Transcriptomic and Proteomic Spatial Profiling of Pediatric and Adult Diffuse Midline Glioma H3 K27-Altered, Reveals Region Specific Differences and Limited Overlap between mRNA and Protein.

Diffuse midline glioma, H3 K27 -altered (DMG-Alt) are highly aggressive malignancies of the central nervous system (CNS) that primarily affect the pediatric population. Large scale spatial transcriptomic studies have implicated that tumor microenvironmental landscape plays an important role in determining the phenotypic differences in tumor presentation and clinical course, however, data connecting overall transcriptomic changes to the protein level is lacking. The NanoString GeoMx™ Digital Spatial Profiler platform was used to determine the spatial transcriptomic and proteomic landscape in a cohort of both pediatric and adult H3 K27 -altered DMG biopsy samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells ( H3 K27M ) and a nucleic acid stain (SYTO-13) were used to establish regions of interest (ROI) for genomic and proteomic analysis. We found genetic alterations within the tumor which can be delineated across patient age and spatial location. We show that the H3 K27M mutation itself has a profound impact on tumor cells transcriptomics and interestingly we found limited fidelity between overall transcriptome and proteome. Our data also validate the previously described OPC like precursor signature at the proteomic level and reveal a special shift in the signature based on the local TME composition.

Genomic analysis of human brain metastases treated with stereotactic radiosurgery reveals unique signature based on treatment failure.

Stereotactic radiosurgery (SRS) has been shown to be efficacious for the treatment of limited brain metastasis (BM); however, the effects of SRS on human brain metastases have yet to be studied. We performed genomic analysis on resected brain metastases from patients whose resected lesion was previously treated with SRS. Our analyses demonstrated for the first time that patients possess a distinct genomic signature based on type of treatment failure including local failure, leptomeningeal spread, and radio-necrosis. Examination of the center and peripheral edge of the tumors treated with SRS indicated differential DNA damage distribution and an enrichment for tumor suppressor mutations and DNA damage repair pathways along the peripheral edge. Furthermore, the two clinical modalities used to deliver SRS, LINAC and GK, demonstrated differential effects on the tumor landscape even between controlled primary sites. Our study provides, in human, biological evidence of differential effects of SRS across BM's.

Spatial transcriptomics in glioblastoma: is knowing the right zip code the key to the next therapeutic breakthrough?

Spatial transcriptomics, the technology of visualizing cellular gene expression landscape in a cells native tissue location, has emerged as a powerful tool that allows us to address scientific questions that were elusive just a few years ago. This technological advance is a decisive jump in the technological evolution that is revolutionizing studies of tissue structure and function in health and disease through the introduction of an entirely new dimension of data, spatial context. Perhaps the organ within the body that relies most on spatial organization is the brain. The central nervous system's complex microenvironmental and spatial architecture is tightly regulated during development, is maintained in health, and is detrimental when disturbed by pathologies. This inherent spatial complexity of the central nervous system makes it an exciting organ to study using spatial transcriptomics for pathologies primarily affecting the brain, of which Glioblastoma is one of the worst. Glioblastoma is a hyper-aggressive, incurable, neoplasm and has been hypothesized to not only integrate into the spatial architecture of the surrounding brain, but also possess an architecture of its own that might be actively remodeling the surrounding brain. In this review we will examine the current landscape of spatial transcriptomics in glioblastoma, outline novel findings emerging from the rising use of spatial transcriptomics, and discuss future directions and ultimate clinical/translational avenues.

Genomic Analysis of Human Brain Metastases Treated with Stereotactic Radiosurgery Under the Phase-II Clinical Trial (NCT03398694) Reveals DNA Damage Repair at the Peripheral Tumor Edge.

