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OBJECTIVE Immune checkpoint inhibitors such as anti–programmed cell death protein 1 (anti-PD-1) have shown promise for the treatment of cancers such as melanoma, but results for glioblastoma (GBM) have been disappointing thus far. It has been suggested that GBM has multiple mechanisms of immunosuppression, indicating a need for combinatorial treatment strategies. It is well understood that GBM increases glutamate in the tumor microenvironment (TME); however, the significance of this is not well understood. The authors posit that glutamate upregulation in the GBM TME is immunosuppressive. The authors utilized a novel glutamate modulator, BHV-4157, to determine synergy between glutamate modulation and the well-established anti-PD-1 immunotherapy for GBM. METHODS C57BL/6J mice were intracranially implanted with luciferase-tagged GL261 glioma cells. Mice were randomly assigned to the control, anti-PD-1, BHV-4157, or combination anti-PD-1 plus BHV-4157 treatment arms, and median overall survival was assessed. In vivo microdialysis was performed at the tumor site with administration of BHV-4157. Intratumoral immune cell populations were characterized with immunofluorescence and flow cytometry. RESULTS The BHV-4157 treatment arm demonstrated improved survival compared with the control arm (p < 0.0001). Microdialysis demonstrated that glutamate concentration in TME significantly decreased after BHV-4157 administration. Immunofluorescence and flow cytometry demonstrated increased CD4+ T cells and decreased Foxp3+ T cells in mice that received BHV-4157 treatment. No survival benefit was observed when CD4+ or CD8+ T cells were depleted in mice prior to BHV-4157 administration (p < 0.05). CONCLUSIONS In this study, the authors showed synergy between anti-PD-1 immunotherapy and glutamate modulation. The authors provide a possible mechanism for this synergistic benefit by showing that BHV-4157 relies on CD4+ and CD8+ T cells. This study sheds light on the role of excess glutamate in GBM and provides a basis for further exploring combinatorial approaches for the treatment of this disease.

OBJECTIVE Atypical teratoid rhabdoid tumors (ATRTs) are aggressive pediatric brain tumors with no current standard of care and an estimated median patient survival of 12 to 18 months. Previous genetic analyses have implicated cyclin D1 and enhancer of zeste homolog 2 (EZH2), a histone methyltransferase that is implicated in many cancers, as key drivers of tumorigenicity in ATRTs. Since the effects of EZH2 and cyclin D1 are facilitated by a host of cyclin-dependent kinases (CDKs), the authors sought to investigate the potential therapeutic effects of targeting CDKs in ATRTs with the multi–CDK inhibitor, TG02. METHODS Human ATRT cell lines BT12, BT37, CHLA05, and CHLA06 were selected for investigation. The effects of TG02 on cell viability, proliferation, clonogenicity, and apoptosis were assessed via Cell Counting Kit-8 assays, cell counting, clonogenic assays, and flow cytometry, respectively. Similar methods were used to determine the effects of TG02 combined with radiation therapy (RT) or cisplatin. Synergism indices for TG02-cisplatin combination therapy were calculated using CompuSyn software. RESULTS TG02 was observed to significantly impair ATRT cell growth in vitro by limiting cell proliferation and clonogenicity, and by inducing apoptosis. TG02 inhibited ATRT cell proliferation and decreased cell viability in a dose-dependent manner with nanomolar half maximal effective concentration (EC50) values (BT12, 207.0 nM; BT37, 127.8 nM; CHLA05, 29.7 nM; CHLA06, 18.7 nM). TG02 (150 nM) dramatically increased the proportion of apoptotic ATRT cells 72 hours posttreatment (TG02 8.50% vs control 1.52% apoptotic cells in BT12, p < 0.0001; TG02 70.07% vs control 15.36%, p < 0.0001). Combination therapy studies revealed that TG02 acted as a potent radiosensitizer in ATRT cells (BT12 surviving fraction, RT 51.2% vs RT + TG02 21.7%). Finally, CompuSyn analysis demonstrated that TG02 acted synergistically with cisplatin against ATRT cells at virtually all therapeutic doses. These findings were consistent in cell lines that cover all three molecular subgroups of ATRTs. CONCLUSIONS The results of this investigation have established that TG02 is an effective therapeutic against ATRTs in vitro. Given the lack of standard therapy for ATRTs, these findings help fill an unmet need and support further study of TG02 as a potential therapeutic option for patients with this deadly disease.

INTRODUCTION: Treatments for brain cancer have radically evolved in the past decade due to a better understanding of the interplay between the immune system and tumors of the central nervous system (CNS). However, glioblastoma multiforme (GBM) remains the most common and lethal CNS malignancy affecting adults. AREAS COVERED: The authors review the literature on glioblastoma pharmacologic therapies with a focus on trials of combination chemo-/immunotherapies and drug delivery platforms from 2015 to 2021. EXPERT OPINION: Few therapeutic advances in GBM treatment have been made since the Food and Drug Administration (FDA) approval of the BCNU-eluting wafer, Gliadel, in 1996 and oral temozolomide (TMZ) in 2005. Recent advances in our understanding of GBM have promoted a wide assortment of new therapeutic approaches including combination therapy, immunotherapy, vaccines, and Car T-cell therapy along with developments in drug delivery. Given promising preclinical data, these novel pharmacotherapies for the treatment of GBM are currently being evaluated in various stages of clinical trials.

OBJECTIVE Medulloblastoma, the most common pediatric brain malignancy, has Sonic Hedgehog (SHH) and group 3 (Myc driven) subtypes that are associated with the activity of eukaryotic initiation factor 4E (eIF4E), a critical mediator of translation, and enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and master regulator of transcription. Recent drug repurposing efforts in multiple solid and hematologic malignancies have demonstrated that eIF4E and EZH2 are both pharmacologically inhibited by the FDA-approved antiviral drug ribavirin. Given the molecular overlap between medulloblastoma biology and known ribavirin activity, the authors investigated the preclinical efficacy of repurposing ribavirin as a targeted therapeutic in cell and animal models of medulloblastoma. METHODS Multiple in vitro assays were performed using human ONS-76 (a primitive SHH model) and D425 (an aggressive group 3 model) cells. The impacts of ribavirin on cellular growth, death, migration, and invasion were quantified using proliferation and Cell Counting Kit-8 (CCK-8) assays, flow cytometry with annexin V (AnnV) staining, scratch wound assays, and Matrigel invasion chambers, respectively. Survival following daily ribavirin treatment (100 mg/kg) was assessed in vivo in immunodeficient mice intracranially implanted with D425 cells. RESULTS Compared to controls, ribavirin treatment led to a significant reduction in medulloblastoma cell growth (ONS-76 proliferation assay, p = 0.0001; D425 CCK-8 assay, p < 0.0001) and a significant increase in cell death (flow cytometry for AnnV, ONS-76, p = 0.0010; D425, p = 0.0284). In ONS-76 cells, compared to controls, ribavirin significantly decreased cell migration and invasion (Matrigel invasion chamber assay, p = 0.0012). In vivo, ribavirin significantly extended survival in an aggressive group 3 medulloblastoma mouse model compared to vehicle-treated controls (p = 0.0004). CONCLUSIONS The authors demonstrate that ribavirin, a clinically used drug known to inhibit eIF4E and EZH2, has significant antitumor effects in multiple preclinical models of medulloblastoma, including an aggressive group 3 animal model. Ribavirin may represent a promising targeted therapeutic in medulloblastoma.

Background Hydrocephalus is managed by surgically implanting flow-diversion technologies such as differential pressure valves and antisiphoning devices; however, such hardware is prone to failure. Extensive research has tested them in flow-controlled settings using saline or de-aerated water, yet little has been done to validate their performance in a setting recreating physiologically relevant parameters, including intracranial pressures, cerebrospinal fluid (CSF) protein content, and body position. Objective To more accurately chart the episodic drainage characteristics of flow-diversion technology. A gravity-driven benchtop model of flow was designed and tested continuously during weeks-long trials. Methods Using a hydrostatic pressure gradient as the sole driving force, interval flow rates of 6 valves were examined in parallel with various fluids. Daily trials in the upright and supine positions were run with fluid output collected from distal catheters placed at alternating heights for extended intervals. Results Significant variability in flow rates was observed, both within specific individual valves across different trials and among multiple valves of the same type. These intervalve and intravalve variabilities were greatest during supine trials and with increased protein. None of the valves showed evidence of overt obstruction during 30 d of exposure to CSF containing 5 g/L protein. Conclusion Day-to-day variability of ball-in-cone differential pressure shunt valves may increase overdrainage risk. Narrow-lumen high-resistance flow control devices as tested here under similar conditions appear to achieve more consistent flow rates, suggesting their use may be advantageous, and did not demonstrate any blockage or trend of decreasing flow over the 3 wk of chronic use.

