The Role of Cellular Immunity and Adaptive Immunity in Pathophysiology of Brain and Spinal Cord Tumors.

Major advances have been made in our understanding of CNS tumors, especially glioma, however, the survival of patients with malignant glioma remains poor. While radiation and chemotherapy have increased overall survival, glioblastoma multiforme (GBM) still has one of the worst 5-year survival rates of all human cancers. Here, in this chapter, the authors review the abrogation of the immune system in the tumor setting, revealing many plausible targets for therapy and the current immunotherapy treatment strategies employed. Notably, glioma has also been characterized as a subset of primary spinal cord tumor and current treatment recommendations are outlined here.

Triptolide reverses helper T cell inhibition and down-regulates IFN-γ induced PD-L1 expression in glioma cell lines.

PURPOSE: Immunosuppression is one of hallmark features in many cancers including glioma. Triptolide, a natural compound purified from the Chinese herb Tripterygium wilfordii, has been reported to inhibit PD-L1 otherwise known as the B7 homolog 1 (B7-H1) expression in breast cancer. The purpose of this paper is to test the effects of Triptolide on T cell inhibition in glioma cells. METHODS: We labeled T cells and cocultured with Interferon-γ (IFN-γ) and Triptolide treated glioma cells. The effect on inhibition of T cells as well as subtypes of T cells was measured by Flow Cytometry. We also tested the expression of PD-L1 in six glioma cell lines. RESULTS: We found that Triptolide could reverse T cell inhibition especially CD4+ T cell and induced IFN-γ secretion. In addition, Triptolide could also induce interleukin-2 secretion and overcome interleukin-10 inhibition caused by glioma cells under IFN-γ treated condition. Triptolide could also down-regulate IFN-γ induced PD-L1 surface expression in glioma cells. CONCLUSIONS: These results suggest that Triptolide may be used to reverse CD4+ T cell inhibition caused by glioma cells and is an alternative candidate for targeting PD-L1, one of the checkpoint inhibitors for the treatment of glioma.

Phase 1 clinical trial of intratumoral reovirus infusion for the treatment of recurrent malignant gliomas in adults.

Reovirus, an oncolytic RNA virus exhibiting antiglioma activity, was shown in a previous single institution phase 1 study found that the inoculation of the virus to be well tolerated in patients with recurrent malignant glioma (MG). The goals of multicenter study reported herein were to determine the dose-limiting toxicity, maximum tolerated dose, and target lesion response rate when reovirus was administered in a novel fashion via intratumoral infusion for 72 hours in patients with recurrent malignant glioma. Fifteen adult patients were treated in a dose escalation study ranging from 1 × 10(8) to 1 × 10(10) tissue culture infectious dose 50, tentimes the dose achieved in the previous trial. Neurological, functional examinations, and imaging studies were completed pre- and postinfusion. There was one grade 3 adverse event (convulsions) felt to be possibly related to treatment, but no grade 4 adverse events considered probably or definitely related to treatment. Dose-limiting toxicity were not identified and a maximum tolerated dose was not reached. Evidence of antiglioma activity was seen in some patients. This first report of intratumoral infusion of reovirus in patients with recurrent malignant glioma demonstrated the approach to be safe and well tolerated, warranting further studies.

Immunobiology and immunotherapeutic targeting of glioma stem cells.

For decades human brain tumors have confounded our efforts to effectively manage and treat patients. In adults, glioblastoma multiforme is the most common malignant brain tumor with a patient survival of just over 14 months. In children, brain tumors are the leading cause of solid tumor cancer death and gliomas account for one-fifth of all childhood cancers. Despite advances in conventional treatments such as surgical resection, radiotherapy, and systemic chemotherapy, the incidence and mortality rates for gliomas have essentially stayed the same. Furthermore, research efforts into novel therapeutics that initially appeared promising have yet to show a marked benefit. A shocking and somewhat disturbing view is that investigators and clinicians may have been targeting the wrong cells, resulting in the appearance of the removal or eradication of patient gliomas only to have brain cancer recurrence. Here we review research progress in immunotherapy as it pertains to glioma treatment and how it can and is being adapted to target glioma stem cells (GSCs) as a means of dealing with this potential paradigm.

Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma.

BACKGROUND: This study evaluated the safety and immune responses to an autologous dendritic cell vaccine pulsed with class I peptides from tumor-associated antigens (TAA) expressed on gliomas and overexpressed in their cancer stem cell population (ICT-107). METHODS: TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. HLA-A1- and/or HLA-A2-positive patients with glioblastoma (GBM) were eligible. Mononuclear cells from leukapheresis were differentiated into dendritic cells, pulsed with TAA peptides, and administered intradermally three times at two-week intervals. RESULTS: Twenty-one patients were enrolled with 17 newly diagnosed (ND-GBM) and three recurrent GBM patients and one brainstem glioma. Immune response data on 15 newly diagnosed patients showed 33 % responders. TAA expression by qRT-PCR from fresh-frozen tumor samples showed all patient tumors expressed at least three TAA, with 75 % expressing all six. Correlations of increased PFS and OS with quantitative expression of MAGE1 and AIM-2 were observed, and a trend for longer survival was observed with gp100 and HER2 antigens. Target antigens gp100, HER1, and IL13Rα2 were downregulated in recurrent tumors from 4 HLA-A2+ patients. A decrease in or absence of CD133 expression was seen in five patients who underwent a second resection. At a median follow-up of 40.1 months, six of 16 ND-GBM patients showed no evidence of tumor recurrence. Median PFS in newly diagnosed patients was 16.9 months, and median OS was 38.4 months. CONCLUSIONS: Expression of four ICT-107 targeted antigens in the pre-vaccine tumors correlated with prolonged overall survival and PFS in ND-GBM patients. The goal of targeting tumor antigens highly expressed on glioblastoma cancer stem cells is supported by the observation of decreased or absent CD133 expression in the recurrent areas of gadolinium-enhanced tumors.

Activated T cell therapy targeting glioblastoma cancer stem cells.

Naïve T cells become effector T cells following stimulation by antigen-loaded dendritic cells (DCs) and sequential cytokine activation. We aimed to develop procedures to efficiently activate T cells with tumor-associated antigens (TAAs) to glioblastoma (GBM) stem cells. To remove antigen presentation outside of the immunosuppressive tumor milieu, three different glioma stem cell (GSC) specific antigen sources to load DCs were compared in their ability to stimulate lymphocytes. An activated T cell (ATC) protocol including cytokine activation and expansion in culture to target GSCs was generated and optimized for a planned phase I clinical trial. We compared three different antigen-loading methods on DCs to effectively activate T cells, which were GBM patient-derived GSC-lysate, acid-eluate of GSCs and synthetic peptides derived from proteins expressed in GSCs. DCs derived from HLA-A2 positive blood sample were loaded with TAAs. Autologous T cells were activated by co-culturing with loaded DCs. Efficiency and cytotoxicity of ATCs were evaluated by targeting TAA-pulsed DCs or T2 cells, GSCs, or autologous PHA-blasts. Characteristics of ATCs were evaluated by Flow Cytometry and ELISpot assay, which showed increased number of ATCs secreting IFN-γ targeting GSCs as compared with non-activated T cells and unloaded target cells. Neither GSC-lysate nor acid-eluate loading showed enhancement in response of ATCs but the synthetic peptide pool showed significantly increased IFN-γ secretion and increased cytotoxicity towards target cells. These results demonstrate that ATCs activated using a TAA synthetic peptide pool efficiently enhance cytotoxicity specifically to target cells including GSC.

The Influence of Modifiable Risk Factors on Postoperative Outcomes in Patients Receiving Surgery for Resection for Acoustic Neuroma.