Stereotactic Radiosurgery (SRS) is one of the leading treatment modalities for oligo brain metastasis (BM), however no comprehensive genomic data assessing the effect of radiation on BM in humans exist. Leveraging a unique opportunity, as part of the clinical trial (NCT03398694), we collected post-SRS, delivered via Gamma-knife or LINAC, tumor samples from core and peripheral-edges of the resected tumor to characterize the genomic effects of overall SRS as well as the SRS delivery modality. Using these rare patient samples, we show that SRS results in significant genomic changes at DNA and RNA levels throughout the tumor. Mutations and expression profiles of peripheral tumor samples indicated interaction with surrounding brain tissue as well as elevated DNA damage repair. Central samples show GSEA enrichment for cellular apoptosis while peripheral samples carried an increase in tumor suppressor mutations. There are significant differences in the transcriptomic profile at the periphery between Gamma-knife vs LINAC.

GM-CSF and IL-7 fusion cytokine engineered tumor vaccine generates long-term Th-17 memory cells and increases overall survival in aged syngeneic mouse models of glioblastoma.

Age-related immune dysfunctions, such as decreased T-cell output, are closely related to pathologies like cancers and lack of vaccine efficacy among the elderly. Engineered fusokine, GIFT-7, a fusion of interleukin 7 (IL-7) and GM-CSF, can reverse aging-related lymphoid organ atrophy. We generated a GIFT-7 fusokine tumor vaccine and employed it in aged syngeneic mouse models of glioblastoma and found that peripheral vaccination with GIFT-7TVax resulted in thymic regeneration and generated durable long-term antitumor immunity specifically in aged mice. Global cytokine analysis showed increased pro-inflammatory cytokines including IL-1ß in the vaccinated group that resulted in hyperactivation of dendritic cells. In addition, GIFT-7 vaccination resulted in increased T-cell trafficking to the brain and robust Th-17 long-term effector memory T-cell formation. TCR-seq analysis showed increased productive frequency among detected rearrangements within the vaccinated group. Overall, our data demonstrate that aging immune system can be therapeutically augmented to generate lasting antitumor immunity.

Ribonucleotide reductase regulatory subunit M2 drives glioblastoma TMZ resistance through modulation of dNTP production.

During therapy, adaptations driven by cellular plasticity are partly responsible for driving the inevitable recurrence of glioblastoma (GBM). To investigate plasticity-induced adaptation during standard-of-care chemotherapy temozolomide (TMZ), we performed in vivo single-cell RNA sequencing in patient-derived xenograft (PDX) tumors of GBM before, during, and after therapy. Comparing single-cell transcriptomic patterns identified distinct cellular populations present during TMZ therapy. Of interest was the increased expression of ribonucleotide reductase regulatory subunit M2 (RRM2), which we found to regulate dGTP and dCTP production vital for DNA damage response during TMZ therapy. Furthermore, multidimensional modeling of spatially resolved transcriptomic and metabolomic analysis in patients' tissues revealed strong correlations between RRM2 and dGTP. This supports our data that RRM2 regulates the demand for specific dNTPs during therapy. In addition, treatment with the RRM2 inhibitor 3-AP (Triapine) enhances the efficacy of TMZ therapy in PDX models. We present a previously unidentified understanding of chemoresistance through critical RRM2-mediated nucleotide production.

The CXCL16-CXCR6 axis in glioblastoma modulates T-cell activity in a spatiotemporal context.

