The Blood-Brain Barrier (BBB) is a highly selective semipermeable border of endothelial cells that prevents soluble molecules in the circulating blood from non-selectively crossing into the extracellular space of the brain where neurons reside. The blood-brain barrier is formed by endothelial cells of the blood vessel capillary wall those are connected by tight junctions, astrocyte end-feet ensheathing the capillary, and pericytes embedded in the capillary basement membrane (Fig. 1, ref 1). The blood-brain barrier restricts the passage of pathogens, diffusion of soluble small molecules in the blood, and large or hydrophilic molecules into the cerebrospinal fluid, while allowing diffusion of hydrophobic molecules such as hormones and O2 or CO2, and small polar molecules. Cells of the barrier actively transport metabolic products such as glucose across the barrier using specific transport proteins. The capillary length extends for about 650 kilometers in human and 0.6 km in mice, thus, capillaries provide the largest endothelial surface for the bidirectional transports of solutes between blood circulation and the brain (1). However, 100% of molecules larger than 500 dalton and 98% of smaller molecules cannot cross BBB.
In a brain tumor, BBB is disrupted during tumor progression and is referred to as the Blood-Tumor Barrier (BTB). Although BTB is more permeable than BBB, its heterogeneous permeability results in unpredictable drug passage and accumulation in brain tumors, thus BTB is one of the rate-limiting factors in clinically effective therapy. In the intact BBB, active transport occurs across the BBB endothelium by polarized expression of ATP-binding cassette transporters (ABC transporters) in vessel walls. These transporters mediate efflux of xenobiotics and toxins from the endothelium away from the neuroparenchymal space. Most anti-cancer low molecular weight drugs are captured by ABC proteins. For example, several targeted therapies for gliomas and brain metastases have affinity for the multidrug-resistant ABC transporters, P-glycoprotein, breast cancer resistance protein (BCRP) and multidrug resistance proteins, which are all expressed in the BBB (2). This transport mechanism is essential for homeostasis as well as affecting delivery of therapeutics. In addition, Microglia, the most abundant innate immune cells in the brain and peripheral immune cells such as leukocytes can increase BBB permeability through interleukin-1B secretion or through the adhesion proteins. The BBB can also be innervated by synaptic nerve endings. GABA-ergic, cholinergic, noradrenergic and serotonergic neurons can directly interact with the brain endothelium to regulate blood flow, neurovascular coupling and BBB permeability.
When tumors grow in the brain, they typically impede blood flow due to the confined space within the brain. During primary brain tumor progression and the development of brain metastasis, the tumor vasculature becomes increasingly heterogeneous. Nutritional demand of proliferating cancer cells recruit existing vessels as well as creating new vessels via angiogenesis. Vascular dysfunction during tumor progression, in part mediated by deregulated expression of angiogenic factors such as VEGF, leads to hypoxia and acidic microenvironment fuels tumor progression through HIF-1a induced transcriptional programs. Blocking VEGF signaling transiently attenuate immature and leaky vessels of brain tumors in mice and actively remodels the remaining vasculature so that it more closely resembles the normal vasculature – called vascular normalization. Researchers have shown the survival benefit of vascular normalization in patients with newly diagnosed as well as recurrent glioblastoma receiving antiangiogenic agents (2). However, an adverse effect of anti-angiogenic therapy is that the resulting hypoxia can increase invasiveness of cancer cells. Furthermore, VEGF itself can regulate BBB permeability; therefore, anti-angiogenics at high doses might decrease BTB permeability.
The structure of BTB is characterized by aberrant pericyte distribution, and loss of astrocytic end-feet and neuronal connections. Furthermore, invading glioma cells can physically disrupt BBB integrity. T cell subpopulations and peripheral monocytes are detected in brain tumors, indicating permeability of the neuro vascular unit. The BTB is generally considered “leakier” than the BBB, however, the BTB retains expression of active efflux transporters. BTB structural integrity is heterogeneous between metastatic lesions and tumor types. For example, among four types of medulloblastoma subtypes, the WNT subtype of medulloblastoma has a fenestrated vasculature allowing higher accumulation of chemo-therapeutic agents, whereas the Sonic Hedge Hog type medulloblastoma has an intact BBB (2). HER2 positive breast cancer brain metastases generally express more GLUT1 and BCRP transporters than other subtypes.
Drug Delivery Across the BTB
Despite emerging knowledge of BTB structures and functions, novel strategies to overcome the BTB and deliver therapeutics to brain tumors are necessary.
A targeting ligand binds to its receptor to trigger endocytosis, using the vesicular trafficking machinery to transport drugs. A drug can be molecularly linked to this ligand enabling transport the BTB. Well characterized receptors for this approach include transferrin, insulin and insulin-like growth factor 1 receptors (3).
Recent study published by one of our CureScience™ colleagues featuring IF7 peptide is also applying existing trafficking machinery, Annexin A1. Annexin A1 is expressed specifically on luminal side of angiogenic vessels, thus IF7 peptide and conjugated drug SN38 was captured by Annexin A1 and crossed BTB, transported to the tumor in mouse model (4). Other targeted approaches used endogenously expressed BTB receptor low-density lipoprotein receptor-related protein 1 (LRP1). Three paclitaxel molecules covalently linked to angiopep-2 peptide targeted LRP1 protein, increased delivery nearly fifty folds into brain metastases and improved median survival in preclinical and clinical studies (5).