Acoustic neuromas are the most common tumor of the cerebellopontine angle that are associated with a number of symptoms that negatively impact a patient's quality of life. While the mainstay of treatment for these benign tumors remains microsurgical resection, there is limited research exploring how certain modifiable risk factors (MRFs) may affect the perioperative course. The purpose of this study was to investigate how MRFs including malnutrition, obesity, dyslipidemia, uncontrolled hypertension, and smoking may affect postoperative rates of readmission and nonroutine discharges. We utilized the 2016 and 2017 Healthcare Cost and Utilization Project Nationwide Readmissions Database. MRFs were queried using appropriate International Classification of Diseases, Tenth Revision (ICD-10) coding for categories including malnutrition, obesity, dyslipidemia, smoking, alcohol, and hypertension. The statistical analysis was done using RStudio (Version 1.3.959). Chi-squared tests were done to evaluate differences between categorical variables. The Mann-Whitney U-testing was utilized to evaluate for statistically significant differences in continuous data. The "Epitools" package was used to develop logistic regression models for postoperative complications and post hoc receiver operating characteristic curves were developed. Pertaining to nonroutine discharge, predictive models using malnutrition outperformed all other MRFs as well as those with no MRFs (P < .05). In the case of readmission, models using malnutrition outperformed those of obesity and smoking (P < .05). Again, an increase in predictive power is seen in models using dyslipidemia when compared to obesity, smoking, or uncontrolled hypertension. Lastly, models using no MRFs outperformed those of obesity, smoking, and uncontrolled hypertension (P < .05). This is the first study of its kind to evaluate the role of MRFs in those undergoing surgical resection of their acoustic neuroma. We concluded that certain MRFs may play a role in complicating a patient's perioperative surgical course.

Glioma immunotherapy enhancement and CD8-specific sialic acid cleavage by isocitrate dehydrogenase (IDH)-1.

The promise of adaptive cancer immunotherapy in treating highly malignant tumors such as glioblastoma multiforme (GBM) can only be realized through expanding its benefits to more patients. Alleviating various modes of immune suppression has so far failed to achieve such expansion, but exploiting endogenous immune enhancers among mutated cancer genes could represent a more direct approach to immunotherapy improvement. We found that Isocitrate Dehydrogenase-1 (IDH1), which is commonly mutated in gliomas, enhances glioma vaccine efficacy in mice and discerns long from short survivors after vaccine therapy in GBM patients. Extracellular IDH1 directly enhanced T cell responses to multiple tumor antigens, and prolonged experimental glioma cell lysis. Moreover, IDH1 specifically bound to and exhibited sialidase activity against CD8. By contrast, mutant IDH1R132H lacked sialidase activity, delayed killing in glioma cells, and decreased host survival after immunotherapy. Overall, our findings identify IDH1 as an immunotherapeutic enhancer that mediates the known T cell-enhancing reaction of CD8 desialylation. This uncovers a new axis for immunotherapeutic improvement in GBM and other cancers, reveals novel physiological and molecular functions of IDH1, and hints at an unexpectedly direct link between lytic T cell function and metabolic activity in target cells.

ZEB1 loss increases glioma stem cell tumorigenicity and resistance to chemoradiation.

OBJECTIVE: Glioblastoma has been known to be resistant to chemotherapy and radiation, whereas the underlying mechanisms of resistance have not been fully elucidated. The authors studied the role of the transcription factor ZEB1 (zinc finger E-box-binding homeobox 1 protein), which is associated with epithelial-mesenchymal transition (EMT) and is central to the stemness of glioblastoma, to determine its role in therapeutic resistance to radiation and chemotherapy. The authors previously demonstrated that ZEB1 is deleted in a majority of glioblastomas. METHODS: The authors explored resistance to therapy in the context of ZEB1 loss and overexpression in glioma stem cells (GSCs) and in patient data. RESULTS: Patients with ZEB1 loss had a shorter survival time than patients with wild-type ZEB1 in both the high- and low-MGMT groups. Consistent with the clinical data, mice implanted with ZEB1 knockdown GSCs showed shortened survival compared with mice inoculated with nonsilencing control (NS) short-hairpin RNA (shRNA) GSC glioblastoma. ZEB1-deleted GSCs demonstrated increased tumorigenicity with regard to proliferation and invasion. Importantly, GSCs that lose ZEB1 expression develop enhanced resistance to chemotherapy, radiotherapy, and combined chemoradiation. ZEB1 loss may lead to increased HER3 expression through the HER3/Akt pathway associated with this chemoresistance. Conversely, overexpression of ZEB1 in GSCs that are ZEB1 null leads to increased sensitivity to chemoradiation. CONCLUSIONS: The study results indicate that ZEB1 loss in cancer stem cells confers resistance to chemoradiation and uncovers a potentially targetable cell surface receptor in these resistant cells.