Interstitial chemotherapy plays a pivotal role in the treatment of glioblastoma multiforme (GBM), an aggressive form of primary brain cancer, by enhancing drug biodistribution to the tumor and avoiding systemic toxicities. The use of new polymer structures that extend the release of cytotoxic agents may therefore increase survival and prevent recurrence. A novel core-sheath fiber loaded with the drug carmustine (BCNU) was evaluated in an in vivo brain tumor model. Three-dimensional discs were formed from coaxially electrospun fiber membranes and in vitro BCNU release kinetics were measured. In vivo survival was assessed following implantation of discs made of compressed core-sheath fibers (NanoMesh) either concurrently with or five days after intracranial implantation of 9L gliosarcoma. Co-implantation of NanoMesh and 9L gliosarcoma resulted in statistically significant long-term survival (>150 days). Empty control NanoMesh confirmed the safety of these novel implants. Similarly, Day 5 studies showed significant median, overall, and long-term survival rates, suggesting optimal control of tumor growth, confirmed with histological and immunohistochemical analyses. Local chemotherapy by means of biodegradable NanoMesh implants is a new treatment paradigm for the treatment for brain tumors. Drug delivery with coaxial core-sheath structures benefits from high drug loading, controlled long-term release kinetics, and slow polymer degradation. This represents a promising evolution for the current treatment of GBM.

Purpose: High-grade glioma (HGG) treatment is limited by the inability of otherwise potentially efficacious drugs to penetrate the blood–brain barrier. We evaluate the unique intracavity delivery mode and translational potential of a blend of poly(DL-lactic acid-co-glycolic acid; PLGA) and poly(ethylene glycol; PEG) paste combining temozolomide and etoposide to treat surgically resected HGG. Experimental Design: To prolong stability of temozolomide prodrug, combined in vitro drug release was quantitatively assessed from low pH–based PLGA/PEG using advanced analytic methods. In vitro cytotoxicity was measured against a panel of HGG cell lines and patient-derived cultures using metabolic assays. In vivo safety and efficacy was evaluated using orthotopic 9L gliosarcoma allografts, previously utilized preclinically to develop Gliadel. Results: Combined etoposide and temozolomide in vitro release (22 and 7 days, respectively) was achieved from a lactic acid–based PLGA/PEG paste, used to enhance stability of temozolomide prodrug. HGG cells from central-enhanced regions were more sensitive to each compound relative to primary lines derived from the HGG-invasive margin. Both drugs retained cytotoxic capability upon release from PLGA/PEG. In vivo studies revealed a significant overall survival benefit in postsurgery 9L orthotopic gliosarcomas, treated with intracavity delivered PLGA/PEG/temozolomide/etoposide and enhanced with adjuvant radiotherapy. Long-term survivorship was observed in over half the animals with histologic confirmation of disease-free brain. Conclusions: The significant survival benefit of intracavity chemotherapy demonstrates clinical applicability of PLGA/PEG paste-mediated delivery of temozolomide and etoposide adjuvant to radiotherapy. PLGA/PEG paste offers a future platform for combination delivery of molecular targeted compounds.

OBJECTIVEVentricular shunt infection remains an issue leading to high patient morbidity and cost, warranting further investigation. The authors sought to create an animal model of shunt infection that could be used to evaluate possible catheter modifications and innovations.METHODSThree dogs underwent bilateral ventricular catheter implantation and inoculation with methicillin-sensitive Staphylococcus aureus (S. aureus). In 2 experimental animals, the catheters were modified with a polymer containing chemical “pockets” loaded with vancomycin. In 1 control animal, the catheters were polymer coated but without antibiotics. Animals were monitored for 9 to 11 days, after which the shunts were explanted. MRI was performed after shunt implantation and prior to catheter harvest. The catheters were sonicated prior to microbiological culture and also evaluated by electron microscopy. The animals’ brains were evaluated for histopathology.RESULTSAll animals underwent successful catheter implantation. The animals developed superficial wound infections, but no neurological deficits. Imaging demonstrated ventriculitis and cerebral edema. Harvested catheters from the control animal demonstrated > 104 colony-forming units (CFUs) of S. aureus. In the first experimental animal, one shunt demonstrated > 104 CFUs of S. aureus, but the other demonstrated no growth. In the second experimental animal, one catheter demonstrated no growth, and the other grew trace S. aureus. Brain histopathology revealed acute inflammation and ventriculitis in all animals, which was more severe in the control.CONCLUSIONSThe authors evaluated an animal model of ventricular shunting and reliably induced features of shunt infection that could be microbiologically quantified. With this model, investigation of pathophysiological and imaging correlates of infection and potentially beneficial shunt catheter modifications is possible.

Glioblastoma multiforme (GBM) has few clinically approved therapeutic regimens. One of these therapeutic options includes placing biodegradable wafers releasing BCNU (Gliadel®) into the tumor bed at the time of surgical removal of the tumor. Due to the significant benefit this polymer technology has had clinically, we have prepared wafers releasing Temozolomide (TMZ), an anticancer drug used systemically for treating GBM. TMZ delivered via polymer wafer could be used as a complementary treatment with or as an alternative to Gliadel®. TMZ is an alkylating agent which is water soluble. To remain comparable with the preclinical studies that led to Gliadel® the same size of wafers were formulated with TMZ. Wafers were loaded with 50% w/w TMZ in poly(lactic acid-glycolic acid) (PLGA) and showed reliable release of high dose TMZ for a period of 4 weeks. To achieve this 30-day release of the highly water soluble drug, we developed an encapsulation method, where the drug powder was first coated with the polymer to form core-shell particles in which the coating shell served as a rate controlling membrane for the drug particles. Wafers were also made with a co-loading of TMZ and BCNU. All wafers were tested in vivo by treating an intracranial 9 L gliosarcoma model in F344 rats. Rats that were either untreated or treated with blank wafer died within 11 days while the median survival for rats treated with systemic TMZ was 18 days. The group that received the BCNU alone wafer had a median survival of 15 days, the group that received the TMZ wafer alone had a median survival of 19 days, and the group treated with the BCNU-TMZ wafer had a median survival of 28 days with 25% of the animals living long term (p .0038 vs. Control; p .001 vs. Blank Polymer). These findings demonstrate the potential of this newly designed wafer for treating GBM. Moreover, this concept, can pave the way for other drug combinations that may improve the clinical application of numerous agents to treat solid tumors.

Neurovascular coupling, cerebrovascular remodeling and hemodynamic changes are critical to brain function, and dysregulated in neuropathologies such as brain tumors. Interrogating these phenomena in freely behaving animals requires a portable microscope with multiple optical contrast mechanisms. Therefore, we developed a miniaturized microscope with: a fluorescence (FL) channel for imaging neural activity (e.g., GCaMP) or fluorescent cancer cells (e.g., 9L-GFP); an intrinsic optical signal (IOS) channel for imaging hemoglobin absorption (i.e., cerebral blood volume); and a laser speckle contrast (LSC) channel for imaging perfusion (i.e., cerebral blood flow). Following extensive validation, we demonstrate the microscope’s capabilities via experiments in unanesthetized murine brains that include: (i) multi-contrast imaging of neurovascular changes following auditory stimulation; (ii) wide-area tonotopic mapping; (iii) EEG-synchronized imaging during anesthesia recovery; and (iv) microvascular connectivity mapping over the life-cycle of a brain tumor. This affordable, flexible, plug-and-play microscope heralds a new era in functional imaging of freely behaving animals., Measuring multiple neurophysiologic variables usually requires bulky benchtop optical systems and working with anesthetized animals. Here the authors present a miniature portable microscope for neurovascular imaging in awake rodents, combining fluorescence, intrinsic optical signals and laser speckle contrast.

Background Captopril is a well-characterized, FDA-approved drug that has demonstrated promise as a repurposed oncology therapeutic. Captopril’s known anti-cancer effects include inhibition of Matrix Metalloproteinase-2 (MMP-2), an endopeptidase which selectively breaks down the extracellular matrix to promote cell migration. MMP-2 is a known therapeutic target in gliomas, tumors with significant clinical need. Using an aggressive gliosarcoma model, we assessed captopril’s effects on MMP-2 expression in vitro and in vivo as well as its efficacy as an adjuvant in combination therapy regimens in vivo. Methods Following captopril treatment, MMP-2 protein expression and migratory capabilities of 9 L gliosarcoma cells were assessed in vitro via western blots and scratch wound assays, respectively. Rats were intracranially implanted with 9 L gliosarcoma tumors, and survival was assessed in the following groups: control; captopril (30 mg/kg/day); temozolomide (TMZ) (50 mg/kg/day), and captopril+TMZ. In vivo experiments were accompanied by immunohistochemistry for MMP-2 from brain tissue. Results In vitro, captopril decreased MMP-2 protein expression and reduced migratory capacity in 9 L gliosarcoma cells. In a gliosarcoma animal model, captopril decreased MMP-2 protein expression and extended survival as a TMZ adjuvant relative to untreated controls, captopril monotherapy, and TMZ monotherapy groups (27.5 versus 14 (p Conclusions Captopril decreases gliosarcoma cell migration, which may be mediated by reduction in MMP-2 protein expression. Captopril provided a survival advantage as a TMZ adjuvant in a rat intracranial gliosarcoma model. Captopril may represent a promising potential adjuvant to TMZ therapy in gliosarcoma as a modulator of the MMP-2 pathway.