Another approach is, blocking the efflux pumps. Efflux pumps actively pump out pharmacological compounds with sufficient binding affinity. Efflux transporters including ABC transporters, P-glycoprotein and BCRP have shown affinity for several FDA approved drugs, including chemotherapeutics. Preclinical studies focused on the co-administration of drugs with transporter inhibitors, for example, inhibitors of ABC transporters. This approach has shown significant enhancement in the brain concentrations of chemotherapeutic agents and targeted therapies for temozolomide, ADP ribose polymerase inhibitor ABT-888, and BRAF inhibitor vemurafenib (6). In addition, structural design of targeted agents such as PI3K or TOR kinase inhibitors with reduced affinity to P-glycoprotein or BCRP is a promising approach.
Adapting the homing ability of stem cells is another approach for therapeutic delivery across BTB. Among a wide range of stem cells, interestingly, neural stem cells (NSCs) and mesenchymal stem cells (MSCs) showed high tropism to tumors and their ability to cross the BTB. This makes them preferred carriers of therapeutics to brain tumors (2). Migration of stem cells are not well characterized, yet several reports showed that stem cells recruit similar steps as immune cell infiltration, such as rolling on and adhesion to the endothelium and transmigrate across the BBB. Stem cells migrate along invasive tumor borders, even at a distant site from the tumor, indicating possible application of stem cells as targeting brain micro-metastases.
Mesenchymal stem cells are also known to secrete growth factors, anti-inflammatory mediators, and immune modulatory cytokines under hypoxic condition. The CureScience™ team is developing stem cell therapy to treat glioblastoma, COVID-19 and other applications (7). NSC-mediated expression of an enzyme that converts a separately administered non-toxic pro-drug into a cytotoxic drug via the bystander effect is another approach that was also found to be well tolerated in a recent phase I trial in glioblastoma (2).
Cancer immunotherapy is another emerging treatment in recent years. Yet because of the BBB/BTB , accessibility to the brain by immune cells are limited. This suppression is called “immune privilege” or immunologically silenced. The BBB limit antigen presentation and immune cell infiltration, in fact unusually extravagated T-cells degrade myelin sheaths and induce inflammation, become a cause of auto-immune disease Multiple Sclerosis (8, 9). In the case of glioblastoma, since the BBB is disrupted, infiltration of CD8+ cytotoxic T-cells are often observed. The compromised BTB may facilitate the tumor-associated antigen presentation, supported by the evidence that administration of immune checkpoint inhibitors before surgery improved response of glioblastoma patients (10). Adoptively transferred T-cell therapy and chimeric antigen receptor (CAR) T cell therapy are applied clinically, but further considerations are necessary. One of invasive approach of administration is, infusing CAR T cells directly into the blood, cerebral spinal fluid, or locally in the tumor cavity, bypassing the BTB blockage (11).
The combination of low-intensity focused ultrasound (FUS) pulses and circulating microbubbles, lipid, albumin or polymer-shelled gas pockets, which scatter sound and vibrate in response to ultrasound waves, provides a physical method to transiently disrupt the BBB/BTB and increase the permeability of the BBB/BTB (2).
Preclinical research has shown that the method of FUS with microbubbles can lead to an increase in the delivery and penetration of intravenously administered anticancer agents in brain tumors, and increase in the median survival of murine tumor models. Additionally several phase I clinical trials utilizing FUS are ongoing in order to demonstrate its safety and efficacy (2).
Our team at CureScience™ is developing novel therapeutics of anti-cancer drugs as well as evaluating effective strategies to deliver those drugs directly to the brain tumors. Further investigation is ongoing.
Written By: Misa S. Anekoji, Ph.D.
November 11, 2020
Key words: Blood-Brain Barrier, Blood-Tumor Barrier, Brain Cancer, Glioblastoma, Drug Delivery
(1)Tatiana Barichello (ed), “Blood-Brain Barrier, Neuromethods,” vol. 142, Springer Nature 2019 (book)
(2) Costas D. Alvanitis, Gino B. Ferraro, and Rakesh K. Jain, Nature Reviews Cancer, vol. 20, p.26-41(2020), Doi: 10.1038/s41568-019-0205-x
(3) Lajoie, J. M. & Shusta, E. V. Targeting receptor- mediated transport for delivery of biologics across the blood–brain barrier. Annu. Rev. Pharmacol. Toxicol. 55, 613–631 (2015).
(4) Nonaka, M., Suzuki-Anekoji, M., Nakayama, J. et al. Overcoming the blood–brain barrier by Annexin A1-binding peptide to target brain tumours. Br J Cancer (2020). Sep 14, 2020. https://doi.org/10.1038/s41416-020-01066-2
(5) Drappatz, J. et al. Phase I study of GRN1005 in recurrent malignant glioma. Clin. Cancer Res. 19, 1567–1576 (2013).
(6) Lin, F. et al. ABCB1, ABCG2, and PTEN determine the response of glioblastoma to temozolomide and ABT-888 therapy. Clin. Cancer Res. 20, 2703–2713 (2014).
(7) Hypoxia Treated Stem Cells for COVID19: A New Weapon in a Deadly Fight. CureScience BLOG, Apr 23, 2020. Tom Ichim.
(8) Neuroscience News, Nov. 22, 2017
(9) Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation. Sarah E. Lutz, Julian R. Smith, Dae Hwan Kim, et al. Cell Reports. Published online November 21 (2017) doi:10.1016/j.celrep.2017.10.094
(10) Cloughesy, T. F. et al. Neoadjuvant anti-PD-1 immunotherapy promotes a survival benefit with intratumoral and systemic immune responses in recurrent glioblastoma. Nat. Med. 25, 477 (2019).
(11) CAR T cell therapy for pediatric brain tumors. John D. Patterson, Jeffrey C. Henson, et al. Front. Oncol., 12 August 2020 | https://doi.org/10.3389/fonc.2020.01582