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ABSTRACTImmunotherapy has revolutionized the treatment of cancers. Reinvigorating lymphocytes with checkpoint blockade has become a cornerstone of immunotherapy for multiple tumor types, but the treatment of glioblastoma has not yet shown clinical efficacy. A major hurdle to treat GBM with checkpoint blockade is the high degree of myeloid-mediated immunosuppression in brain tumors that limits CD8 T-cell activity. A potential strategy to improve anti-tumor efficacy against glioma is to use myeloid-modulating agents to target immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. We found that the co-inhibition of the chemokine receptors CCR2 and CCR5 in murine model of glioma improves the survival and synergizes robustly with anti-PD-1 therapy. Moreover, the treatment specifically reduced the infiltration of monocytic-MDSCs (M-MDSCs) into brain tumors and increased lymphocyte abundance and cytokine secretion by tumor-infiltrating CD8 T cells. The depletion of T-cell subsets and myeloid cells abrogated the effects of CCR2 and CCR5 blockade, indicating that while broad depletion of myeloid cells does not improve survival, specific reduction in the infiltration of immunosuppressive myeloid cells, such as M-MDSCs, can boost the anti-tumor immune response of lymphocytes. Our study highlights the potential of CCR2/CCR5 co-inhibition in reducing myeloid-mediated immunosuppression in GBM patients.

Abstract Glioma is one of the most common primary malignant brain tumors. Despite progress in therapeutic approaches, the median survival of patients with glioma remains less than 2 years, generating the need for new therapeutic approaches. Ultrasound (US) is widely used in medical fields and is used as a therapeutic tool mainly for improving the performance of therapeutic entities. In this study, we examined a novel approach using low frequency US (20 kHz) (LFUS) as an independent treatment tool for malignant glioma, since primary studies showed that cancer cells are more susceptible to LFUS than healthy cells. LFUS safety and efficacy were examined in a 9L gliosarcoma‐bearing female Fischer 344 rats. Two LFUS protocols were examined: a one‐time treatment (US1X), and two treatments 24 h apart (US2X). For safety evaluation, rats were monitored for weight change and pain measurements. For efficacy, tumor volume was measured as a function of time and the tumor structural chances were examined histopathologically. LFUS treatment showed rapid inhibition of tumor growth, seen as soon as 12 h after US application. In addition, LFUS was found to affect the tumor structure, which was more extensive (>60% of tumor area) in smaller tumors. In US2X, the tumor tissue was completely destroyed, and an extensive immune response was observed. Importantly, the treatment was highly selective, keeping the healthy tissue surrounding the tumor unharmed. We developed a highly efficient and selective therapeutic protocol for treating malignant glioma with minimal side effects based solely on LFUS.

Abstract Background Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of research, the survival rates for GBM remain effectively stagnant. A defining hallmark of GBM is a highly acidic tumor microenvironment, which is thought to activate pro-tumorigenic pathways. This acidification is the result of altered tumor metabolism favoring aerobic glycolysis, a phenomenon known as the Warburg effect. Low extracellular pH confers radioresistant tumors to glial cells. Notably GPR68, an acid sensing GPCR, is upregulated in radioresistant GBM. Usage of Lorazepam, which has off target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. However, the role of tumor microenvironment acidification in GPR68 activation has not been assessed in cancer. Here we interrogate the role of GPR68 specifically in GBM cells using a novel highly specific small molecule inhibitor of GPR68 named Ogremorphin (OGM) to induce the iron mediated cell death pathway: ferroptosis. Method OGM was identified in a non-biased zebrafish embryonic development screen and validated with Morpholino and CRISPR based approaches. Next, A GPI-anchored pH reporter, pHluorin2, was stably expressed in U87 glioblastoma cells to probe extracellular acidification. Cell survival assays, via nuclei counting and cell titer glo, were used to demonstrate sensitivity to GPR68 inhibition in twelve immortalized and PDX GBM lines. To determine GPR68 inhibition’s mechanism of cell death we use DAVID pathway analysis of RNAseq. Our major indication, ferroptosis, was then confirmed by western blotting and qRT-PCR of reporter genes including TFRC. This finding was further validated by transmission electron microscopy and liperfluo staining to assess lipid peroxidation. Lastly, we use siRNA and CRISPRi to demonstrate the critical role of ATF4 suppression via GPR68 for GBM survival. Results We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68. Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested, irrespective of genetic and phenotypic heterogeneity, or resistance to the mainstay GBM chemotherapeutic temozolomide. We use U87 and U138 glioblastoma cell lines to show how selective induction of ferroptosis occurs in an ATF4-dependent manner. Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells. Conclusion These results indicate GPR68 emerges as a critical sensor for an autocrine pro-tumorigenic signaling cascade triggered by extracellular acidification in glioblastoma cells. In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death. These findings provide a promising therapeutic approach to selectively induce ferroptosis in glioblastoma cells while sparing healthy neural tissue.