Introduction: Glioblastoma multiforme (GBM) pathobiology is characterized by its significant induction of immunosuppression within the tumor microenvironment, predominantly mediated by immunosuppressive tumor-associated myeloid cells (TAMCs). Myeloid cells play a pivotal role in shaping the GBM microenvironment and influencing immune responses, with direct interactions with effector immune cells critically impacting these processes. Methods: Our study investigates the role of the CXCR6/CXCL16 axis in T-cell myeloid interactions within GBM tissues. We examined the surface expression of CXCL16, revealing its limitation to TAMCs, while microglia release CXCL16 as a cytokine. The study explores how these distinct expression patterns affect T-cell engagement, focusing on the consequences for T-cell function within the tumor environment. Additionally, we assessed the significance of CXCR6 expression in T-cell activation and the initial migration to tumor tissues. Results: Our data demonstrates that CXCL16 surface expression on TAMCs results in predominant T-cell engagement with these cells, leading to impaired T-cell function within the tumor environment. Conversely, our findings highlight the essential role of CXCR6 expression in facilitating T-cell activation and initial migration to tumor tissues. The CXCL16-CXCR6 axis exhibits dualistic characteristics, facilitating the early stages of the T-cell immune response and promoting T-cell infiltration into tumors. However, once inside the tumor, this axis contributes to immunosuppression. Discussion: The dual nature of the CXCL16-CXCR6 axis underscores its potential as a therapeutic target in GBM. However, our results emphasize the importance of carefully considering the timing and context of intervention. While targeting this axis holds promise in combating GBM, the complex interplay between TAMCs, microglia, and T cells suggests that intervention strategies need to be tailored to optimize the balance between promoting antitumor immunity and preventing immunosuppression within the dynamic tumor microenvironment.

Alcohol's effects during uncertain and uncontrollable stressors in the laboratory.

Alcohol's effects on reactivity to stressors depends on the nature of the stressor and the reactivity being assessed. Research identifying characteristics of stressors that modulate reactivity and clarifies the neurobehavioral, cognitive, and affective components of this reactivity may help prevent, reduce or treat the negative impacts of acute and chronic alcohol use with implications for other psychopathology involving maladaptive reactivity to stressors. We used a novel, multi-measure, cued electric shock stressor paradigm in a greater university community sample of adult recreational drinkers to test how alcohol (N=64), compared to No-alcohol (N=64), effects reactivity to stressors that vary in both their perceived certainty and controllability. Preregistered analyses suggested alcohol significantly dampened subjective anxiety (self-report) and defensive reactivity (startle potentiation) more during uncertain than during certain stressors regardless of controllability, suggesting that stressor uncertainty -but not uncontrollability- may be sufficient to enhance alcohol's stress reactivity dampening and thus negative reinforcement potential.

Sexual dimorphism of the immune system predicts clinical outcomes in glioblastoma immunotherapy: A systematic review and meta-analysis.

Background: Biological differences based on sex have been documented throughout the scientific literature. Glioblastoma (GBM), the most common primary malignant brain tumor in adults, has a male sex incidence bias, however, no clinical trial data examining differential effects of treatment between sexes currently exists. Method: We analyzed genomic data, as well as clinical trials, to delineate the effect of sex on the immune system and GBM outcome following immunotherapy. Results: We found that in general females possess enriched immunological signatures on gene set enrichment analysis, which also stratified patient survival when delineated by sex. Female GBM patients treated with immunotherapy had a statistically significant survival advantage at the 1-year compared to males (relative risk [RR] = 1.15; P = .0241). This effect was even more pronounced in vaccine-based immunotherapy (RR = 1.29; P = .0158). Conclusions: Our study shows a meaningful difference in the immunobiology between males and females that also influences the overall response to immunotherapy in the setting of GBM.

Surgical Neuro-Oncology: Management of Glioma.

Gliomas are the most common intrinsic brain tumor in adults. Although maximal tumor resection improves survival, this must be balanced with preservation of neurologic function. Technological advancements have greatly expanded our ability to safely maximize tumor resection and design innovative therapeutic trials that take advantage of intracavitary delivery of therapeutic agents after resection. In this article, we review the role of surgical intervention for both low-grade and high-grade gliomas and the innovations that are driving and expanding the role of surgery in this therapeutically challenging group of malignancies.

Neuroinflammation in Autoimmune Disease and Primary Brain Tumors: The Quest for Striking the Right Balance.