Background Medulloblastoma (MB) is the most common brain malignancy in children, and is still responsible for significant mortality and morbidity. The aim of this study was to assess the safety and efficacy of Disulfiram (DSF), an FDA-approved inhibitor of Aldehyde-Dehydrogenase (ALDH), and Copper (Cu++) in human SSH-driven and Group 3 MB. The molecular mechanisms, effect on cancer-stem-cells (CSC) and DNA damage were investigated in xenograft models. Methods The cytotoxic and anti-CSC effects of DSF/Cu++ were evaluated with clonogenic assays, flow-cytometry, immunofluorescence, western-blotting. ONS76, UW228 (SHH-driven with Tp53m), D425med, D283 and D341 (Group 3) cell-lines were used. In vivo survival and nuclear protein localization protein-4 (NPL4), Ki67, Cleaved-Caspase-3, GFAP and NeuN expression were assessed in two Group 3 MB xenografts with immunohistochemistry and western-blotting. Results Significant in vitro cytotoxicity was demonstrated at nanomolar concentrations. DSF/Cu++ induced cell-death through NPL4 accumulation in cell-nucleus and buildup of poly-ubiquitylated proteins. Flow-cytometry demonstrated a significant decrease in ALDH+, Nestin+ and CD133+ following treatment, anti-CSC effect was confirmed in vitro and in vivo. DSF/Cu++ prolonged survival, and increased nuclear NPL4 expression in vivo. Conclusions Our data suggest that this combination may serve as a novel treatment, as monotherapy or in combination with existing therapies, for aggressive subtypes of pediatric MB.

Malignant gliomas are the most common and aggressive form of primary brain tumors, with a median survival of 15–20 months for patients receiving maximal interventions. Advances in nanomedicine have provided tumor‐specific delivery of chemotherapeutics to potentially overcome their off‐target toxicities. Recent advances in dendrimer‐based nanomedicines have established that hydroxyl‐terminated poly(amidoamine) dendrimers can intrinsically target neuroinflammation and brain tumors from systemic administration without the need for targeting moieties. The size of nanocarriers is a critical parameter that determines their tumor‐targeting efficiency, intratumor distribution, and clearance mechanism. In this study, we explore the dendrimer size effects on brain tumor targeting capability in two clinically relevant orthotopic brain tumor models, the 9L rat and GL261 mouse models, which capture differing aspects of gliomas. We show that increasing dendrimers from Generation 4 to Generation 6 significantly enhances their tumor accumulation (~10‐fold greater at 24 hr), tumor specificity (~2–3 fold higher), and tumor retention. The superior tumor targeting effect of G6 dendrimers is associated with its reduced renal clearance rate, resulting in longer circulation time compared to G4 dendrimers. Additionally, the increase in dendrimer generation does not compromise its homogeneous tumor distribution and intrinsic targeting of tumor‐associated macrophages. These results validate the potential for these dendrimers as an effective, clinically translatable platform for effectively targeting tumor‐associated macrophages in malignant gliomas.

Spinal cord injury (SCI) affects approximately 2.5 million people worldwide. The primary phase of SCI is initiated by mechanical trauma to the spinal cord, while the secondary phase involves the ensuing tissue swelling and ischemia that worsen tissue damage and functional outcome. Optimizing blood flow to the spinal cord after SCI can mitigate injury progression and improve outcome. Accurate, sensitive, real-time monitoring is critical to assessing the spinal cord perfusion status and optimizing management, particularly in those with injuries severe enough to require surgery. However, the complex anatomy of the spinal cord vasculature and surrounding structures present significant challenges to such a monitoring strategy. In this study, Doppler ultrasound was hypothesized to be a potential solution to detect and monitor spinal cord tissue perfusion in SCI patients who required spinal decompression and/or stabilization surgeries. This approach could provide real-time visual blood flow information and pulsatility of the spinal cord as biomarkers of tissue perfusion. Importantly, Doppler ultrasound could be readily integrated into the surgical workflow, because the spinal cord was exposed during surgery, thereby allowing easy access for Doppler deployment, while keeping the dura intact. Doppler ultrasound successfully measured blood flow in single and bifurcated microfluidic channels at physiologically relevant flow rates and dimensions in both in-vitro and in-vivo porcine SCI models. Furthermore, perfusion was quantified from the obtained images. Our results provide a promising and viable solution to intraoperatively assess and monitor blood flow at the SCI site to optimize tissue perfusion and improve functional recovery in SCI patients.

Introduction: Malignant gliomas are the most common and aggressive primary brain tumor with current available therapies increasing median survival to a modest 20 months. Multiple preclinica...

Glioblastoma multiforme (GBM) is highly invasive and uniformly fatal, with median survival < 20 months after diagnosis even with the most aggressive treatment that includes surgery, radiation, and systemic chemotherapy. Cisplatin is a particularly potent chemotherapeutic agent, but its use to treat GBM is limited by severe systemic toxicity and inefficient penetration of brain tumor tissue even when it is placed directly in the brain within standard delivery systems. We describe the development of cisplatin-loaded nanoparticles that are small enough (70 nm in diameter) to move within the porous extracellular matrix between cells and that possess a dense polyethylene glycol (PEG) corona that prevents them from being trapped by adhesion as they move through the brain tumor parenchyma. As a result, these “brain penetrating nanoparticles” penetrate much deeper into brain tumor tissue compared to nanoparticles without a dense PEG corona following local administration by either manual injection or convection enhanced delivery. The nanoparticles also provide controlled release of cisplatin in effective concentrations to kill the tumor cells that they reach without causing toxicity-related deaths that were observed when cisplatin was infused into the brain without a delivery system. Median survival time of rats bearing orthotopic glioma was significantly enhanced when cisplatin was delivered in brain penetrating nano-particles (median survival not reached; 80% long-term survivors) compared to cisplatin in conventional un-PEGylated particles (median survival = 40 days), cisplatin alone (median survival = 12 days) or saline-treated controls (median survival = 28 days).

OBJECTIVEChordoma is a slow-growing, locally aggressive cancer that is minimally responsive to conventional chemotherapy and radiotherapy and has high local recurrence rates after resection. Currently, there are no rodent models of spinal chordoma. In the present study, the authors sought to develop and characterize an orthotopic model of human chordoma in an immunocompromised rat.METHODSThirty-four immunocompromised rats were randomly allocated to 4 study groups; 22 of the 34 rats were engrafted in the lumbar spine with human chordoma. The groups were as follows: UCH1 tumor–engrafted (n = 11), JHC7 tumor–engrafted (n = 11), sham surgery (n = 6), and intact control (n = 6) rats. Neurological impairment of rats due to tumor growth was evaluated using open field and locomotion gait analysis; pain response was evaluated using mechanical or thermal paw stimulation. Cone beam CT (CBCT), MRI, and nanoScan PET/CT were performed to evaluate bony changes due to tumor growth. On Day 550, rats were killed and spines were processed for H & E–based histological examination and immunohistochemistry for brachyury, S100β, and cytokeratin.RESULTSThe spine tumors displayed typical chordoma morphology, that is, physaliferous cells filled with vacuolated cytoplasm of mucoid matrix. Brachyury immunoreactivity was confirmed by immunostaining, in which samples from tumor-engrafted rats showed a strong nuclear signal. Sclerotic lesions in the vertebral body of rats in the UCH1 and JHC7 groups were observed on CBCT. Tumor growth was confirmed using contrast-enhanced MRI. In UCH1 rats, large tumors were observed growing from the vertebral body. JHC7 chordoma–engrafted rats showed smaller tumors confined to the bone periphery compared with UCH1 chordoma–engrafted rats. Locomotion analysis showed a disruption in the normal gait pattern, with an increase in the step length and duration of the gait in tumor-engrafted rats. The distance traveled and the speed of rats in the open field test was significantly reduced in the UCH1 and JHC7 tumor–engrafted rats compared with controls. Nociceptive response to a mechanical stimulus showed a significant (p < 0.001) increase in the paw withdrawal threshold (mechanical hypalgesia). In contrast, the paw withdrawal response to a thermal stimulus decreased significantly (p < 0.05) in tumor-engrafted rats.CONCLUSIONSThe authors developed an orthotopic human chordoma model in rats. Rats were followed for 550 days using imaging techniques, including MRI, CBCT, and nanoScan PET/CT, to evaluate lesion progression and bony integrity. Nociceptive evaluations and locomotion analysis were performed during follow-up. This model reproduces cardinal signs, such as locomotor and sensory deficits, similar to those observed clinically in human patients. To the authors’ knowledge, this is the first spine rodent model of human chordoma. Its use and further study will be essential for pathophysiology research and the development of new therapeutic strategies.