Racial and gender disparities and the role of primary tumor type on inpatient outcomes following craniotomy for brain metastases.

BACKGROUND: Race and gender disparities in outcomes have been documented in many cancers. Our study evaluated the role of race, gender, and tumor primary site in predicting in-hospital mortality, discharge disposition, and complications among patients with brain metastases. METHODS: Using Nationwide Inpatient Sample (NIS) data from 1998 to 2007, we evaluated in-patient outcomes of brain metastases patients who underwent a craniotomy in U.S. hospitals. Univariate and multivariate analyses were used to assess the effect of patient/tumor characteristics in predicting the proposed outcomes. RESULTS: NIS estimated 78,170 patients with metastatic brain tumors underwent craniotomy between 1998 and 2007 in the United States. Median age was 59.2 years, 52.1 % were women, and 6.4 % were black. In-hospital mortality rate was 2.2 % with an average length of stay of 7.6 days. Black patients had significantly higher morbidity and nonroutine discharges than whites/others (p < .0001). Black women had almost twice the mortality (3.4 vs 1.8 %, p < .0001), a higher complication rate (24.6 vs 18.8 %, p < .0001), longer hospital stays (10.0 vs 7.3 days, p < .0001), and more nonroutine discharges (45.1 vs 36.8 %, p < .0001), compared with white/other women. Tumor histology was a significant predictor of outcomes, with female lung cancer patients having the highest odds of mortality and primary gastrointestinal tumors having the highest number of complications. CONCLUSIONS: Evidence of race and gender disparities in outcomes were found in black patients, especially in black females who underwent surgical resection for brain metastases. These findings highlight an opportunity to reduce the gap of outcome disparities in brain metastasis patients.

Glioma stem cell research for the development of immunotherapy.

Malignant gliomas are characterized by its invasiveness and dissemination, resulting in frequent tumor recurrence after surgical resection and/or conventional chemotherapy and radiation therapy. Various strategies of active and passive immunotherapy in developing stages have shown promise to increase patient survival time with little severe side effects. In recent years, glioma stem cells had been isolated from patient tumor specimens. Biochemical and biological characterization of these cancer initiating cells implicated their critical roles in cancer growth, malignancy and resistance to conventional treatments. In this chapter, we review recent research progress in targeting brain cancer using neural stem cells delivered cytotoxic factors and immune regulation factor, dendritic cell based vaccination, with special emphasis on targeting glioma stem cells. We present evidence supporting the notion that glioma stem cells may be preferred therapeutic targets not only for conventional therapies, but also for immunotherapies. Future progress in glioma stem cell research may fundamentally improve the prospect of malignant glioma treatments.

Development of a TCR-like antibody and chimeric antigen receptor against NY-ESO-1/HLA-A2 for cancer immunotherapy.