According to classical dogma, the central nervous system (CNS) is defined as an immune privileged space. The basis of this theory was rooted in an incomplete understanding of the CNS microenvironment, however, recent advances such as the identification of resident dendritic cells (DC) in the brain and the presence of CNS lymphatics have deepened our understanding of the neuro-immune axis and revolutionized the field of neuroimmunology. It is now understood that many pathological conditions induce an immune response in the CNS, and that in many ways, the CNS is an immunologically distinct organ. Hyperactivity of neuro-immune axis can lead to primary neuroinflammatory diseases such as multiple sclerosis and antibody-mediated encephalitis, whereas immunosuppressive mechanisms promote the development and survival of primary brain tumors. On the therapeutic front, attempts are being made to target CNS pathologies using various forms of immunotherapy. One of the most actively investigated areas of CNS immunotherapy is for the treatment of glioblastoma (GBM), the most common primary brain tumor in adults. In this review, we provide an up to date overview of the neuro-immune axis in steady state and discuss the mechanisms underlying neuroinflammation in autoimmune neuroinflammatory disease as well as in the development and progression of brain tumors. In addition, we detail the current understanding of the interactions that characterize the primary brain tumor microenvironment and the implications of the neuro-immune axis on the development of successful therapeutic strategies for the treatment of CNS malignancies.

Aging- and Tumor-Mediated Increase in CD8+CD28- T Cells Might Impose a Strong Barrier to Success of Immunotherapy in Glioblastoma.

Clinical use of various forms of immunotherapeutic drugs in glioblastoma (GBM), has highlighted severe T cell dysfunction such as exhaustion in GBM patients. However, reversing T cell exhaustion using immune checkpoint inhibitors in GBM clinical trials has not shown significant overall survival benefit. Phenotypically, CD8+ T cells with downregulated CD28 coreceptors, low CD27 expression, increased CD57 expression, and telomere shortening are classified as senescent T cells. These senescent T cells are normally seen as part of aging and also in many forms of solid cancers. Absence of CD28 on T cells leads to several functional irregularities including reduced TCR diversity, incomplete activation of T cells, and defects in Ag-induced proliferation. In the context of GBM, presence and/or function of these CD8+CD28- T cells is unknown. In this clinical correlative study, we investigated the effect of aging as well as tumor microenvironment on CD8+ T cell phenotype as an indicator of its function in GBM patients. We systematically analyzed and describe a large population of CD8+CD28- T cells in both the blood and tumor-infiltrating lymphocytes of GBM patients. We found that phenotypically these CD8+CD28- T cells represent a distinct population compared with exhausted T cells. Comparative transcriptomic and pathway analysis of CD8+CD28- T cell populations in GBM patients revealed that tumor microenvironment might be influencing several immune related pathways and thus further exaggerating the age associated immune dysfunction in this patient population.

A Comparison Between Chemo-Radiotherapy Combined With Immunotherapy and Chemo-Radiotherapy Alone for the Treatment of Newly Diagnosed Glioblastoma: A Systematic Review and Meta-Analysis.

BACKGROUND: Immunotherapy for GBM is an emerging field which is increasingly being investigated in combination with standard of care treatment options with variable reported success rates. OBJECTIVE: To perform a systematic review of the available data to evaluate the safety and efficacy of combining immunotherapy with standard of care chemo-radiotherapy following surgical resection for the treatment of newly diagnosed GBM. METHODS: A literature search was performed for published clinical trials evaluating immunotherapy for GBM from January 1, 2000, to October 1, 2020, in PubMed and Cochrane using PICOS/PRISMA/MOOSE guidelines. Only clinical trials with two arms (combined therapy vs. control therapy) were included. Outcomes were then pooled using weighted random effects model for meta-analysis and compared using the Wald-type test. Primary outcomes included 1-year overall survival (OS) and progression-free survival (PFS), secondary outcomes included severe adverse events (SAE) grade 3 or higher. RESULTS: Nine randomized phase II and/or III clinical trials were included in the analysis, totaling 1,239 patients. The meta-analysis revealed no statistically significant differences in group's 1-year OS [80.6% (95% CI: 68.6%-90.2%) vs. 72.6% (95% CI: 65.7%-78.9%), p = 0.15] or in 1-year PFS [37% (95% CI: 26.4%-48.2%) vs. 30.4% (95% CI: 25.4%-35.6%) p = 0.17] when the immunotherapy in combination with the standard of care group (combined therapy) was compared to the standard of care group alone (control). Severe adverse events grade 3 to 5 were more common in the immunotherapy and standard of care group than in the standard of care group (47.3%, 95% CI: 20.8-74.6%, vs 43.8%, 95% CI: 8.7-83.1, p = 0.81), but this effect also failed to reach statistical significance. CONCLUSION: Our results suggests that immunotherapy can be safely combined with standard of care chemo-radiotherapy without significant increase in grade 3 to 5 SAE; however, there is no statistically significant increase in overall survival or progression free survival with the combination therapy.

