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Study of human monocytic Myeloid-Derived Suppressor cells Mo-MDSC (CD14(+) HLA-DR(neg/low)) has been hampered by the lack of positive cell-surface markers. In order to identify positive markers for Mo-MDSC, we performed microarray analysis comparing Mo-MDSC cells from healthy subjects versus CD14(+) HLA-DR(high) monocytes. We have identified the surface ectoenzyme Vanin-2(VNN2) protein as a novel biomarker highly-enriched in healthy subjects Mo-MDSC. Indeed, healthy subjects Mo-MDSC cells expressed 68% VNN2, whereas only 9% VNN2 expression was observed on CD14(+) HLA-DR(high) cells (n=4 p

Background The interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, for example, glioblastoma (GBM) where metastasis is rare. Histologically, it is known that glioma stem cells (GSCs) are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcomes in many cancers. While platelets are known to promote the progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Here, we aimed to understand how the bidirectional interaction between platelets and GSCs drives GBM oncogenesis. Methods Male and female NSG mice were transplanted with GSC lines and treated with antiplatelet and anti-thrombin inhibitors. Immunofluorescence, qPCR, and Western blots were used to determine expression of coagulation mechanism in GBM tissue and subsequent GSC lines. Results We show that GSCs activate platelets by endogenous production of all the factors of the intrinsic and extrinsic coagulation cascades in a plasma-independent manner. Therefore, GSCs produce thrombin resulting in platelet activation. We further demonstrate that the endogenous coagulation cascades of these cancer stem cells are tumorigenic: they activate platelets to promote stemness and proliferation in vitro and pharmacological inhibition delays tumor growth in vivo. Conclusions Our findings uncover a specific preferential relationship between platelets and GSCs that drive GBM malignancies and identify a therapeutically targetable novel interaction.

IntroductionGlioblastoma multiforme (GBM) constitutes one of the deadliest tumors to afflict humans, although it is still considered an orphan disease. Despite testing multiple new and innovative therapies in ongoing clinical trials, the median survival for this type of malignancy is less than two years after initial diagnosis, regardless of therapy. One class of promising new therapies are chimeric antigen receptor T cells or CAR-T which have been shown to be very effective at treating refractory liquid tumors such as B-cell malignancies. However, CAR-T effectivity against solid tumors such as GBM has been limited thus far.MethodsA Pubmed, Google Scholar, Directory of Open Access Journals, and Web of Science literature search using the terms chimeric antigen receptor or CAR-T, GBM, solid tumor immunotherapy, immunotherapy, and CAR-T combination was performed for publication dates between January 1987 and November 2021.ResultsIn the current review, we present a comprehensive list of CAR-T cells developed to treat GBM, we describe new possible T-cell engineering strategies against GBM while presenting a short introductory history to the reader regarding the origin(s) of this cutting-edge therapy. We have also compiled a unique list of anti-GBM CAR-Ts with their specific protein sequences and their functions as well as an inventory of clinical trials involving CAR-T and GBM.ConclusionsThe aim of this review is to introduce the reader to the field of T-cell engineering using CAR-Ts to treat GBM and describe the obstacles that may need to be addressed in order to significantly delay the relentless growth of GBM.

INTRODUCTION Glioblastoma multiforme (GBM) is the most common primary central nervous system malignancy associated with a 12-15 month survival after surgery and radio-chemotherapy. Utilizing adoptive cellular immunotherapy using natural killer (NK) cells has developed over the past two decades for a variety of hematologic malignancies. This approach in solid malignancies is limited by questions of cell dose versus tumor burden, insufficient tumor infiltration, and a tumor microenvironment that suppresses NK cell function. METHODS We isolated NK cells from healthy volunteers and activated them using IL-2, -15, -12, -18, then perform cytotoxic assays in the presence of glioma stem cells. We also tested the efficacy of the NK cells with intracranial delivery in a pre-clinical murine model of glioma. We tested various concentrations of IL-2 and IL-15 on the cytokine culture platform. RESULTS In this study, we demonstrate human NK cells, activated using a cytokine cocktail of interleukins-2, -15, -12, and -18, exert strong cytotoxic events against glioma cell lines. To further examine the efficacy of activated NK cells in vitro, we utilized intracranially xenografted glioma lines and demonstrated a survival benefit with tumor bed injections of these cytokine-activated NK cells (p=0.0089). We were able to confirm that NK cells cultured with low doses (200u IL2; 50ng/ml IL15) of both cytokines are just as effective as higher doses. This is important, as in vivoexhaustion of NK cells stimulated with high doses of either cytokine has been well validated. We also found that low-dose irradiation (4Gy) of glioma cells prior to co-culture with cytokine-activated NK cells promoted increased targeted glioma cell killing within 4 hours(32% cell killing). CONCLUSIONS These findings suggest that in a clinical study, injection of cytokine-activated NK cells into the glioblastoma tumor bed could be used as adjuvant treatment following either stereotactic radiation or surgical resection.