Glioblastoma (GBM) is an aggressive and highly malignant primary brain tumor characterized by rapid growth and a poor prognosis for patients. Despite advancements in treatment, the median survival time for GBM patients remains low. One of the crucial challenges in understanding and treating GBMs involves its remarkable cellular heterogeneity and adaptability. Central to the survival and proliferation of GBM cells is their ability to undergo metabolic reprogramming. Metabolic reprogramming is a process that allows cancer cells to alter their metabolism to meet the increased demands of rapid growth and to survive in the often oxygen- and nutrient-deficient tumor microenvironment. These changes in metabolism include the Warburg effect, alterations in several key metabolic pathways including glutamine metabolism, fatty acid synthesis, and the tricarboxylic acid (TCA) cycle, increased uptake and utilization of glutamine, and more. Despite the complexity and adaptability of GBM metabolism, a deeper understanding of its metabolic reprogramming offers hope for developing more effective therapeutic interventions against GBMs.

Glioblastoma multiforme (GBM) is a deadly and aggressive malignant brain cancer that is highly resistant to treatments. A particular challenge of treatment is caused by the blood–brain barrier (BBB), the relatively impermeable vasculature of the brain. The BBB prevents large molecules from entering the brain parenchyma. This protective characteristic of the BBB, however, also limits the delivery of therapeutic drugs for the treatment of brain tumors. To address this limitation, focused ultrasound (FUS) has been safely utilized to create transient openings in the BBB, allowing various high molecular weight drugs access to the brain. We performed a systematic review summarizing current research on treatment of GBMs using FUS-mediated BBB openings in in vivo mouse and rat models. The studies gathered here highlight how the treatment paradigm can allow for increased brain and tumor perfusion of drugs including chemotherapeutics, immunotherapeutics, gene therapeutics, nanoparticles, and more. Given the promising results detailed here, the aim of this review is to detail the commonly used parameters for FUS to open the BBB in rodent GBM models.

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.

Abstract 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.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with an extremely poor prognosis. There is a dire need to develop effective therapeutics to overcome the intrinsic and acquired resistance of GBM to current therapies. The process of developing novel anti-neoplastic drugs from bench to bedside can incur significant time and cost implications. Drug repurposing may help overcome that obstacle. A wide range of drugs that are already approved for clinical use for the treatment of other diseases have been found to target GBM-associated signaling pathways and are being repurposed for the treatment of GBM. While many of these drugs are undergoing pre-clinical testing, others are in the clinical trial phase. Since GBM stem cells (GSCs) have been found to be a main source of tumor recurrence after surgery, recent studies have also investigated whether repurposed drugs that target these pathways can be used to counteract tumor recurrence. While several repurposed drugs have shown significant efficacy against GBM cell lines, the blood–brain barrier (BBB) can limit the ability of many of these drugs to reach intratumoral therapeutic concentrations. Localized intracranial delivery may help to achieve therapeutic drug concentration at the site of tumor resection while simultaneously minimizing toxicity and side effects. These strategies can be considered while repurposing drugs for GBM.

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.

Background Glioblastoma (GBM) is a poorly immunogenic neoplasm treated with focused radiation. Immunotherapy has demonstrated synergistic survival effects with stereotactic radiosurgery (SRS) in murine GBM. GITR is a co-stimulatory molecule expressed constitutively on regulatory T-cells and by effector T-cells upon activation. We tested the hypothesis that anti-GITR monoclonal antibody (mAb) and SRS together would confer an immune-mediated survival benefit in glioma using the orthotopic GL261 glioma model.Methods Mice received SRS and anti-GITR 10 days after implantation. The anti-GITR mAbs tested were formatted as mouse IgG1 D265A (anti-GITR (1)) and IgG2a (anti-GITR (2a)) isotypes. Mice were randomized to four treatment groups: (1) control; (2) SRS; (3) anti-GITR; (4) anti-GITR/SRS. SRS was delivered to the tumor in one fraction, and mice were treated with mAb thrice. Mice were euthanized on day 21 to analyze the immunologic profile of tumor, spleen, and tumor draining lymph nodes.Results Anti-GITR (1)/SRS significantly improved survival over either treatment alone (p