BACKGROUND: The current therapeutic antibodies and chimeric antigen receptor (CAR) T cells are capable of recognizing surface antigens, but not of intracellular proteins, thus limiting the target coverage for drug development. To mimic the feature of T-cell receptor (TCR) that recognizes the complex of major histocompatibility class I and peptide on the cell surface derived from the processed intracellular antigen, we used NY-ESO-1, a cancer-testis antigen, to develop a TCR-like fully human IgG1 antibody and its derivative, CAR-T cells, for cancer immunotherapy. METHODS: Human single-chain variable antibody fragment (scFv) phage library (~10∧11) was screened against HLA-A2/NY-ESO-1 (peptide 157-165) complex to obtain target-specific antibodies. The specificity and affinity of those antibodies were characterized by flow cytometry, ELISA, biolayer interferometry, and confocal imaging. The biological functions of CAR-T cells were evaluated against target tumor cells in vitro. In vivo antitumor activity was investigated in a triple-negative breast cancer (TNBC) model and primary melanoma tumor model in immunocompromised mice. RESULTS: Monoclonal antibody 2D2 identified from phage-displayed library specifically bound to NY-ESO-1157-165 in the context of human leukocyte antigen HLA-A*02:01 but not to non-A2 or NY-ESO-1 negative cells. The second-generation CAR-T cells engineered from 2D2 specifically recognized and eliminated A2+/NY-ESO-1+tumor cells in vitro, inhibited tumor growth, and prolonged the overall survival of mice in TNBC and primary melanoma tumor model in vivo. CONCLUSIONS: This study showed the specificity of the antibody identified from human scFv phage library and demonstrated the potential antitumor activity by TCR-like CAR-T cells both in vitro and in vivo, warranting further preclinical and clinical evaluation of the TCR-like antibody in patients. The generation of TCR-like antibody and its CAR-T cells provides the state-of-the-art platform and proof-of-concept validation to broaden the scope of target antigen recognition and sheds light on the development of novel therapeutics for cancer immunotherapy.

A Phase I Study of Autologous Dendritic Cell Vaccine Pulsed with Allogeneic Stem-like Cell Line Lysate in Patients with Newly Diagnosed or Recurrent Glioblastoma.

PURPOSE: Glioblastoma (GBM) is a heterogeneous malignancy with multiple subpopulations of cancer cells present within any tumor. We present the results of a phase I clinical trial using an autologous dendritic cell (DC) vaccine pulsed with lysate derived from a GBM stem-like cell line. PATIENTS AND METHODS: Patients with newly diagnosed and recurrent GBM were enrolled as separate cohorts. Eligibility criteria included a qualifying surgical resection or minimal tumor size, ≤ 4-mg dexamethasone daily dose, and Karnofsky score ≥70. Vaccine treatment consisted of two phases: an induction phase with vaccine given weekly for 4 weeks, and a maintenance phase with vaccines administered every 8 weeks until depletion of supply or disease progression. Patients with newly diagnosed GBM also received standard-of-care radiation and temozolomide. The primary objective for this open-label, single-institution trial was to assess the safety and tolerability of the autologous DC vaccine. RESULTS: For the 11 patients with newly diagnosed GBM, median progression-free survival (PFS) was 8.75 months, and median overall survival was 20.36 months. For the 25 patients with recurrent GBM, median PFS was 3.23 months, 6-month PFS was 24%, and median survival was 11.97 months. A subset of patients developed a cytotoxic T-cell response as determined by an IFNγ ELISpot assay. CONCLUSIONS: In this trial, treatment of newly diagnosed and recurrent GBM with autologous DC vaccine pulsed with lysate derived from an allogeneic stem-like cell line was safe and well tolerated. Clinical outcomes add to the body of evidence suggesting that immunotherapy plays a role in the treatment of GBM.

A Proposed Brain-, Spine-, and Mental- Health Screening Methodology (NEUROSCREEN) for Healthcare Systems: Position of the Society for Brain Mapping and Therapeutics.