De novo purine biosynthesis is a major driver of chemoresistance in glioblastoma.

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.

LNX1 Modulates Notch1 Signaling to Promote Expansion of the Glioma Stem Cell Population during Temozolomide Therapy in Glioblastoma.

Glioblastoma (GBM) is the most common primary brain malignancy in adults, with a 100% recurrence rate and 21-month median survival. Our lab and others have shown that GBM contains a subpopulation of glioma stem cells (GSCs) that expand during chemotherapy and may contribute to therapeutic resistance and recurrence in GBM. To investigate the mechanism behind this expansion, we applied gene set expression analysis (GSEA) to patient-derived xenograft (PDX) cells in response to temozolomide (TMZ), the most commonly used chemotherapy against GBM. Results showed significant enrichment of cancer stem cell and cell cycle pathways (False Discovery Rate (FDR) < 0.25). The ligand of numb protein 1 (LNX1), a known regulator of Notch signaling by targeting negative regulator Numb, is strongly upregulated after TMZ therapy (p < 0.0001) and is negatively correlated with survival of GBM patients. LNX1 is also upregulated after TMZ therapy in multiple PDX lines with concomitant downregulations in Numb and upregulations in intracellular Notch1 (NICD). Overexpression of LNX1 results in Notch1 signaling activation and increased GSC populations. In contrast, knocking down LNX1 reverses these changes, causing a significant downregulation of NICD, reduction in stemness after TMZ therapy, and resulting in more prolonged median survival in a mouse model. Based on this, we propose that during anti-GBM chemotherapy, LNX1-regulated Notch1 signaling promotes stemness and contributes to therapeutic resistance.

Temozolomide Treatment Increases Fatty Acid Uptake in Glioblastoma Stem Cells.

Among all cancers, glioblastoma (GBM) remains one of the least treatable. One key factor in this resistance is a subpopulation of tumor cells termed glioma stem cells (GSCs). These cells are highly resistant to current treatment modalities, possess marked self-renewal capacity, and are considered key drivers of tumor recurrence. Further complicating an understanding of GBM, evidence shows that the GSC population is not a pre-ordained and static group of cells but also includes previously differentiated GBM cells that have attained a GSC state secondary to environmental cues. The metabolic behavior of GBM cells undergoing plasticity remains incompletely understood. To that end, we probed the connection between GSCs, environmental cues, and metabolism. Using patient-derived xenograft cells, mouse models, transcriptomics, and metabolic analyses, we found that cell state changes are accompanied by sharp changes in metabolic phenotype. Further, treatment with temozolomide, the current standard of care drug for GBM, altered the metabolism of GBM cells and increased fatty acid uptake both in vitro and in vivo in the plasticity driven GSC population. These results indicate that temozolomide-induced changes in cell state are accompanied by metabolic shifts-a potentially novel target for enhancing the effectiveness of current treatment modalities.