Neurofibromatosis type 2 (NF2), a neurogenetic condition manifest by peripheral nerve sheath tumors (PNST) throughout the neuroaxis for which there are no approved therapies. In vitro and in vivo studies presented here examine agents targeting signaling pathways, angiogenesis, and DNA repair mechanisms. In vitro dose response assays demonstrated potent activity of lapatinib and nilotinib against the mouse schwannoma SC4 (Nf2 −/−) cell line. We then examined the efficacy of everolimus, nilotinib, lapatinib, bevacizumab and radiation (RT) as mono- and combination therapies in flank and sciatic nerve in vivo NF2-PNST models. Data were analyzed using generalized linear models, two sample T-tests and paired T-tests, and linear regression models. SC4(Nf2 −/−) cells implanted in the flank or sciatic nerve showed similar rates of growth (p = 0.9748). Lapatinib, nilotinib and RT significantly reduced tumor growth rate versus controls in the in vivo flank model (p = 0.0025, 0.0062, and 0.009, respectively) whereas bevacizumab and everolimus did not. The best performers were tested in the in vivo sciatic nerve model of NF2 associated PNST, where chemoradiation outperformed nilotinib or lapatinib as single agents (nilotinib vs. nilotinib + RT, p = 0.0001; lapatinib versus lapatinib + RT, p

// Avadhut D. Joshi 1,* , Rachel C. Botham 2,* , Lisa J. Schlein 3 , Howard S. Roth 2 , Antonella Mangraviti 1 , Alexandra Borodovsky 1 , Betty Tyler 1 , Steve Joslyn 4 , Jayme S. Looper 5 , Michael Podell 6 , Timothy M. Fan 7 , Paul J. Hergenrother 2 and Gregory J. Riggins 1 1 Department of Neurosurgery, School of Medicine, Johns Hopkins University, Baltimore, MD, USA 2 Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA 3 Department of Pathobiology, University of Illinois Urbana-Champaign, Urbana, IL, USA 4 VetCT Australia, Fremantle WA, Australia 5 Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA, USA 6 Department of Neurology, MedVet Chicago, Chicago, IL, USA 7 Department of Veterinary Clinical Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA * These authors have contributed equally to this work Correspondence to: Gregory J. Riggins, email: // Paul J. Hergenrother, email: // Timothy M. Fan, email: // Keywords : PAC-1, procaspase-3 activator, glioblastoma, small molecule therapy Received : March 29, 2017 Accepted : June 09, 2017 Published : July 07, 2017 Abstract Purpose: Glioblastoma is a deadly brain cancer with a median survival time of ~15 months. Ionizing radiation plus the DNA alkylator temozolomide (TMZ) is the current standard therapy. PAC-1, a procaspase-3 activating small molecule, is blood-brain barrier penetrant and has previously demonstrated ability to synergize with diverse pro-apoptotic chemotherapeutics. We studied if PAC-1 could enhance the activity of TMZ, and whether addition of PAC-1 to standard treatment would be feasible in spontaneous canine malignant gliomas. Experimental Design: Using cell lines and online gene expression data, we identified procaspase-3 as a potential molecular target for most glioblastomas. We investigated PAC-1 as a single agent and in combination with TMZ against glioma cells in culture and in orthotopic rodent models of glioma. Three dogs with spontaneous gliomas were treated with an analogous human glioblastoma treatment protocol, with concurrent PAC-1. Results: Procaspase-3 is expressed in gliomas, with higher gene expression correlating with increased tumor grade and decreased prognosis. PAC-1 is cytotoxic to glioma cells in culture and active in orthotopic rodent glioma models. PAC-1 added to TMZ treatments in cell culture increases apoptotic death, and the combination significantly increases survival in orthotopic glioma models. Addition of PAC-1 to TMZ and radiation was well-tolerated in 3 out of 3 pet dogs with spontaneous glioma, and partial to complete tumor reductions were observed. Conclusions: Procaspase-3 is a clinically relevant target for treatment of glioblastoma. Synergistic activity of PAC-1/TMZ in rodent models and the demonstration of feasibility of the combined regime in canine patients suggest potential for PAC-1 in the treatment of glioblastoma.

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma with a proclivity for systemic dissemination, leading many patients to present with advanced stage disease and fail available treatments. There is a notable lack of targeted therapies for NPC, despite working knowledge of multiple proteins with integral roles in NPC cancer biology. These proteins include EZH2, Snail, eIF4E, and IMPDH, which are all overexpressed in NPC and correlated with poor prognosis. These proteins are known to be modulated by ribavirin, an FDA-approved hepatitis C antiviral that has recently been repurposed as a promising therapeutic in several solid and hematologic malignancies. Here, we investigated the potential of ribavirin as a targeted anticancer agent in five human NPC cell lines. Using cellular growth assays, flow cytometry, BrdU cell proliferation assays, scratch wound assays, and invasion assays, we show in vitro that ribavirin decreases NPC cellular proliferation, migration, and invasion and promotes cell-cycle arrest and cell death. Modulation of EZH2, Snail, eIF4E, IMPDH, mTOR, and cyclin D1 were observed in Western blots and enzymatic activity assays in response to ribavirin treatment. As monotherapy, ribavirin reduced flank tumor growth in multiple NPC xenograft models in vivo. Most importantly, we demonstrate that ribavirin enhanced the effects of radiotherapy, a central component of NPC treatment, both in vitro and in vivo. Our work suggests that NPC responds to ribavirin-mediated EZH2, Snail, eIF4E, IMPDH, and mTOR changes and positions ribavirin for clinical evaluation as a potential addition to our NPC treatment armamentarium.

Primary or metastatic brain tumors are challenging to treat due to their aggressive and heterogenic characteristics and the limited availability of drugs and tools capable of permeating the blood–brain barrier (BBB). A vast array of methods has been explored to bypass these physical limitations, but over the past few decades only a small portion of these techniques and compounds have been translated into clinical practice. In this chapter, we focus our attention on different ways to efficiently deliver drugs across the BBB and enumerate some of the roadblocks to efficacious treatment. First, we briefly review the anatomic and functional barriers that prevent the diffusion of therapeutics in the central nervous system, which will clarify the unique challenges of brain tumor therapy. We then describe preclinical models and the clinical experience of polymeric drug delivery and practical applications for both primary and metastatic brain tumors.

Despite recent technological advancements and promising preclinical experiments, brain tumor patients are still met with limited treatment options. Some of the barriers to clinical improvements include the systemic toxicity of cytotoxic compounds, the impedance of the blood brain barrier (BBB), and the lack of therapeutic agents that can selectively target the intracranial tumor environment. To overcome such barriers, a number of chemotherapeutic agents and nucleic acid-based therapies are rapidly being synthesized and tested as new brain tumor-targeted delivery strategies. Novel carriers include liposomal and polymeric nanoparticles, wafers, microchips, microparticle-based nanoplatforms and cells-based vectors. Strong preclinical results suggest that these nanotechnologies are set to transform the therapeutic paradigm for brain tumor treatment. In addition to new tumoricidal agents, parallel work is also being conducted on the BBB front. Preclinical testing of chemical and physical modulation strategies is yielding improved intracranial concentrations. New diagnostic and therapeutic imaging techniques, such as high-intensity focused ultrasound and MRI-guided focused ultrasound, are being used to modulate the BBB in a more precise and non-invasive manner. This review details some of the tremendous advances that are being explored in current brain tumor targeted therapies, including local implant development, nanobiotechnology-based delivery strategies, and techniques of BBB manipulation.

BACKGROUND Medulloblastoma (MB), the most common brain malignancy among children, is classified into four molecular subtypes, WNT-driven, Shh-driven, Group 3 and 4. The aim of this study was to assess the effects of the combination of Disulfiram, an inhibitor of Aldehyde Dehydrogenase, and Copper in Shh-driven and Group 3 MB, in vitro and in vivo. METHODS The mechanisms of action and anti-cancer stem-cell effects of disulfiram were evaluated with clonogenic assays, flow-cytometry (FC), western blotting (WB), and immunofluorescence (IF) using established MB cell-lines - ONS76, UW228, D425med, D341 and D283- representing the main molecular subtypes. Survival, tumor volume, nuclear protein localization protein-4-expression (NPL4) and markers of proliferation/apoptosis were assessed in multiple models of Group 3 MB in vivo and ex vivo with WB and immunohistochemistry (IHC). RESULTS Significant in vitro cytotoxicity was demonstrated at nanomolar concentrations of DSF in all lines. DSF/Cu++ induced cell death (increased AnnV/PI, cleaved-Poly(ADP-ribose)polymerase fraction, and Apoptosis Inducing Factor on WB/FC/IF, in vitro and ex vivo) through NPL4 accumulation in cell nucleus and intracellular buildup of poly-ubiquitylated proteins. DNA damage was also detected with WB and H2AX foci on immunofluorescence. Flow-cytometry analysis demonstrated a significant reduction in ALDH, Nestin- and CD133-positive cells in Group 3 lines, confirmed with WB in vitro and ex vivo. DSF/Cu++-toxicity was tested in vivo in multiple dose-escalating trials, and the combination significantly prolonged survival and reduced tumor volume in D425 and D341 xenografts. IHC showed lower Ki67 and increased Cleaved-Caspase-3 expression, higher NPL4-positive cells and no difference in NeuN and GFAP-positive cells after treatment. CONCLUSIONS DSF/Cu++ demonstrated a potent therapeutic effect on Shh-driven and Group 3 MB cell lines by determining apoptosis, targeting cancer stem cells and inducing DNA damage. Our data suggest that this combination may serve as a novel treatment, alone or with existing therapies, for pediatric MB.