The COVID-19 pandemic has accelerated neurological, mental health disorders, and neurocognitive issues. However, there is a lack of inexpensive and efficient brain evaluation and screening systems. As a result, a considerable fraction of patients with neurocognitive or psychobehavioral predicaments either do not get timely diagnosed or fail to receive personalized treatment plans. This is especially true in the elderly populations, wherein only 16% of seniors say they receive regular cognitive evaluations. Therefore, there is a great need for development of an optimized clinical brain screening workflow methodology like what is already in existence for prostate and breast exams. Such a methodology should be designed to facilitate objective early detection and cost-effective treatment of such disorders. In this paper we have reviewed the existing clinical protocols, recent technological advances and suggested reliable clinical workflows for brain screening. Such protocols range from questionnaires and smartphone apps to multi-modality brain mapping and advanced imaging where applicable. To that end, the Society for Brain Mapping and Therapeutics (SBMT) proposes the Brain, Spine and Mental Health Screening (NEUROSCREEN) as a multi-faceted approach. Beside other assessment tools, NEUROSCREEN employs smartphone guided cognitive assessments and quantitative electroencephalography (qEEG) as well as potential genetic testing for cognitive decline risk as inexpensive and effective screening tools to facilitate objective diagnosis, monitor disease progression, and guide personalized treatment interventions. Operationalizing NEUROSCREEN is expected to result in reduced healthcare costs and improving quality of life at national and later, global scales.

CD133 mRNA-transfected dendritic cells induce coordinated cytotoxic and helper T cell responses against breast cancer stem cells.

Breast cancer, a leading cause of death yearly, has been shown to be initiated and propagated by cancer stem cells. CD133, a cell surface antigen, has been shown to be present on cancer stem cells of many solid tumors, including breast cancer. A limitation to targeting CD133 is major histocompatibility complex (MHC)-restricted presentation of epitopes, leading to activation of only one arm of the immune system: either CD4+ helper T cells or CD8+ cytotoxic T cells. Thus, we hypothesized that by creating an MHC-independent vaccination, we would give rise to a sustained immune response against CD133 in triple-negative breast cancer (TNBCs). We transfected CD133 mRNA into dendritic cells and then tested this in animal models of TNBC. We showed in these models the activation of both CD8+ cytotoxic T cells and CD4+ helper T cells by dendritic cell vaccination with modified CD133 mRNA, with subsequent decrease in tumor growth. This study for the first time demonstrates in a syngeneic mouse model of TNBC that targeting CD133, in an MHC-independent manner, is an effective strategy against the cancer stem cell population, leading to tumor abrogation.

Elevated Asparagine Biosynthesis Drives Brain Tumor Stem Cell Metabolic Plasticity and Resistance to Oxidative Stress.

Asparagine synthetase (ASNS) is a gene on the long arm of chromosome 7 that is copy-number amplified in the majority of glioblastomas. ASNS copy-number amplification is associated with a significantly decreased survival. Using patient-derived glioma stem cells (GSC), we showed that significant metabolic alterations occur in gliomas when perturbing the expression of ASNS, which is not merely restricted to amino acid homeostasis. ASNS-high GSCs maintained a slower basal metabolic profile yet readily shifted to a greatly increased capacity for glycolysis and oxidative phosphorylation when needed. This led ASNS-high cells to a greater ability to proliferate and spread into brain tissue. Finally, we demonstrate that these changes confer resistance to cellular stress, notably oxidative stress, through adaptive redox homeostasis that led to radiotherapy resistance. Furthermore, ASNS overexpression led to modifications of the one-carbon metabolism to promote a more antioxidant tumor environment revealing a metabolic vulnerability that may be therapeutically exploited. IMPLICATIONS: This study reveals a new role for ASNS in metabolic control and redox homeostasis in glioma stem cells and proposes a new treatment strategy that attempts to exploit one vulnerable metabolic node within the larger multilayered tumor network.

A Crowdsourced Consensus on Supratotal Resection Versus Gross Total Resection for Anatomically Distinct Primary Glioblastoma.

