miR-644a is a tumor cell-intrinsic mediator of sex bias in glioblastoma.

Background: Biological sex is an important risk factor for glioblastoma (GBM), with males having a higher incidence and poorer prognosis. The mechanisms for this sex bias are thought to be both tumor intrinsic and tumor extrinsic. MicroRNAs (miRNAs), key post-transcriptional regulators of gene expression, have been previously linked to sex differences in various cell types and diseases, but their role in the sex bias of GBM remains unknown. Methods: We leveraged previously published paired miRNA and mRNA sequencing of 39 GBM patients (22 male, 17 female) to identify sex-biased miRNAs. We further interrogated a separate single-cell RNA sequencing dataset of 110 GBM patients to examine whether differences in miRNA target gene expression were tumor cell intrinsic or tumor cell extrinsic. Results were validated in a panel of patient-derived cell models. Results: We identified 10 sex-biased miRNAs (adjusted < 0.1), of which 3 were more highly expressed in males and 7 more highly expressed in females. Of these, miR-644a was higher in females, and increased expression of miR-644a target genes was significantly associated with decreased overall survival (HR 1.3, p = 0.02). Furthermore, analysis of an independent single-cell RNA sequencing dataset confirmed sex-specific expression of miR-644a target genes in tumor cells (p < 10-15). Among patient derived models, miR-644a was expressed a median of 4.8-fold higher in females compared to males. Conclusions: Our findings implicate miR-644a as a candidate tumor cell-intrinsic regulator of sex-biased gene expression in GBM.

Androgen loss weakens anti-tumor immunity and accelerates brain tumor growth.

Many cancers, including glioblastoma (GBM), have a male-biased sex difference in incidence and outcome. The underlying reasons for this sex bias are unclear but likely involve differences in tumor cell state and immune response. This effect is further amplified by sex hormones, including androgens, which have been shown to inhibit anti-tumor T cell immunity. Here, we show that androgens drive anti-tumor immunity in brain tumors, in contrast to its effect in other tumor types. Upon castration, tumor growth was accelerated with attenuated T cell function in GBM and brain tumor models, but the opposite was observed when tumors were located outside the brain. Activity of the hypothalamus-pituitary-adrenal gland (HPA) axis was increased in castrated mice, particularly in those with brain tumors. Blockade of glucocorticoid receptors reversed the accelerated tumor growth in castrated mice, indicating that the effect of castration was mediated by elevated glucocorticoid signaling. Furthermore, this mechanism was not GBM specific, but brain specific, as hyperactivation of the HPA axis was observed with intracranial implantation of non-GBM tumors in the brain. Together, our findings establish that brain tumors drive distinct endocrine-mediated mechanisms in the androgen-deprived setting and highlight the importance of organ-specific effects on anti-tumor immunity.

Tumor cell-derived spermidine promotes a pro-tumorigenic immune microenvironment in glioblastoma via CD8+ T cell inhibition.

The glioblastoma microenvironment is enriched in immunosuppressive factors that potently interfere with the function of cytotoxic T lymphocytes. Cancer cells can directly impact the immune system, but the mechanisms driving these interactions are not completely clear. Here we demonstrate that the polyamine metabolite spermidine is elevated in the glioblastoma tumor microenvironment. Exogenous administration of spermidine drives tumor aggressiveness in an immune-dependent manner in pre-clinical mouse models via reduction of CD8+ T cell frequency and phenotype. Knockdown of ornithine decarboxylase, the rate-limiting enzyme in spermidine synthesis, did not impact cancer cell growth in vitro but did result in extended survival. Furthermore, glioblastoma patients with a more favorable outcome had a significant reduction in spermidine compared to patients with a poor prognosis. Our results demonstrate that spermidine functions as a cancer cell-derived metabolite that drives tumor progression by reducing CD8+T cell number and function.

SerpinB3 drives cancer stem cell survival in glioblastoma.

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. Knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a GBM CSC-specific survival mechanism involving a cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of GBM therapies against therapeutically resistant CSCs.

Identifying conserved molecular targets required for cell migration of glioblastoma cancer stem cells.

Glioblastoma (GBM) is the most prevalent primary malignant brain tumor and is associated with extensive tumor cell infiltration into the adjacent brain parenchyma. However, there are limited targeted therapies that address this disease hallmark. While the invasive capacity of self-renewing cancer stem cells (CSCs) and their non-CSC progeny has been investigated, the mode(s) of migration used by CSCs during invasion is currently unknown. Here we used time-lapse microscopy to evaluate the migratory behavior of CSCs, with a focus on identifying key regulators of migration. A head-to-head migration assay demonstrated that CSCs are more invasive than non-CSCs. Time-lapse live cell imaging further revealed that GBM patient-derived CSC models either migrate in a collective manner or in a single cell fashion. To uncover conserved molecular regulators responsible for collective cell invasion, we utilized the genetically tractable Drosophila border cell collective migration model. Candidates for functional studies were generated using results from a targeted Drosophila genetic screen followed by gene expression analysis of the human homologs in GBM tumors and associated GBM patient prognosis. This strategy identified the highly conserved small GTPase, Rap1a, as a potential regulator of cell invasion. Alteration of Rap1a activity impaired the forward progress of Drosophila border cells during development. Rap1a expression was elevated in GBM and associated with higher tumor grade. Functionally, the levels of activated Rap1a impacted CSC migration speed out of spheres onto extracellular matrix. The data presented here demonstrate that CSCs are more invasive than non-CSCs, are capable of both collective and single cell migration, and express conserved genes that are required for migration and invasion. Using this integrated approach, we identified a new role for Rap1a in the migration of GBM CSCs.