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma with a propensity for metastasis, leading many patients to fail available treatments and/or present with advanced-stage disease. There is currently a scarcity of targeted therapies for NPC, despite working knowledge of several proteins with key roles in NPC cancer biology. These include EZH2, Snail, eIF4E, and IMPDH, which are all overexpressed in NPC and correlated with poor patient prognosis. These proteins are known to be targeted by ribavirin, a well-characterized anti-viral drug that has recently been repurposed as an anti-cancer agent in several solid and hematologic malignancies. In the present study, we investigated the potential of ribavirin as a targeted therapy and radiosensitizing agent in five human NPC cell lines. We show in vitro, using cellular growth assays, flow cytometry, BrdU cell proliferation assays, scratch wound assays, and invasion assays, that ribavirin decreases NPC cellular proliferation, migration, and invasion and promotes cell cycle arrest and cell death. Modulation of EZH2, Snail, eIF4E, IMPDH, and mTOR were observed in western blots and enzymatic activity assays in response to ribavirin treatment. In vivo, monotherapy with ribavirin reduced flank tumor growth in multiple NPC xenograft models. Given that radiation therapy is a mainstay of NPC treatment, we next investigated the effects of combining ribavirin with radiation. Using clonogenic assays and flow cytometry, we demonstrate that ribavirin enhanced the cytotoxic effects of radiation on NPC cells in vitro. Most importantly, using a flank tumor xenograft model, we show that pre-treatment with ribavirin potentiated the effects of radiation therapy in vivo. Our work suggests that NPC responds to ribavirin-mediated EZH2, Snail, eIF4E, IMPDH, and mTOR modulation and positions ribavirin for clinical evaluation as a targeted therapy and radiosensitizing agent in this cancer. Citation Format: Sakibul Huq, Joshua Casaos, Riccardo Serra, Michael Peters, Yuanxuan Xia, Andy Ding, Jeffrey Ehresman, Jayanidhi Kedda, Manuel Morales, Noah Gorelick, Tianna Zhao, Wataru Ishida, Alexander Perdomo-Pantoja, Arba Cecia, Chenchen Ji, Ian Suk, David Sidransky, Mariana Brait, Henry Brem, Nicolas Skuli, Betty Tyler. Use of the anti-viral drug ribavirin as a radiosensitizing agent in nasopharyngeal carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6268.

Purpose: Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that is often diagnosed at an advanced stage, leading to poor disease-free and overall survival. Accumulating literature suggests that elevated protein expression of enhancer of zeste homolog 2 (EZH2), eukaryotic initiation factor 4E (eIF4E), and inosine-5’-monophosphate dehydrogenase (IMPDH)—proteins implicated in myriad cancers—correlates with poor prognosis in NPC. These three proteins are modulated by the Food and Drug Administration-approved antiviral drug ribavirin, which has recently been repositioned by our laboratory and others as a promising anticancer agent. Based on this intersection of molecular signature and drug targets, we investigated the potential of ribavirin as a therapeutic agent for NPC. Experimental Design: We assessed antineoplastic efficacy of ribavirin on six human NPC cell lines in vitro using cellular growth assays, flow cytometry, and scratch wound assays. Mechanistic pathways involved were investigated using genomic expression datasets, Western blots, and enzymatic activity assays. The effects of combining ribavirin with radiation were assessed using clonogenic assays and flow cytometry. Finally, we evaluated the effects of ribavirin on tumor growth in vivo using two human cell line-derived xenograft models. Results: Ribavirin significantly decreased NPC cellular proliferation and migratory capacity in addition to promoting cell cycle arrest and cell death. Modulation of the EZH2, Snail, eIF4E, and IMPDH pathways was observed in response to ribavirin treatment. Ribavirin significantly enhanced the cytotoxic effects of radiation therapy in NPC. Most importantly, ribavirin significantly reduced flank tumor growth in two NPC xenograft models. Conclusions: Our work suggests that ribavirin has potent anticancer effects in NPC and could represent a safe and promising addition to current NPC treatment regimens. Citation Format: Sakibul Huq, Joshua Casaos, Michael Peters, Yuanxuan Xia, Andy Ding, Manuel Morales, Noah Gorelick, Riccardo Serra, Tianna Zhao, Wataru Ishida, Alexander Perdomo-Pantoja, Arba Cecia, Chenchen Ji, Ian Suk, David Sidransky, Mariana Brait, Henry Brem, Nicolas Skuli, Betty Tyler. Repositioning the FDA-approved antiviral drug ribavirin as targeted therapy for nasopharyngeal carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B06.

Gliomas are the most common solid tumors and the greatest cause of cancer-related deaths among children in the U.S. Treatment for this group of heterogeneous malignancies involves surgery and chemotherapy; however, tumor recurrence is inevitable. Immunotherapy is a treatment modality that can stimulate the intrinsic immune defenses of the body to eliminate tumor cells. This has been challenging for gliomas, though, due to the exclusionary anatomic barriers and the immunologically quiescent environment of the brain. This project aims to develop an injectable hydrogel patch for controlled local delivery of combination immunotherapy directly to the postsurgical formed cavity of the brain to treat residual disease and to prevent tumor recurrence. Using the hydrogel patch, we seek to overcome the delivery and immunosuppressive barriers of the brain by locally releasing, as a programmed regimen, the following immunomodulatory entities: i) C-X-C motif chemokine 10 (CXCL10; to promote T-lymphocyte recruitment), ii), a new generation of programmed death-ligand 1 (PD-L1) inhibitors (to prevent T-cell exhaustion), iii) FMS-like tyrosine kinase 3 ligand (FLT3L; to induce differentiation and expansion of dendritic cells), and iv) a stimulator of interferon genes (STING) agonist (to trigger cross-priming of CD8+ cytotoxic T cells). Our novel technology possesses the flexibility to personalize an array of antigens/adjuvants or other components of immunotherapy and enables the prevention of brain metastasis by creating an immunologically inhospitable setting for circulating tumor cells. As pediatric gliomas are notoriously resistant to treatment, our hydrogel formulation seeks to address an unmet clinical need for more effective therapeutic modalities. Note:This abstract was not presented at the conference. Citation Format: Shiran Ferber, Alexander M. Cryer, Noah Gorelick, Betty Tyler, Henry Brem, Robert S. Langer, Natalie Artzi. Training an immuno-army: Exploiting immunoengineering for the treatment of glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A27.

Together, medulloblastoma (MB) and atypical teratoid/rhabdoid tumors (AT/RT) represent two of the most prevalent pediatric brain malignancies. Current treatment involves radiation, which has high risks of developmental sequelae for patients under the age of three. New safer and more effective treatment modalities are needed. Cancer gene therapy is a promising alternative, but there are challenges with using viruses in pediatric patients. We developed a library of poly(beta-amino ester) (PBAE) nanoparticles and evaluated their efficacy for plasmid delivery of a suicide gene therapy to pediatric brain cancer models-specifically herpes simplex virus type I thymidine kinase (HSVtk), which results in controlled apoptosis of transfected cells. In vivo, PBAE-HSVtk treated groups had a greater median overall survival in mice implanted with AT/RT (P = 0.0083 vs. control) and MB (P 0.0001 vs. control). Our data provide proof of principle for using biodegradable PBAE nanoparticles as a safe and effective nanomedicine for treating pediatric CNS malignancies.

The growing cost of medical care worldwide, particularly in oncology, has incentivized researchers and physicians to repurpose clinically used drugs to alleviate the financial burden of drug development and offer potential new therapeutics. Recent works have demonstrated anticancer properties of the FDA-approved drug ribavirin, a synthetic guanosine analogue and antiviral molecule used over the past four decades for the treatment of hepatitis C. The efficacy of ribavirin in cancer has been explored through several preclinical models and ongoing clinical trials in multiple cancers, including acute myeloid leukemia, oropharyngeal squamous cell carcinoma, and metastatic breast cancer. In this review, we summarize the role of ribavirin as an antiviral medication and focus our attention on its recent use as an antitumoral agent. We highlight current knowledge of the potential use and mechanisms of action of ribavirin in cancer. Because current therapeutics for patients with cancer still fail to cure, introducing new forms of treatment is essential. Converging evidence suggests that ribavirin represents a promising addition to a generation of newly repurposed safe and effective anticancer agents.