BACKGROUND There are conflicting reports regarding the prognostic value of platelet and other blood counts in glioblastoma. However, few series have looked at all hematologic parameters simultaneously. METHODS We performed a retrospective chart review of patients diagnosed with supratentorial glioblastoma from 2014-2019 who started conventional chemoradiation following initial surgical biopsy and/or resection. Hematologic parameters were collected at baseline, in the preoperative and postoperative periods and at the initiation and completion of chemoradiation. This included platelet counts, hemoglobin levels, white blood cell counts (WBC), neutrophil and lymphocyte counts with neutrophil:lymphocyte (NLR) and platelet:lymphocyte ratios (PLR) calculated at each time point. Cox regression was performed to assess the association between each hematologic parameter and both overall survival (OS) and progression free survival (PFS). A multivariate Cox proportional hazards model adjusted for all hematologic parameters, age, sex, race and KPS was generated for each time point. All hematologic parameters were modeled as continuous variables. RESULTS A total of 58 patients met inclusion criteria. 18 were female and 40 male. The median age was 59.5 (range 43-82). Median follow up for all patients was 15.3 months. A total of 52 patients completed radiation therapy and 18 completed 6 cycles of adjuvant chemotherapy. Hemoglobin and neutrophil counts at the conclusion of chemoradiation were associated with OS and PFS on univariate and multivariate analyses. The HR for OS were 0.74 (95% CI 0.5807-0.9313) and 1.28 (1.143-1.441) respectively. The HR for PFS were 0.70 (0.5531-0.8881) and 1.16 (1.05-1.271) respectively. Postoperative lymphocyte and platelet counts at initiation of chemoradiation were both associated with OS with unadjusted HR of 3.2 (1.037-9.960) and HR of 0.99 (0.9898-0.9999) respectively, which remained significant on multivariate analysis. However, neither were associated with PFS. CONCLUSION Several hematologic parameters are associated with glioblastoma outcomes in these initial analyses. Further analyses with additional patients are ongoing.

Glioblastoma (GBM) is recognized as one of the deadliest forms of cancer, despite aggressive therapy consisting of maximal surgical resection followed by concurrent radiation and chemotherapy, the median survival remains ~12 months. Glioma stem cells (GSCs) possess potent tumor-initiating properties and comprise a cellular hierarchy that is responsible for treatment resistance and progression. Specifically targeting GSCs has been considered a promising therapeutic approach, however no clear method has been identified. Histologically, it is known that GSCs are found in perivascular and pseudsopalisading regions of GBM. Similarly, platelet aggregates are often found in pseudsopalisading necrotic regions, suggesting a potential interaction between platelets and GSCs due to their spatial locations. High platelet counts have been associated with poor clinical outcome in many cancers including ovarian and endometrial cancer. While platelets are known to affect progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Our work aimed to understand the crosstalk between GSCs and platelets within GBM solid tumors that work to enhance disease progression and treatment resistance. Our clinical studies suggest elevated platelet counts positively correlate with tumor growth and negatively correlate to overall patient survival. We found platelets and GSC co-localization in GBM solid tissue; platelet exposure to GSCs results in increased proliferation of GSCs specifically, by increasing the self-renewing capacity of GSCs in a dose dependent manner, and resulted in an increased “Stem-like” transcriptional pattern. Inhibiting the GSC-platelet interaction results in a decrease in GSC renewal and stemness. These results introduce a novel interaction between GSCs and platelets and elucidate a novel therapeutic approach specifically targeting GSCs by disrupting the GSC-platelet interaction.