Glioblastoma (GBM) is the most common malignant brain tumor in adults and is associated with a poor prognosis. Cytotoxic T lymphocyte antigen -4 (CTLA-4) blocking antibodies have demonstrated an ability to generate robust antitumor immune responses against a variety of solid tumors. 4-1BB (CD137) is expressed by activated T lymphocytes and served as a co-stimulatory signal, which promotes cytotoxic function. Here, we evaluate a combination immunotherapy regimen involving 4-1BB activation, CTLA-4 blockade, and focal radiation therapy in an immune-competent intracranial GBM model.GL261-luciferace cells were stereotactically implanted in the striatum of C57BL/6 mice. Mice were treated with a triple therapy regimen consisted of 4-1BB agonist antibodies, CTLA-4 blocking antibodies, and focal radiation therapy using a small animal radiation research platform and mice were followed for survival. Numbers of brain-infiltrating lymphocytes were analyzed by FACS analysis. CD4 or CD8 depleting antibodies were administered to determine the relative contribution of T helper and cytotoxic T cells in this regimen. To evaluate the ability of this immunotherapy to generate an antigen-specific memory response, long-term survivors were re-challenged with GL261 glioma en B16 melanoma flank tumors.Mice treated with triple therapy had increased survival compared to mice treated with focal radiation therapy and immunotherapy with 4-1BB activation and CTLA-4 blockade. Animals treated with triple therapy exhibited at least 50% long-term tumor free survival. Treatment with triple therapy resulted in a higher density of CD4+ and CD8+ tumor infiltrating lymphocytes. Mechanistically, depletion of CD4+ T cells abrogated the antitumor efficacy of triple therapy, while depletion of CD8+ T cells had no effect on the treatment response.Combination therapy with 4-1BB activation and CTLA-4 blockade in the setting of focal radiation therapy improves survival in an orthotopic mouse model of glioma by a CD4+ T cell dependent mechanism and generates antigen-specific memory.

Glioblastoma multiforme (GBM) is the most common intracranial cancer but despite recent advances in therapy the overall survival remains about 20 months. Whole genome exon sequencing studies implicate mutations in the receptor tyrosine kinase pathways (RTK) for driving tumor growth in over 80% of GBMs. In spite of various RTKs being mutated or altered in the majority of GBMs, clinical studies have not been able to demonstrate efficacy of molecular targeted therapies using tyrosine kinase inhibitors in GBMs. Activation of multiple downstream signaling pathways has been implicated as a possible means by which inhibition of a single RTK has been ineffective in GBM. In this study, we sought a combination of approved drugs that would inhibit in vitro and in vivo growth of GBM oncospheres. A combination consisting of gefitinib and sunitinib acted synergistically in inhibiting growth of GBM oncospheres in vitro. Sunitinib was the only RTK inhibitor that could induce apoptosis in GBM cells. However, the in vivo efficacy testing of the gefitinib and sunitinib combination in an EGFR amplified/PTEN wild type GBM xenograft model revealed that gefitinib alone could significantly improve survival in animals whereas sunitinib did not show any survival benefit. Subsequent testing of the same drug combination in a different syngeneic glioma model that lacked EGFR amplification but was more susceptible to sunitinib in vitro demonstrated no survival benefit when treated with gefitinib or sunitinib or the gefitinib and sunitinib combination. Although a modest survival benefit was obtained in one of two animal models with EGFR amplification due to gefitinib alone, the addition of sunitinib, to test our best in vitro combination therapy, did not translate to any additional in vivo benefit. Improved targeted therapies, with drug properties favorable to intracranial tumors, are likely required to form effective drug combinations for GBM.

Abstract Objective  Intramedullary spinal cord tumors (IMSCT) pose significant challenges given their recurrence rate and limited treatment options. Using our previously described rat model of IMSCT, we describe a technique for microsurgical tumor resection and present the functional and histopathological analysis of tumor progression. Methods  Twenty-four Fischer 344 rats were randomized into two groups. All animals received a 5-μl intramedullary injection of 9L gliosarcoma cells. Animals were evaluated daily for signs of paralysis using the Basso, Beattie, and Bresnahan (BBB) scale. Group 1 continued with daily assessments using the BBB scale following tumor implantation, but received no further treatment. Group 2 underwent surgical removal of intramedullary tumor on postoperative day five. At a BBB score less than 5 (e.g., functional paraplegia), all animals of both groups were killed and sent for histopathological analysis. Results  Group 1 had a median onset of functional hind limb paraplegia at 15 ± 1.0 days. Group 2 had a median onset of hind limb paresis at 53 ± 0.46 days. Hematoxylin–eosin cross-sections confirmed the presence of intramedullary 9L tumor invading the spinal cord in both groups. Conclusion  Animals with 9L IMSCTs consistently developed hind limb paraplegia in a reliable and reproducible manner. Animals undergoing microsurgical resection of IMSCT had a significant delay in the onset of functional paraplegia compared to the untreated controls. These findings suggest that this model may mimic the behavior of IMSCTs following operative resection in humans and thus may be used to examine efficacy of new treatment options for high-grade intramedullary tumors. [ABSTRACT FROM AUTHOR]