The use of inhibitory checkpoint blockade in the management of glioblastoma has been studied in both preclinical and clinical settings. TIGIT is a novel checkpoint inhibitor recently discovered to play a role in cancer immunity. In this study, we sought to determine the effect of anti-PD-1 and anti-TIGIT combination therapy on survival in a murine glioblastoma (GBM) model, and to elucidate the underlying immune mechanisms. Using mice with intracranial GL261-luc+ tumors, we found that TIGIT expression was upregulated on CD8+ and regulatory T cells (Tregs) in the brain compared to draining cervical lymph nodes (CLN) and spleen. We then demonstrated that treatment using anti-PD-1 and anti-TIGIT dual therapy significantly improved survival compared to control and monotherapy groups. The therapeutic effect was correlated with both increased effector T cell function and downregulation of suppressive Tregs and tumor-infiltrating dendritic cells (TIDCs). Clinically, TIGIT expression on tumor-infiltrating lymphocytes was shown to be elevated in patient GBM samples, suggesting that the TIGIT pathway may be a valuable therapeutic target. Expression of the TIGIT ligand, PVR, further portended a poor survival outcome in patients with low-grade glioma. We conclude that anti-TIGIT is an effective treatment strategy against murine GBM when used in combination with anti-PD-1, improving overall survival via modifications of both the T cell and myeloid compartments. Given evidence of PVR expression on human GBM cells, TIGIT presents as a promising immune therapeutic target in the management of these patients.

Studies have demonstrated that buprenorphine, a front line drug for veterinary analgesia, may alleviate symptoms of chronic pain. A cage side observation protocol was used to record behavioral signs in a mouse clinical trial of extended release buprenorphine. A retrospective review of the observations for signs of pain and stress revealed that mice given a fivefold overdose of buprenorphine (16.25 mg/kg) showed lethargy and facial signs associated with stress. However, similar signs were observed in the drug-free control mice as early as Day 3 of single-cage housing. This appears to be the first report of cage effects in a clinical trial for a veterinary drug.

The blood–brain barrier (BBB) significantly reduces the delivery of many systemically administered agents to the central nervous system. Although temozolomide is the only chemotherapy to improve survival in patients with glioblastoma, its concentration in brain is only 20 % of that in blood. Regadenoson, an FDA approved adenosine receptor agonist used for cardiac stress testing, transiently disrupts rodent BBB allowing high molecular weight dextran (70 kD) to enter the brain. This study was conducted to determine if regadenoson could facilitate entry of temozolomide into normal rodent brain. Temozolomide (50 mg/kg) was administered by oral gavage to non-tumor bearing F344 rats. Two-thirds of the animals received a single dose of intravenous regadenoson 60–90 min later. All animals were sacrificed 120 or 360 min after temozolomide administration. Brain and plasma temozolomide concentrations were determined using HPLC/MS/MS. Brain temozolomide concentrations were significantly higher at 120 min when it was given with regadenoson versus alone (8.1 ± 2.7 and 5.1 ± 3.5 µg/g, P

Purpose: Radiotherapy remains a major treatment method for malignant tumors. Magnetic resonance imaging (MRI) is the standard modality for assessing glioma treatment response in the clinic. Compared to MRI, ultrasound imaging is low-cost and portable and can be used during intraoperative procedures. The purpose of this study was to quantitatively compare contrast-enhanced ultrasound (CEUS) imaging and MRI of irradiated gliomas in rats and to determine which quantitative ultrasound imaging parameters can be used for the assessment of early response to radiation in glioma. Methods: Thirteen nude rats with U87 glioma were used. A small thinned skull window preparation was performed to facilitate ultrasound imaging and mimic intraoperative procedures. Both CEUS and MRI with structural, functional, and molecular imaging parameters were performed at preradiation and at 1 day and 4 days postradiation. Statistical analysis was performed to determine the correlations between MRI and CEUS parameters and the changes between pre- and postradiation imaging. Results: Area under the curve (AUC) in CEUS showed significant difference between preradiation and 4 days postradiation, along with four MRI parameters, T 2, apparent diffusion coefficient, cerebral blood flow, and amide proton transfer-weighted (APTw) (all p < 0.05). The APTw signal was correlated with three CEUS parameters, rise time (r = − 0.527, p < 0.05), time to peak (r = − 0.501, p < 0.05), and perfusion index (r = 458, p < 0.05). Cerebral blood flow was correlated with rise time (r = − 0.589, p < 0.01) and time to peak (r = − 0.543, p < 0.05). Conclusions: MRI can be used for the assessment of radiotherapy treatment response and CEUS with AUC as a new technique and can also be one of the assessment methods for early response to radiation in glioma.

Biodegradable polymeric nanoparticles have the potential to be safer alternatives to viruses for gene delivery; however, their use has been limited by poor efficacy in vivo. In this work, we synthesize and characterize polymeric gene delivery nanoparticles and evaluate their efficacy for DNA delivery of herpes simplex virus type I thymidine kinase (HSVtk) combined with the prodrug ganciclovir (GCV) in a malignant glioma model. We investigated polymer structure for gene delivery in two rat glioma cell lines, 9L and F98, to discover nanoparticle formulations more effective than the leading commercial reagent Lipofectamine 2000. The lead polymer structure, poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-modified with 1-(3-aminopropyl)-4-methylpiperazine, is a poly(β-amino ester) (PBAE) and formed nanoparticles with HSVtk DNA that were 138 ± 4 nm in size and 13 ± 1 mV in zeta potential. These nanoparticles containing HSVtk DNA showed 100% cancer cell killing in vitro in the two glioma cell lines when combined with GCV exposure, while control nanoparticles encoding GFP maintained robust cell viability. For in vivo evaluation, tumor-bearing rats were treated with PBAE/HSVtk infusion via convection-enhanced delivery (CED) in combination with systemic administration of GCV. These treated animals showed a significant benefit in survival (p = 0.0012 vs control). Moreover, following a single CED infusion, labeled PBAE nanoparticles spread completely throughout the tumor. This study highlights a nanomedicine approach that is highly promising for the treatment of malignant glioma.

Background: We compared cutting and coagulation of a novel ferromagnetic tool (FMwand) with modalities currently used in the clinical setting. Methods: 24 F344 rats with 9L gliosarcoma flank tumours were randomized into 2 groups (n = 12): 1) Five parallel incisions were made into the tumor of each rat using monopolar electrosurgery (MES) cut mode, MES coagulation (coag) mode, FMwand, carbon dioxide (CO2) laser and cold scalpel. 2) Two parallel incisions were made comparing the MES and the FMwand, both with resecting loop tips. The study was then repeated by a second surgeon. The surgeons applied a grading scale (1 = worst, 5 = best) based on their observations. Results: Average scores for FMwand were superior in ease of tissue dissection (3.58), distortion upon tissues (3.67), and smoke production (2.87). CO2 laser led in effectiveness of hemostasis (4.32). MES cut mode had the highest scores for ease of cleaning of tip (3.17) and speed of dissection (3.92). The FMwand loop device led in all attributes except for ease of cleaning. Conclusions: The FMwand outperformed CO2 laser significantly in ease and speed. It was superior compared to MES cut mode for hemostasis and superior compared to coag mode in ease and speed, distortion upon tissues and smoke production. The FMwand loop was significantly better compared to MES loop for hemostasis, distortion, ease and speed. The FMwand was shown to be safe and effective for hemostatic soft tissue cutting and coagulation.

We assessed the antiangiogenic effects of subconjunctival injection of Fc-endostatin (FcE) using a human vascular endothelial growth factor-induced rabbit corneal neovascularization model. Angiogenesis was induced in rabbit corneas through intrastromal implantations of VEGF polymer implanted 2 mm from the limbus. NZW rabbits were separated into groups receiving twice weekly subconjunctival injections of either saline; 25 mg/mL bevacizumab; 2 mg/mL FcE; or 20 mg/mL FcE. Corneas were digitally imaged at 5 time points. An angiogenesis index (AI) was calculated (vessel length (mm) × vessel number score) for each observation. All treatment groups showed a significant decrease in the vessel length and AI compared to saline on all observation days (P<0.001). By day 15, FcE 2 inhibited angiogenesis significantly better than FcE 20 (P<0.01). There was no significant difference between FcE 2 and BV, although the values trended towards significantly increased inhibition by BV. BV was a significantly better inhibitor than FcE 20 by day 8 (P<0.01). FcE was safe and significantly inhibited new vessel growth in a rabbit corneal neovascularization model. Lower concentration FcE 2 exhibited better inhibition than FcE 20, consistent with previous FcE studies referencing a biphasic dose-response curve. Additional studies are necessary to further elucidate the efficacy and clinical potential of this novel angiogenesis inhibitor.

