ultrasound appears to be relatively safe when tested in monkeys (Cancer Res 2012;72:3652-3663). Additionally, due to the EPR effect, microbubbles accumulation in tumors upon administration followed by ultrasound treatment allows for further selectivity in BBB opening and enhancement of therapeutic delivery within the tumor(s). In other areas of research, due to unknown long-term effects of BBB modulation, scientists have taken a different approach to overcome the BBB by developing “Trojan horses” that can hijack the transport systems of the BBB, including the insulin receptor, transferrin receptor, and low-density lipoprotein receptor to deliver chemotherapy into the brain (Curr Pharm Des 2016;22:1177-1193). Drug formulations that have been designed to cross the BBB include attachment of monoclonal antibodies or peptides that recognize these receptors (and then become endocytosed) to targeted liposomes that contain toxic anti-tumor agents, or even directly to the drug itself (Clin Cancer Res 2007;13:1663-1674). Even with successful BBB penetration, drug targeting to specifically kill tumor cells is necessary to achieve efficacy without harming healthy brain tissues, which can lead to severe neurotoxicity. BBB Modeling for Therapeutic Development In vitro screening platforms have played crucial roles in facilitating the discovery of new brain therapeutics and optimization of existing drugs with enhanced BBB penetration. In 1983, Bowman and colleagues show that brain endothelial cells can be grown in tissue culture to model the BBB (Ann Neurol 1983;14:396-402). This finding has led to a wave of in vitro BBB mimetic model development, including the mid-throughput static transwell system, which involves the culture of brain endothelial cells within a transwell insert followed by culture of astrocytes (and/or pericytes) at the basal compartment of the transwell (J Cereb Blood Flow Metab 2016;36:862-890). Although the transwell model is highly versatile, simple, and currently the most widely used BBB model, it has been criticized for many well-known limitations (Eur J Nanomedicine 2014;6:185). Efforts to improve BBB modeling through simulation of a more realistic representation of the BBB environment in a living brain have led to the development of the dynamic in vitro BBB model and other microfluidic BBB systems, which account for blood flow and shear stress (Brain Res 2006;1109:1-13, Lab Chip 2012;12:1784-1792, Lab Chip 2013;13:1093-1101). More recently, multicellular BBB organoids spawned through the co-culture of human brain endothelial cells, pericytes, and astrocytes under low-adhesion condition have been shown to closely reproduce key BBB elements and functions (Nat Commun 2017;8:15623). The ease of culture and robustness, along with the offer of a high-throughput capacity for drug screening make this model particularly attractive compared to preceding models. Although culture-based models play a critical role in advancing neuroscience, they are not a substitute for in vivo models and should always be complemented with results gained from animal (or clinical) studies. CHOI-FONG CHO, PHD, is Instructor in Neurosurgery at the Brigham and Women's Hospital and Harvard Medical School, Boston, and Research Fellow in the Department of Chemistry, Massachusetts Institute of Technology.Choi-Fong Cho, PhD: Choi-Fong Cho, PhDCan't Find a Past Issue? Go online to http://bit.ly/2wCfymG where you can view all the articles from past issues of Oncology Times.

In vitro models of the blood-brain barrier (BBB) are critical tools for the study of BBB transport and the development of drugs that can reach the CNS. Brain endothelial cells grown in culture are often used to model the BBB; however, it is challenging to maintain reproducible BBB properties and function. 'BBB organoids' are obtained following coculture of endothelial cells, pericytes and astrocytes under low-adhesion conditions. These organoids reproduce many features of the BBB, including the expression of tight junctions, molecular transporters and drug efflux pumps, and hence can be used to model drug transport across the BBB. This protocol provides a comprehensive description of the techniques required to culture and maintain BBB organoids. We also describe two separate detection approaches that can be used to analyze drug penetration into the organoids: confocal fluorescence microscopy and mass spectrometry imaging. Using our protocol, BBB organoids can be established within 2-3 d. An additional day is required to analyze drug permeability. The BBB organoid platform represents an accurate, versatile and cost-effective in vitro tool. It can easily be scaled to a high-throughput format, offering a tool for BBB modeling that could accelerate therapeutic discovery for the treatment of various neuropathologies.