Abstract  Docetaxel (Taxotere) is a hemisynthetic, anti-cancer compound with good preclinical and clinical activity in a variety of systemic neoplasms. We tested its activity against malignant gliomas using local delivery methods. Antitumor activity was assessed in vitro against human (U87 and U80 glioma) and rat brain-tumor (9L gliosarcoma and F98 glioma) cell lines. For in vivo evaluation, we incorporated docetaxel into a biodegradable polymer matrix, determined associated toxicity in the rat brain, and measured efficacy at extending survival in a rat model of malignant glioma. Also, we examined the combined local delivery of docetaxel with carmustine (BCNU) against the experimental intracranial glioma. Rats bearing intracranial 9L gliosarcomas were treated 5 days after tumor implantation with various polymers (placebo, 5% docetaxel, 3.8% BCNU, or 5% docetaxel and 3.8% BCNU combination). Animals receiving docetaxel polymers (n = 15, median survival 39.1 days) had significantly improved survival over control animals (n = 12, median survival 22.5 days, P = 0.01). Similarly, animals receiving BCNU polymers (n = 15, median survival 39.3 days, 13.3% long-term survivors) demonstrated an increase in survival compared to the controls (P = 0.04). Animals receiving the combination polymers demonstrated a modest increase in survival compared to either chemotherapeutic agent alone (n = 14, median survival 54.9 days, 28.6% long-term survivors) with markedly improved survival over controls (P = 0.003). We conclude that locally delivered docetaxel shows promise as a novel anti-glioma therapy and that the combination of drug regimens via biodegradable polymers may be a great therapeutic benefit to patients with malignant glioma. [ABSTRACT FROM AUTHOR]

Lactacystin, a proteasome-inhibitor, has been shown to induce apoptosis of experimental gliomas in vitro. However, its systemic toxicity prevents further clinical use. To circumvent this problem, lactacystin can be delivered intratumorally. We tested the efficacy of lactacystin incorporated into controlled-release polymers for treating experimental gliomas. 9L-gliosarcoma and F98-glioma cell lines were treated with lactacystin (10–100 μg/ml) for 72 h in vitro. Cell-viability was measured with MTT-assays. Toxicity of lactacystin/polycarboxyphenoxypropane-sebacic-acid (pCPP : SA) polymers was tested in vivo using Fischer-344 rats intracranially implanted with lactacystin polymers loaded from 0.1 to 2% lactacystin by weight. The efficacy of 1, 1.3, 1.5 and 1.7% lactacystin/pCPP : SA polymers was determined in Fischer-344 rats intracranially challenged with 9L and treated either simultaneously or 5 days after tumor implantation. Lactacystin was cytotoxic in 9L cells, causing a 16 ± 8% growth inhibition at 10-μg/ml that increased to 78 ± 4% at 100-μg/ml. Similarly, lactacystin inhibited growth of F98 by 18 ± 8% at 10-μg/ml and 74 ± 2% at 100-μg/ml in vitro. Polymers released lactacystin for 21 days and intracranial implantation in rats neither generate local nor systemic toxicity at doses lower than 2%. Treatment with lactacystin/pCPP : SA polymers with loading concentrations of 1.0, 1.3, and 1.5% prolonged survival of animals intracranially challenged with 9L when polymers where inserted in the day of tumor implantation. In conclusion, lactacystin exhibits potent cytotoxic-activity against 9L and F98 in vitro, it can be efficiently incorporated and delivered using controlled-release polymers, and at the proposed concentrations lactacystin polymers are safe for CNS delivery and prolong survival in the 9L model. [ABSTRACT FROM AUTHOR]

Summary Introduction: Local delivery of adriamycin (ADR) via biodegradable polymers has been shown to improve survival in rats challenged intracranially with 9L gliosarcoma. Likewise, local delivery of interleukin-2 (IL-2) has been shown to extend survival in experimental brain tumor models. In the current study, we hypothesized that local delivery of ADR and IL-2 might act synergistically against experimental intracranial glioma. [ABSTRACT FROM AUTHOR]