Glioblastoma (GBM) is recognized as one of the deadliest forms of cancer, despite aggressive therapy consisting of maximal surgical resection followed by concurrent radiation and chemotherapy, the median survival remains ~15 months. Glioma stem cells (GSCs) possess potent tumor-initiating properties and comprise a cellular hierarchy that is responsible for treatment resistance and progression. Specifically targeting GSCs has been considered a promising therapeutic approach, however no clear method has been identified. Histologically, it is known that GSCs are found in perivascular and pseudsopalisading regions of GBM. Similarly, platelet aggregates are often found in pseudsopalisading necrotic regions, suggesting a potential interaction between platelets and GSCs due to their spatial locations. High platelet counts have been associated with poor clinical outcome in many cancers including ovarian and endometrial cancer. While platelets are known to affect progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Our work aimed to understand the crosstalk between GSCs and platelets within GBM solid tumors that work to enhance disease progression and treatment resistance. Our clinical studies suggest elevated platelet counts positively correlate with tumor growth and negatively correlate to overall patient survival. We found platelets and GSC co-localization in GBM solid tissue; platelet exposure to GSCs results in increased proliferation of GSCs specifically, by increasing the self-renewing capacity of GSCs in a dose dependent manner, and resulted in an increased “Stem-like” transcriptional pattern. Consequently, inhibiting the GSC-platelet interaction results in a decrease in GSC renewal and stemness. These results introduce a novel interaction between GSCs and platelets and elucidate a novel therapeutic approach specifically targeting GSCs by disrupting the GSC-platelet interaction.

INTRODUCTION Glioblastomas (GBM) are the most common and malignant primary brain tumors. Their rapid growth and invasion into neuronal parenchyma is devastating, with limited treatment options. Genomic alterations have been extensively studied in gliomas tumors, including aberrations of the Slit-Robo pathway genes. The Slit family of secreted proteins modulates migration of somatic cells during development and mediate their effect by binding to its receptor Roundabout (Robo). Genes in the Slit-Robo pathways have been shown to be inactivated by promoter hypermethylation in a number of human cancers. We hypothesize that Slit-Robo signaling pathways are modulated via promotor methylation, controlling GBM malignancy and regulating the invasive capacity of glioma stem cells (GSCs). METHODS We characterized expression of mRNA and protein via real-time PCR and Western blots in primary GBM tissue. We then assessed whether epigenetic alterations correlate with Slit-Robo expression by conducting in vitro demethylation assays in patient derived GSCs followed by real-time PCR. We conducted invasion assays to elucidate the role of Slit in GSC invasion. RESULTS The Cancer Genome Atlas indicates a negative correlation between Robo2 expression and glioma patient survival (17.1 months versus 37.4 months; p < 1.4E-4). We discovered differential expression of the Slit (1-3) and Robo (2, 3) genes and protein of up to 8-fold between grades 3 and 4 astrocytomas (8.34 versus 4.73 density). Treating patient derived glioma stem cells with 5-aza-2-deoxycytidine results in induction in Slit-Robo gene expression ranging from 5 – 40-fold (p < 0.01). Addition of Slit 2 and 3 in an invasion assay induced migration of GSCs in a differential and concentration dependent manner of 10 - 25% (p < 0.05). CONCLUSIONS Our results suggest that promoter methylation and gene expression of the Slit-Robo genes correlates with protein expression, tumor invasiveness, and prognosis and a potential therapeutic target.

The effect of platelets on oncogenesis has been studied extensively in cancer metastasis, but not in glioblastoma (GBM), where metastasis is rare. Here we identify the unique crosstalk between glioma stem cells (GSCs) and platelets within GBM solid tumors that enhance disease progression. Targeting GSCs is considered a promising therapeutic approach; however, no clear method has been identified. High platelet counts have been associated with poor clinical outcome in many cancers including ovarian and endometrial cancer. While platelets are known to affect progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Immunofluorescence, qPCR, and western blot were used to evaluate the presence of GSCs and platelets and their colocalization in GBM patient tissue at University Hospitals-Seidman Cancer Center. Functional assays followed by RNA sequencing were conducted to determine the functional effect of healthy and GBM platelets on growth of patient derived, autologous GSCs. Our clinical studies suggest elevated platelet counts positively correlate with GSC proliferation and negatively correlate with overall survival in patients with GBM. Patients with high platelet counts ( >350k/µl) had a median survival time of 9 months compared to 16 months median survival for patients with normal platelet count (150-350/µl) (p