INTRODUCTION: The blood brain barrier is a critical limiting step to achieving therapeutic CNS drug concentrations. We have developed a poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG)-based Platform Technology to interstitially deliver multiple chemotherapy agents from a single thermo-setting paste, potentially filling the 3-week therapy gap that currently exists between surgery and commencement of radiotherapy in the treatment of high grade gliomas. Here we evaluate mouldable PLGA/PEG paste for combined temozolomide (TMZ) and etoposide (ETOP) delivery in an orthotopic high grade glioma model. METHODS: Drug release and preserved stability of the active TMZ molecule (AIC) was evaluated by fluoroscopic and LC-mass spectrometer based methods. In vitro cytotoxicity of released TMZ/ETOP was evaluated against high grade glioma cell lines and patient-derived primary cultures using metabolic assays. In vivo efficacy and overall survival were evaluated in the syngenic orthotopic 9L glioma rat model of intra cavity chemotherapy. RESULTS: TMZ and ETOP were released from PLGA/PEG alone or in combination over 2 weeks in vitro. Cytotoxicity of released drugs in vitro is comparable to directly applied agents, demonstrating the retained molecular integrity of TMZ/ETOP upon loading and releasing from PLGA/PEG. Both high (20% w/w TMZ / 50% w/w ETOP) and low (10% w/w TMZ / 25% w/w ETOP) doses were well tolerated in vivo, with no observable weight loss nor neurological deficits. Significant survival benefits from PLGA/PEG/TMZ/ETOP therapy were observed compared to surgery alone (49 vs. 14 days; p < 0.001), surgery with blank paste (49 vs. 14 days; p < 0.001) or surgery with daily oral TMZ (49 vs. 33 days; p < 0.004). CONCLUSIONS: This study demonstrates that interstitial delivery of TMZ/ETOP achieves significant glioma survival benefits. As such, PLGA/PEG paste applied to the post-surgical resection cavity offers a realistic opportunity for localised control of residual disease in pediatric and adult high grade gliomas.

Tumor progression, limited efficacy of current standard treatments, and the rise in patient mortality are associated with gene expression caused by the synergistic action of intratumoral hypoxia and HIF-1α activation. For this reason, recent investigations have focused on HIF-targeting therapeutic agents, with encouraging preclinical and clinical results in solid tumors. Here we describe the efficacy of a HIF-1α inhibitor, Acriflavine, and demonstrate its potency against brain cancer. This safe antibacterial dye induces cell death and apoptosis in several glioma cell lines, targets HIF-1α–mediated pathways, and decreases the level of PGK1, VEGF and HIF-1α in vitro and in vivo. Administered locally via biodegradable polymers, Acriflavine provides significant benefits in survival resulting in nearly 100% long term survival, confirmed by MRI and histological analyses. This study reports preclinical evidence that this safe, small molecule can contribute to brain tumor therapy and highlights the significance of HIF-1α-targeting molecules.

Dramatic improvements in cancer survival rates have been achieved over the past 40 years, with a one-year median survival for all cancers in 1971 climbing to a predicted 6-year median survival today.1 Colon cancer has proven to have the greatest improvement, from a median survival of 7 months in 1971 to a current estimate of 10 years. Encouraging improvements in survival for primary brain malignancies have also been noted, but not to such a profound extent. Despite significant advances in neuroimaging, surgical technique, radiation therapy, and conventional chemotherapy, median survival for WHO grade IV astrocytoma (glioblastoma multiforme [GBM]) remains 30 ATPs produced through oxidative phosphorylation.7 Theories have been proposed regarding the evolutionary advantage to malignant cells of the additional role of glycolytic enzymes as regulators of transcription and apoptosis.8 The breakdown of glucose also provides many of the cellular building blocks required for production of the proteins, nucleic acids, and lipids required for replication.7 In addition, aerobic glycolysis facilitates extracellular acidosis, selecting for cancer cells with more resistant phenotypes that can withstand anaerobic conditions once the oxygen supply has been outgrown.9 Some glycolytic enzymes have also been noted to play a role in regulating apoptosis. With the upregulation of glycolysis, therefore, malignant cells would be less likely to undergo cell death. Hexokinase (HK) is the first regulatory enzyme within glycolysis that catalyzes the conversion of glucose to glucose-6-phosphate, capturing glucose molecules within the cell. HK has 4 isoforms: I, II, III, and IV. HK II is found to be upregulated in many tumor cell lines10 and primarily promotes anabolic functions such as glycogen and lipid synthesis.11 Wolf et al recently found HK II to be a key mediator of aerobic glycolysis in GBMs, as opposed to predominant HK I expression in normal brain tissue and low-grade gliomas.12 3-Brompyruvate (3-BrPA) is a potent ATP inhibitor and direct inhibitor of HK II.13,14 3-BrPA has been shown to be well tolerated in vivo at doses required to significantly inhibit tumor ATP production, decrease lactic acid production, and promote apoptosis, with little noted toxicity to normal tissue at therapeutic doses.15–17 While shown to be effective in glioma cell lines,16 a limitation of the applicability of 3-BrPA to GBM therapy is its inability to cross the blood-brain barrier.18 A second molecule for targeting aerobic glycolysis is dichloroacetate (DCA), which inhibits the mitochondrial enzyme pyruvate dehydrogenase kinase (PDK).19 PDK is a direct inhibitor of pyruvate dehydrogenase (PDH), which regulates the passage of pyruvate into mitochondria. Through the inhibition of an inhibitor, therefore, DCA promotes mitochondrial oxidation and decreases lactic acid formation.19 In essence, DCA promotes the aerobic metabolism of glucose through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, in place of the conversion of pyruvate into lactate. Due to the widespread distribution of oral DCA in the central nervous system,20,21 its efficacy at inhibiting growth in several GBM cell lines has been investigated.6 DCA led to rapid reversal of mitochondrial hyperpolarization in GBM cells (a marker of an apoptosis-resistant state), promoted p53 activation, and suppressed angiogenesis. In a trial of 3 participants with recurrent GBMs and 2 with primary GBMs, DCA was administered, in addition to standard chemotherapeutic and radiation protocols, and found to possibly be associated with prolonged radiological regression and stabilization. The dose of DCA, however, was limited by reversible peripheral neuropathy. Peripheral nerve toxicity had previously been noted during in vivo and human studies involving disorders of lactic acidosis.22,23 Due to the reported inability of 3-BrPA to cross the blood-brain barrier and the dose-limiting toxicity of DCA, we set out to establish whether these molecules could be safely and effectively delivered locally at the tumor site. To maximize clinical relevancy, we selected a proven, FDA-approved method of local drug release using the biodegradable polyanhydride, poly-(1,3 bis[p-carboxyphenoxy] propane-co-sebacic acid) (p[CPP:SA, 20:80]).24 P(CPP:SA, 20:80) has been shown to be biocompatible in the brain with no evidence of systemic or local toxicity. In multiple glioma studies no difference in survival has been noted between groups receiving empty p(CPP:SA) wafers and untreated animals.25,26 We present the tolerability and efficacy of locally delivered 3-BrPA and DCA via p(CPP:SA, 20:80) polymer wafer. We then investigate the combination of 3-BrPA and DCA with temozolomide and radiation therapy.

Object The aim of this study was to demonstrate that paclitaxel could function as a radiosensitizer for malignant glioma in vitro and in vivo. Methods The radiosensitizing effect of paclitaxel was tested in vitro using the human U373MG and rat 9L glioma cell lines. Cell cycle arrest in response to paclitaxel exposure was quantified by flow cytometry. Cells were subsequently irradiated, and toxicity was measured using the clonogenic assay. In vivo studies were performed in Fischer 344 rats implanted with intracranial 9L gliosarcoma. Rats were treated with control polymer implants, paclitaxel controlled-release polymers, radiotherapy, or a combination of the 2 treatments. The study end point was survival. Results Flow cytometry demonstrated G2-M arrest in both U373MG and 9L cells following 6–12 hours of paclitaxel exposure. The order in which the combination treatment was administered was significant. Exposure to radiation treatment (XRT) during the 6–12 hours after paclitaxel treatment resulted in a synergistic reduction in colony formation. This effect was greater than the effect from either treatment alone and was also greater than the effect of radiation exposure followed by paclitaxel. Rats bearing 9L gliosarcoma tumors treated with paclitaxel polymer administration followed by single-fraction radiotherapy demonstrated a synergistic improvement in survival compared with any other treatment, including radiotherapy followed by paclitaxel treatment. Median survival for control animals was 13 days; for those treated with paclitaxel alone, 21 days; for those treated with XRT alone, 21 days; for those treated with XRT followed by paclitaxel, 45 days; and for those treated with paclitaxel followed by XRT, more than 150 days (p < 0.0001). Conclusions These results indicate that paclitaxel is an effective radiosensitizer for malignant gliomas because it renders glioma cells more sensitive to ionizing radiation by causing G2-M arrest, and induces a synergistic response to chemoradiotherapy.