Chemotherapeutic Stress Induces Transdifferentiation of Glioblastoma Cells to Endothelial Cells and Promotes Vascular Mimicry.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor affecting adults, with a median survival of approximately 21 months. One key factor underlying the limited efficacy of current treatment modalities is the remarkable plasticity exhibited by GBM cells, which allows them to effectively adapt to changes induced by anticancer therapeutics. Moreover, GBM tumors are highly vascularized with aberrant vessels that complicate the delivery of antitumor agents. Recent research has demonstrated that GBM cells have the ability to transdifferentiate into endothelial cells (ECs), illustrating that GBM cells may use plasticity in concert with vascularization leading to the creation of tumor-derived blood vessels. The mechanism behind this transdifferentiation, however, remains unclear. Here, we show that treatment with temozolomide (TMZ) chemotherapy induces time-dependent expression of markers for glioma stem cells (GSCs) and immature and mature ECs. In addition, GBM tumors growing as orthotopic xenografts in nude mice showed increased expression of GSC and EC markers after TMZ treatment. Ex vivo FACS analysis showed the presence of immature and mature EC populations. Furthermore, immunofluorescence analysis revealed increased tumor-derived vessels in TMZ-recurrent tumors. Overall, this study identifies chemotherapeutic stress as a new driver of transdifferentiation of tumor cells to endothelial cells and highlights cellular plasticity as a key player in therapeutic resistance and tumor recurrence.

Regionally specified human pluripotent stem cell-derived astrocytes exhibit different molecular signatures and functional properties.

Astrocytes display diverse morphologies in different regions of the central nervous system. Whether astrocyte diversity is attributable to developmental processes and bears functional consequences, especially in humans, is unknown. RNA-seq of human pluripotent stem cell-derived regional astrocytes revealed distinct transcript profiles, suggesting differential functional properties. This was confirmed by differential calcium signaling as well as effects on neurite growth and blood-brain barrier formation. Distinct transcriptional profiles and functional properties of human astrocytes generated from regionally specified neural progenitors under the same conditions strongly implicate the developmental impact on astrocyte diversity. These findings provide a rationale for renewed examination of regional astrocytes and their role in the pathogenesis of psychiatric and neurological disorders.

Interleukin-8/CXCR2 signaling regulates therapy-induced plasticity and enhances tumorigenicity in glioblastoma.

Emerging evidence reveals enrichment of glioma-initiating cells (GICs) following therapeutic intervention. One factor known to contribute to this enrichment is cellular plasticity-the ability of glioma cells to attain multiple phenotypes. To elucidate the molecular mechanisms governing therapy-induced cellular plasticity, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression analysis (gene microarray analysis) during treatment with standard of care temozolomide (TMZ) chemotherapy. Analysis revealed significant enhancement of open-chromatin marks in known astrocytic enhancers for interleukin-8 (IL-8) loci as well as elevated expression during anti-glioma chemotherapy. The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project data demonstrated that IL-8 transcript expression is negatively correlated with GBM patient survival (p = 0.001) and positively correlated with that of genes associated with the GIC phenotypes, such as KLF4, c-Myc, and HIF2α (p < 0.001). Immunohistochemical analysis of patient samples demonstrated elevated IL-8 expression in about 60% of recurrent GBM tumors relative to matched primary tumors and this expression also positively correlates with time to recurrence. Exposure to IL-8 significantly enhanced the self-renewing capacity of PDX GBM (average threefold, p < 0.0005), as well as increasing the expression of GIC markers in the CXCR2 population. Furthermore, IL-8 knockdown significantly delayed PDX GBM tumor growth in vivo (p < 0.0005). Finally, guided by in silico analysis of TCGA data, we examined the effect of therapy-induced IL-8 expression on the epigenomic landscape of GBM cells and observed increased trimethylation of H3K9 and H3K27. Our results show that autocrine IL-8 alters cellular plasticity and mediates alterations in histone status. These findings suggest that IL-8 signaling participates in regulating GBM adaptation to therapeutic stress and therefore represents a promising target for combination with conventional chemotherapy in order to limit GBM recurrence.