Blood Alcohol Concentration Is Associated With Improved AIS Motor Score After Spinal Cord Injury.

OBJECTIVE: To investigate the relationship between blood alcohol concentration (BAC) and neurologic recovery after traumatic spinal cord injury (TSCI) using standardized outcome measures from the International Standards for the Neurological Classification of Spinal Cord Injury (ISNCSCI) examination. METHOD: This is a retrospective review of merged, prospectively collected, multicenter data from the Spinal Cord Injury Model Systems Database and institutional trauma databases from five academic medical centers across the United States. Patients with SCI and a documented BAC were analyzed for American Spinal Injury Association Impairment Scale (AIS) motor score, FIM, sensory light touch score, and sensory proprioception score upon admission and discharge from rehabilitation. Linear regression was used for the analysis. RESULTS: The study identified 210 patients. Mean age at injury was 47 ± 20.5 years, 73% were male, 31% had an AIS grade A injury, 56% had ≥1 comorbidity, mean BAC was 0.42 ± 0.9 g/dL, and the mean Glasgow Coma Score upon arrival was 13.27 ± 4.0. ISNCSCI motor score gain positively correlated with higher BAC (4.80; confidence interval [CI], 2.39-7.22; p < .0001). FIM motor gain showed a trend toward correlation with higher BAC, although it did not reach statistical significance (3.27; CI, -0.07 to 6.61; p = .055). ISNCSCI sensory light touch score gain and sensory proprioception score gain showed no correlation with BAC (p = .44, p = .09, respectively). CONCLUSION: The study showed a positive association between higher BAC and neurologic recovery in patients with SCI as measured by ISNCSCI motor score gain during rehabilitation. This finding has not been previously reported in the literature and warrants further study to better understand possible protective physiological mechanisms underlying the relationship between BAC and SCI.

Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune-suppression signatures associated with prognosis.

Glioblastoma (GBM) remains uniformly lethal, and despite a large accumulation of immune cells in the microenvironment, there is limited antitumor immune response. To overcome these challenges, a comprehensive understanding of GBM systemic immune response during disease progression is required. Here, we integrated multiparameter flow cytometry and mass cytometry TOF (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing flow cytometry analysis in a cohort of 259 patients ranging from benign to malignant primary and metastatic brain tumors, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in peripheral blood but not immunosuppressive Tregs. In GBM patient tissue, we found that increased MDSC levels in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from newly diagnosed GBM patients revealed that reduced MDSCs over time were accompanied by a concomitant increase in DCs. GBM patients with extended survival also had reduced MDSCs, similar to the levels of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis.

Subsequent Pulse Generator Replacement Surgery Does Not Increase the Infection Rate in Patients With Deep Brain Stimulator Systems: A Review of 1537 Unique Implants at a Single Center.

INTRODUCTION: Deep brain stimulation (DBS) is a well-recognized treatment for patients with movement disorders and other neurological diseases. The implantable pulse generator (IPG) is a fundamental component of the DBS system. Although IPG implantation and replacement surgeries are comparatively minor procedures relative to the brain lead insertion, patients often require multiple IPG replacements during their lifetime with each operation carrying a small but possibly cumulative risk of complications. To better educate our patients and improve surgical outcomes, we reviewed our series of patients at our institution. METHODS: Using electronic health record data, we retrospectively reviewed all initial and subsequent IPG surgeries from patients who underwent at least one IPG surgery between the years of 2010 and 2015 at the Cleveland Clinic main campus. We calculated infection rates for initial IPG implantation surgeries and the infection rate for subsequent replacements. Fisher's exact tests were used to evaluate the chance of an infection between the initial implantation and replacement. Fisher's exact tests and simple logistic regression analyses were used to determine the predictive ability of selected demographic and clinical variables RESULTS: Our final sample included 697 patients and 1537 surgeries. For all patients, the infection rate at the first surgery was 2.01%; at the second surgery, it was 0.44%; and at the third surgery, it was 1.83%. When considering only patients that underwent at least three replacement surgeries (n = 114) the infection rate did not change in a significant manner with subsequent interventions compared to the first replacement. No other variable of interest was a significant predictor of infection. CONCLUSION: We did not find increasing rates of infection with subsequent IPG replacement procedures.

A chemical screen identifies small molecules that regulate hepcidin expression.

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1 standard deviation below the mean of the other chemicals (z-score >3 or <1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.