Object The poor outcome of malignant gliomas is largely due to local invasiveness. Previous studies suggest that gliomas secrete excess glutamate and destroy surrounding normal peritumoral brain by means of excitotoxic mechanisms. In this study the authors assessed the effect on survival of 2 glutamate modulators (riluzole and memantine) in rodent glioma models. Methods In an in vitro growth inhibition assay, F98 and 9L cells were exposed to riluzole and memantine. Mouse cerebellar organotypic cultures were implanted with F98 glioma cells and treated with radiation, radiation + riluzole, or vehicle and assessed for tumor growth. Safety and tolerability of intracranially implanted riluzole and memantine CPP:SA polymers were tested in F344 rats. The efficacy of these drugs was tested against the 9L model and riluzole was further tested with and without radiation therapy (RT). Results In vitro assays showed effective growth inhibition of both drugs on F98 and 9L cell lines. F98 organotypic cultures showed reduced growth of tumors treated with radiation and riluzole in comparison with untreated cultures or cultures treated with radiation or riluzole alone. Three separate efficacy experiments all showed that localized delivery of riluzole or memantine is efficacious against the 9L gliosarcoma tumor in vivo. Systemic riluzole monotherapy was ineffective; however, riluzole given with RT resulted in improved survival. Conclusions Riluzole and memantine can be safely and effectively delivered intracranially via polymer in rat glioma models. Both drugs demonstrate efficacy against the 9L gliosarcoma and F98 glioma in vitro and in vivo. Although systemic riluzole proved ineffective in increasing survival, riluzole acted synergistically with radiation and increased survival compared with RT or riluzole alone.

There has been considerable interest in repurposing the poly ADP ribose polymerase inhibitor and purported radiosensitiser olaparib (Lynparza), with a recent dose escalation study of olaparib plus temozolomide in recurrent GBM showing good tolerance (Fulton et al 2018). Due to systemic therapy-associated caveats such as dose-limiting toxicities and blood-brain-barrier penetration, here we assess localised post-surgical delivery of olaparib from our previously developed poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) (PLGA/PEG) thermo-sensitive biodegradable paste. Metabolic and clonogenic assays revealed impaired proliferation and clonal growth respectively, upon acute exposure of high-grade glioma cells to olaparib (3–5µM), an effect dramatically potentiated with 3Gy radiation. Flow cytometry of Annexin V+/Propidium iodide+ rodent and human high-grade glioma cells, revealed a significant cell proportion increase at late stage apoptosis when exposed to 2–3µM olaparib and 3Gy radiation (relative to untreated, olaparib alone or radiation alone). A high-grade glioma orthotopic allograft study is ongoing where we already observe a significant overall survival benefit of locally-delivered 10% and 20% w/w (drug:polymer ratio) olaparib via PLGA/PEG paste post-surgery with adjuvant radiotherapy, compared to surgery/oral temozolomide/radiotherapy (GBM standard-of-care) and surgery/systemic olaparib (120 days vs. 44 vs 30 respectively). A more pronounced survival benefit, as determined by the total number of long-term surviving animals, was observed with combined PLGA/PEG/olaparib/temozolomide/radiotherapy or PLGA/PEG/olaparib/etoposide/radiotherapy. RNAseq data from 10 GBM patients show significantly elevated levels of apoptosis-inducing factor-1 in 5-aminolevulinic acid (5ALA)+ fluorescence–activated cell sorted populations (i.e. purified tumour cells from the invasive margin), relative to 5ALA- cells, confirming PARP-1 activity in infiltrative tumour cells. Collectively our data supports a clinical rationale for localised olaparib delivery with adjuvant radiotherapy.

Medulloblastoma (MB), an aggressive pediatric brain malignancy, is commonly classified into four subtypes. Although survival has improved remarkably, Group 3 and Shh-driven tumors still experience high rates of recurrent and metastatic disease. Disulfiram (DSF), a safe and inexpensive drug, has shown an antitumor effect when administered with copper (Cu++) against other brain malignancies, and its repurposing to MB could benefit current standards of treatment. ONS76 (Shh-driven Tp53 wild-type), UW228 (Shh-driven Tp53-mutated), D425med and D341med (Group 3) lines were treated with DSF-Cu++and tested with viability, proliferation and clonogenic assays. Flow cytometry (FACS) with AnnexinV-PI, Ki67, PI/RNAse for necrosis and cell cycle, and CD133 and Aldefluor for cell-stemness were carried out. MB lines were tested with western blotting (WB) and immunofluorescence (IF) for NPL4(Nuclear protein localization protein 4 homolog), AIF (apoptosis-inducing factor), cleaved-PARP(Poly-ADP-ribose-polymerase), H2AX and other molecular targets. In vivo safety (n=8) and efficacy studies (n=16) were carried out on Nu/Nu athymic mice implanted intracranially (IC) with D425med cells, along with Hematoxylin/Eosin and immunohistochemistry analyses on brain samples. DSF-Cu++showed remarkable cytotoxic and anti-proliferative activity against all MB lines. In particular, NPL4, a protein involved in the ubiquitin signaling, appears to be the primary DSF target, as previously shown in other malignancies. Apoptosis quickly follows the Heat-Shock Response, as confirmed by FACS, WB and IF, and cell-stemness is decreased significantly by nanomolar doses of DSF-Cu++(p=0.002). Oral administration of DSF-Cu++was proven safe at doses of 100 mg/kg and significantly prolonged survival in IC xenografts (median 28.5 days; p=0.02) compared to controls (median 24 days). DSF, a compound used in the treatment of chronic alcoholism, has recently shown a remarkable effect in a number of malignancies when co-administered with Cu++. We provide proof of its potential as a chemotherapeutic for pediatric MB, and describe its molecular mechanism of action.

Anti-angiogenic agents, such as bevacizumab (BEV), can induce normalization of the blood brain barrier, which may influence the penetration and activity of a co-administered cytotoxic drug. However, it is unknown whether this effect is associated with a benefit in overall survival. This study employed intracranial human glioma models to evaluate the effect of BEV alone and in combination with temozolomide (TMZ) and/or radiation therapy (XRT) on overall survival. One hundred eight male athymic rats were intracranially injected with either U251 or U87 human glioma. Ten or eleven days after tumor inoculation, animals bearing U251 and U87, respectively, were treated with: TMZ alone (50 mg/kg for 5 consecutive days, P.O.), BEV alone (15 mg/kg, I.V.), a combination of TMZ and BEV, or a combination of TMZ, BEV, and a single fraction of XRT (20 Gy). Controls received no treatment. The U87 experiment was repeated and the relationship between survival and the extent of anti-angiogenesis via anti-laminin antibodies for the detection of blood vessels was assessed. In both U87 glioma experiments, all of the treatment groups had a statistically significant increase in survival as compared to the control groups. Also, for both U87 experiments the combination groups of TMZ and BEV had significantly better survival when compared to either treatment administered alone, with 75 % of animals demonstrating long-term survival (LTS) (defined as animals alive 120 days after tumor implantation) in one experiment and 25 % LTS in the repeat experiment. In the U251 glioma experiment, all treated groups (except BEV alone) had significantly improved survival as compared to controls with minimal statistical variance among groups. The percent vessel area was lowest in the group of animals treated with BEV alone. The addition of BEV to TMZ and/or XRT had variable effect on prolonging survival in the two human glioma models tested with reduced tumor vascularity in groups treated with BEV. These results indicate that BEV has anti-angiogenic activity and does not seem to hinder the effect of TMZ.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and radiation is one of the main treatment modalities. However, cure rates remain low despite best available therapies. Immunotherapy is a promising modality that could work synergistically with radiation, which has been shown to increase antigen presentation and promote a proinflammatory tumor microenvironment. Programmed-death-1 (PD-1) is a surface receptor expressed on activated and exhausted T cells, which mediate T cell inhibition upon binding with its ligand PD-L1, expressed on many tumor types including human GBMs. We tested the combination of anti-PD-1 immunotherapy with stereotactic radiosurgery in a mouse orthotopic GBM model.We performed intracranial implantation of mouse glioma cell line GL261 transfected with luciferase into C57BL/6 mice. Mice were stratified into 4 treatment groups: (1) control; (2) radiation only; (3) anti-PD-1 antibody only; and (4) radiation plus anti-PD-1 antibody. Overall survival was quantified. The mice were killed on day 21 after implantation to assess immunologic parameters in the brain/tumor, cervical lymph nodes, and spleen.Improved survival was demonstrated with combination anti-PD-1 therapy plus radiation compared with either modality alone: median survival was 25 days in the control arm, 27 days in the anti-PD-1 antibody arm, 28 days in the radiation arm, and 53 days in the radiation plus anti-PD-1 therapy arm (P.05 by log-rank Mantle-Cox). Long-term survival was seen only in the combined treatment arm, with a fraction (15%-40%) of animals alive at day 180+ after treatment. Immunologic data on day 21 after implantation showed increased tumor infiltration by cytotoxic T cells (CD8+/interferon-γ+/tumor necrosis factor-α+) and decreased regulatory T cells (CD4+/FOXP3) in the combined treatment group compared with the single modality arms.The combination of PD-1 blockade and localized radiation therapy results in long-term survival in mice with orthotopic brain tumors. These studies provide strong preclinical evidence to support combination trials in patients with GBM.