INTRODUCTION Glioblastoma (GBM) is the most common primary malignant brain tumor with a median overall survival of 12-15months. GBM aggressiveness and poor response to treatment are often attributed to a small population of stem-like cells referred to as glioma stem cells (GSCs). Human Beta-Defensins (HBDs), a family of small molecules initially thought to function as anti-microbials, have been implicated in various cancers with functions that are cancer type specific, including proinflammatory and immunosuppressive roles. GOAL: This study aimed to elucidate HBDs expression in GSCs and differentiated gioma cells (DGSs). METHODS We identified HBD2 and HBD3 in glioma and GSCs and DGSs by immunofluorescence (IF) and immunohistochemistry (IHC). We also assessed the role HBD2/3 play in GBM oncogenesis by investigating their effects on the self-renewal capacity of GSCs. RESULTS HBD2 and HBD3 are found in both bulk tumor and GSCs by IHC and IF. Expression of HBD2 and HBD3 mRNA levels are elevated in GBM patient tissue in comparison to lower grade gliomas by 16.2 & 1.9 fold (respectively; p < 0.0001). Additionally, transcriptional expression of HBD2 and HBD3 are increased by 0.68 and 1.15 fold respectively in GSCs versus autologous DGCs (p < 0.05; p< 0.005 respectively), suggesting a role for HBD 2/3 in oncogenesis. Interestingly however, HBD2 and HBD3 pretreatment of GSC cell lines showed decreased self-renewal capacity by 34.2 and 66.4% (p < 0.001), determined by the reduced number of large neurospheres ( >250mm). CONCLUSIONS Our results demonstrate the presence of HBD2/3 in GBM samples by IHC and IF with increased HBD2/3 mRNA expression in GBM samples. Interestingly DGCs contain less HBD2/3 than their GCS counterparts, and clonogenicity assays demonstrate a decrease in oncogenesis, suggesting that HBD’s role in proliferation and clonogenicity of DGCs and GSCs may be context dependent, leading to additional questions and future studies.

Glioblastoma (GBM) is the most aggressive and lethal type of brain cancer with a median survival of less than two years even following aggressive treatment (Stupp et al., N Engl J Med 352:987-996, 2005). Among the many challenges in treating patients with this devastating disease is the ability to differentiate Magnetic Resonance Imaging (MRI) images that appear following radiation therapy, often termed "radiation necrosis" from true GBM recurrence. Radiation necrosis (RN) and GBM are very difficult to distinguish and currently only a brain biopsy can conclusively differentiate these pathologies. In the present study, we introduce a differential diagnostic approach using a newly identified Myeloid-Derived Suppressor Cell (MDSC) biomarker, vascular non-inflammatory molecule 2 (VNN2+), in combination with expression of traditional HLA-DR on peripheral blood CD14+ monocytes isolated from GBM and/or RN patients. We performed proof-of-principle experiments confirming the sensitivity and specificity of this approach based upon the combined expression levels of HLA-DR and VNN2 among CD14+ Mo-MDSC, which we called the DR-Vanin Index or DVI. The DVI was able to distinguish GBM from RN patients with a high degree of certainty (n = 18 and n = 6 respectively; p = 0.0004). This novel, quick and inexpensive blood-based liquid biopsy could potentially replace invasive brain biopsies in differentiating GBM from RN patients using a minimally-invasive technique.

Glioblastoma Multiforme (GBM) is the most common and lethal malignant primary adult brain tumor. Therapy resistance and tumor recurrence in GBM have been attributed to glioma stem cells (GSCs), which are found in hypoxic but perivascular niches. We hypothesized that the clinically documented and prognostically relevant increase in platelets in GBM promotes formation of functional hypoxic but perivascular niches that support GSCs due to formation of intravascular thromboses, a hallmark of GBM. Our preliminary in silico analysis from TCGA GBM samples indicates significant correlation of platelet and stemness signature expression (P < 0.001). High expression of platelet gene signatures also inversely correlates with overall survival in GBM patients (P < 0.02). Furthermore, we found significant co-localization of known platelet and stemness markers in primary GBM specimens, supporting our hypothesis and motivating further interrogation of GSC-platelet crosstalk. Our preliminary data suggest that GSCs exposed to either tumor or healthy platelets demonstrate increased self-renewal and stemness, as determined by a significant increase in OCT4, NANOG, and OLIG2 expression compared to unexposed GSCs. This increase in stemness and self-renewal markers was not seen in platelet-exposed differentiated glioma cell (DGC) counterparts. Conversely, platelets are stimulated by GSCs and GSC-conditioned media while DGCs elicit no stimulatory effects as measured by ATP release and aggregation assays. Our results implicate a functional role for platelet-GSC interactions in the maintenance of tumor cellular hierarchy that ultimately contributes to poor clinical outcomes in GBM. RNA-Seq of platelet-privileged GSCs is currently underway to elucidate the mechanism by which platelets impact GSC self-renewal. Successful characterization of potential crosstalk between platelets and GSCs may offer new clinical perspectives into GBM and inform development of a novel treatment paradigm to target these specific cell-to-cell interactions.