BACKGROUND: Gross total resection (GTR) of contrast-enhancing tumor is associated with increased survival in primary glioblastoma. Recently, there has been increasing interest in performing supratotal resections (SpTRs) for glioblastoma. OBJECTIVE: To address the published results, which have varied in part due to lack of consensus on the definition and appropriate use of SpTR. METHODS: A crowdsourcing approach was used to survey 21 neurosurgical oncologists representing 14 health systems nationwide. Participants were presented with 11 definitions of SpTR and asked to rate the appropriateness of each definition. Participants reviewed T1-weighed postcontrast and fluid-attenuated inversion-recovery magnetic resonance imaging for 22 anatomically distinct glioblastomas. Participants were asked to assess the tumor location's eloquence, the perceived equipoise of enrolling patients in a randomized trial comparing gross total to SpTR, and their personal treatment plans. RESULTS: Most neurosurgeons surveyed (n = 18, 85.7%) agree that GTR plus resection of some noncontrast enhancement is an appropriate definition for SpTR. Overall, moderate inter-rater agreement existed regarding eloquence, equipoise, and personal treatment plans. The 4 neurosurgeons who had performed >10 SpTRs for glioblastomas in the past year were more likely to recommend it as their treatment plan (P < .005). Cases were divided into 3 anatomically distinct groups based upon perceived eloquence. Anterior temporal and right frontal glioblastomas were considered the best randomization candidates. CONCLUSION: We established a consensus definition for SpTR of glioblastoma and identified anatomically distinct locations deemed most amenable to SpTR. These results may be used to plan prospective trials investigating the potential clinical utility of SpTR for glioblastoma.

Topography of transcriptionally active chromatin in glioblastoma.

Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer-driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM.

Antigen-specific T-cell response from dendritic cell vaccination using cancer stem-like cell-associated antigens.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, with current treatment remaining palliative. Immunotherapies harness the body's own immune system to target cancers and could overcome the limitations of conventional treatments. One active immunotherapy strategy uses dendritic cell (DC)-based vaccination to initiate T-cell-mediated antitumor immunity. It has been proposed that cancer stem-like cells (CSCs) may play a key role in cancer initiation, progression, and resistance to current treatments. However, whether using human CSC antigens may improve the antitumor effect of DC vaccination against human cancer is unclear. In this study, we explored the suitability of CSCs as sources of antigens for DC vaccination again human GBM, with the aim of achieving CSC-targeting and enhanced antitumor immunity. We found that CSCs express high levels of tumor-associated antigens as well as major histocompatibility complex molecules. Furthermore, DC vaccination using CSC antigens elicited antigen-specific T-cell responses against CSCs. DC vaccination-induced interferon-gamma production is positively correlated with the number of antigen-specific T cells generated. Finally, using a 9L CSC brain tumor model, we demonstrate that vaccination with DCs loaded with 9L CSCs, but not daughter cells or conventionally cultured 9L cells, induced cytotoxic T lymphocytes (CTLs) against CSCs, and prolonged survival in animals bearing 9L CSC tumors. Understanding how immunization with CSCs generates superior antitumor immunity may accelerate development of CSC-specific immunotherapies and cancer vaccines.

Oxidative stimuli-responsive nanoprodrug of camptothecin kills glioblastoma cells.

The purpose of this study was to prepare and characterize nanometer-sized prodrug (nanoprodrug) of camptothecin. The camptothecin prodrug was synthesized using tetraethylene glycol spacer linked via carbonate bond to camptothecin and via ester bond to alpha-lipoic acid. The nanoprodrug was prepared through the spontaneous emulsification mechanism using the mixture of camptothecin prodrug and alpha-tocopherol which served as a structural matrix. The nanoprodrug was activated readily by porcine liver esterase and, with a much slower rate, by hydrolytic degradation. Upon longterm storage, the alpha-lipoic acid moiety of the camptothecin prodrug gradually oxidized without loss of structural integrity and therapeutic efficacy. Interestingly, the hydrolytic activation was negligible before the oxidation, but was significantly accelerated after the oxidation of the alpha-lipoic acid moiety, suggesting an oxidative stimuli-responsive activation of the prodrug. The camptothecin nanoprodrug was found to possess significant inhibitory effect on the proliferation of U87-MG glioma cells with an IC(50) of 20 nM.