Abstract The inability of most systemically delivered therapeutics to cross the blood-brain-barrier (BBB) is considered a major barrier to effective brain cancer treatment. In malignant glioma the tumor neovasculature is known to be leaky, however invasive cells remain protected behind an intact BBB, and continue to thrive within the brain, ultimately leading to tumor regrowth and patient death. This leaves us with an urgent unmet need for the development of next-generation therapeutics with improved brain delivery. For the first time, we describe here the utility of 3D multicellular BBB spheroids made of human brain endothelial cells (ECs), pericytes and astrocytes as a screening tool for brain-penetrating agents. We show that the outer surface of the spheroids, composed primarily of ECs and pericytes, form a tight barrier which is permeabilized in the presence of VEGF. The barrier is characterized by the presence of intact tight junctions and efflux-pump activity (i.e., P-glycoprotein). Furthermore, we have used this model to successfully demonstrate the transport of angiopep-2 (a well-known brain delivery vector) and its conjugates (containing cargoes of various sizes such as peptide, protein and affibody), thereby displaying the versatility of this model to screen and study a wide range of therapeutic agents. We demonstrate that this model is superior to the conventional transwell model in maintaining essential BBB characteristics (i.e., tight/adherens junctions and P-glycoprotein expression) and as a drug-screening tool. We have utilized the spheroid model to screen a panel of cell-penetrating peptides (CPPs) to identify several candidates with high brain-penetration potential. We then verified the ability of the top 4 candidate CPPs to cross the BBB in mice. This high-throughput model can lead to better design and analysis of first-in-class glioma therapeutics, and improve prediction of drug penetration in a living model, paving the way for breakthrough discoveries in brain cancer. Citation Format: Choi-Fong Cho. Blood-brain-barrier spheroids: Next-generation screening platform for brain-penetrating agents [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 4014. doi:10.1158/1538-7445.AM2017-4014

High-grade gliomas are deadly cancers, and current standard-of-care has demonstrated limited success. The ability to specifically target glioma cells can allow for the development of safer and more efficacious brain cancer therapy strategies. Brevican (BCAN), a CNS-specific extracellular matrix protein is upregulated in glioma cells and its expression correlates with tumor progression. Particularly, a membrane-bound BCAN isoform lacking normal glycosylation, called B/bΔg is a unique glioma marker and is not expressed in non-cancerous tissues. Therefore, B/bΔg represents a valuable target for anti-cancer strategies. Here, we describe the utilization of state-of-the-art technologies that we have recently developed to discover novel "B/bΔg-Targeting Peptides" (BTP). Briefly, small magnetic beads displaying B/bΔg were used to screen a one-bead-one-compound combinatorial library, enabling high-throughput labeling of library beads presenting peptide candidates with high affinity for B/bΔg. The "hit" beads were rapidly sorted using a microfluidic magnetic-activated sorter of our own design. The hits were then, exposed to cells expressing B/bΔg, and beads with the highest cell association were isolated and sequenced. Characterization of the purified peptides through kinetic binding analysis and cell uptake studies revealed BTP-7 as the lead candidate for B/bΔg binding. BTP-7 displayed 260 nanomolar affinity for recombinant B/bΔg protein, and had little association with the fully glycosylated isoform of BCAN. Scrambling of the BTP-7 sequence led to complete abrogation of B/bΔg binding. For in vivo evaluation, GBM-6 tumors derived from human patients were established intracranially in nude mice, and tumor formation was verified by MRI. Upon systemic administration, BTP-7 displayed approximately 10x greater binding to GBM-6 tumors than the control, as well as 4x higher tumor uptake compared to normal brain tissue. We are currently working on conjugating BTP-7 to a variety of toxic payload to selectively target gliomas, with the goal of improving brain cancer therapeutic efficacy that can ultimately benefit patient.

Here we describe the utility of peptide macrocyclization through perfluoroaryl-cysteine SNAr chemistry to improve the ability of peptides to cross the blood-brain barrier. Multiple macrocyclic analogues of the peptide transportan-10 were investigated that displayed increased uptake in two different cell lines and improved proteolytic stability. One of these analogues (M13) exhibited substantially increased delivery across a cellular spheroid model of the blood-brain barrier. Through ex vivo imaging of mouse brains, we demonstrated that this perfluoroarene-based macrocycle of TP10 exhibits increased penetration of the brain parenchyma following intravenous administration in mice. Finally, we evaluated macrocyclic analogues of the BH3 domain of the BIM protein to assess if our approach would be applicable to a peptide of therapeutic interest. We identified a BIM BH3 analogue that showed increased penetration of the brain tissue in mice.

Angiogenesis is a dynamic process fundamental to the development of solid tumors. Epidermal growth factor-like domain 7 (EGFL7) is a protein whose expression is restricted to endothelial cells undergoing active remodeling that has emerged as a key mediator of this process. EGFL7 expression is associated with poor outcome in several cancers, making it a promising target for imaging or therapeutic strategies. Here, EGFL7 is explored as a molecular target for active neovascularization. Using a combinatorial peptide screening approach, we describe the discovery and characterization of a novel high affinity EGFL7-binding peptide, E7p72, that specifically targets human endothelial cells. Viral nanoparticles decorated with E7p72 peptides specifically target tumor-associated neovasculature with high specificity as assessed by intravital imaging. This work highlights the value of EGFL7 as a target for angiogenic vessels and opens the door for novel targeted therapeutic approaches.