Despite aggressive multi-modality treatment with surgery, radiation and chemotherapies, malignant glioma inevitably recurs and has dismal survival rates. Recent progress in immunotherapy has led to a resurgence of interest, and immunotherapies are being investigated for treatment of glioma. However, the unique brain anatomy and a highly immunosuppressive glioma microenvironment pose significant challenges to achieving efficacy. Thus, there is a critical need for assessment of next-generation immunotherapies for glioma. In this study, we have investigated the efficacy of the nanoparticle platform technology based on plant-derived Cowpea mosaic virus like particles (empty CPMV or eCPMV) to instigate a potent immune response against intracranial glioma. CPMV immunotherapy has been shown to efficiently reverse the immunosuppressive tumor microenvironments in pre-clinical murine models of dermal melanoma and metastatic melanoma, metastatic breast cancer, intraperitoneal ovarian cancer and in canine patients with oral melanoma. In the present study, we demonstrate that in situ administration of CPMV immunotherapy in the setting of glioma can effectively recruit unique subset of effector innate and adaptive immune cells to the brain parenchyma while reducing immune suppressive cellular population, leading to regression of intracranial glioma. The in situ CPMV nanoparticle vaccine offers a potent yet safe and localized immunotherapy for intracranial glioma.

Glioblastoma multiforme (GBM) is the most malignant primary brain cancer affecting adults. Therapy options for GBM have remained the same for over a decade with no significant advancements. In vitro cell line models have minimal success for screening potential therapies. Many therapies that were successful in culture have failed in the clinic, likely due to the complex microenvironment in the brain, which has yet to be reproduced in any culture model. Furthermore, high passage number of cultured cells and clonal selection fail to recapitulate the molecular and genomic signatures of GBM. We have been able to successfully establish 37 patient derived xenografts (PDX) intracranially from GBM patient tissue. Tumor derived glial stem cells isolated from patient resected tissue and implanted intracranially into an immunosuppressed mouse strain. Of the 69 patient samples analyzed, we were successful in passaging three or more generations through mice 53.6% of the time. After characterization of the xenografted tumor tissue, two different tumor growth patterns emerged: highly invasive or localized. This phenotype was likely dependent on malignancy, and previous treatment of the patient from which the xenograft was derived. Physiologically, mice exhibited symptoms more quickly with each subsequent passage, particularly in the localized tumors. Development of these physiologically relevant mice, will enable therapy screenings in a microenvironment that more closely resembles GBM and may allow development of individualized patient models which can be used for simulating treatment effectors. Once established, intracranial PDX may also be useful for personalizing medicine.

Glioblastoma accounts for 54.3% of newly diagnosed glioma in the United States each year. Rapid growth and invasion confers a devastating phenotype with very limited treatment options at recurrence. Numerous studies of the molecular alterations in GBM have been conducted but few have completely characterized the invasiveness characteristic of GBM. The roundabout (Robo) family of transmembrane receptors and their Slit protein ligands, have been demonstrated to be involved in neuronal migration and outgrowth during CNS development and implicated in various cancers. We hypothesize that Slit-Robo pathway may be involved in GBM cell invasion. We reviewed numerous databases and analyzed protein and RNA from GBM samples. Analysis of the NCI Rembrandt database indicated poorer survival among glioma patients with a greater than two-fold overexpression of the Robo2 gene (17.1 months versus 37.4 months; p=1.42E-4). GBM tumor was identified prospectively on pre-operative MRI and sampled using stereotactic image-guided resection from various regions within the tumor. In tissue samples from 6 GBM and 3 control patients, indicted a 2.5 fold difference in the expression of Robo2 protein. Quantitative PCR and Western blot confirmed a 101- fold increase in mRNA expression. Knockdown of the mRNA using siRNA directed against Robo2 in U87 cells resulted in survival advantage (65.5 days versus 52.5 days; p=0.011). These data suggest a complex and heterogenous tumor microenvironment and implicates Robo axis signaling in GBM as a potential therapeutic target.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) inthe regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically thehighly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modificationas a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N