Abstract
Cellular senescence (CS) is a state of irreversible cell cycle arrest, and the expression of p16INK4a in cells is one of the reliable markers for CS. However, senescent cells are ...metabolically active with the senescence-associated secretory phenotype (SASP), which can influence the tissue microenvironment by paracrine signaling to the adjacent tumor, non-tumor, and immune cells. In the present study, we evaluated p16INK4a expression in glioblastoma and investigated its association with CS and SASPs. We analyzed the expression of p16INK4a in 73 glioblastomas by immunostaining. To examine the association of p16INK4a expression and CS, we performed senescence-associated β-galactosidase (SA-β-Gal) staining, a standard marker of CS, using glioblastoma cell lines and fresh frozen tumor tissues. For SASPs analysis, RNA sequencing with Gene Set Enrichment Analysis (GSEA) was performed. Among 73 glioblastomas, twenty-eight cases (38.4%) revealed diffuse strong p16INK4a expression in tumor cells. The glioblastoma with diffuse p16INK4a expression (GMDP) patients were younger (52.4 vs. 59.2 years) and showed prolonged overall survival (median: 25.5 vs. 12.3 months) compared to those harboring negative expression. In vitro analysis, p16INK4a over-expressed glioblastoma cell line showed increased expression of SA-β-Gal which indicates CS. In addition, fresh frozen tissues from GMDP also revealed SA-β-Gal positivity. RNA sequencing analysis revealed that splicing or protein biosynthetic genes were enriched in GMDP, and GSEA showed significant enrichment of SASP genes (false discovery rate < 0.05), especially chemokines associated with monocytes and macrophages. Our data suggest that increased expression of p16INK4a in glioblastoma is associated with tumor cell senescence, and SASPs from senescent tumor cells could be one of the crucial modulators in the tumor immune microenvironment.
Abstract
Glioblastomas (GBM) account for ~60% of adult primary brain tumors. With few advances in therapeutics, median overall survival remains 15-months post-diagnosis. Immunotherapies may provide ...therapeutic benefit; however, no predictive immune features have informed therapeutic stratification. Radiographic tumor contact with the lateral ventricle (C-GBM) correlates with 7-months worse prognosis compared to patients with ventricle non-contacting GBM (NC-GBM), yet the influence of ventricle contact on anti-tumor immunity is unknown. This study characterized the GBM immune microenvironment and identified targetable mechanisms of immunosuppression correlating with worse outcomes in C-GBM patients.Primary glioblastoma tissue was provided with written informed consent in accordance with the Declaration of Helsinki and with approval of the Vanderbilt Institutional Review Board (IRB #131870). Seventeen patients presented with primary, IDH-wildtype C-GBM and 15 with NC-GBM. Machine learning integrated mass cytometry and matched multiplex immunohistochemistry on FFPE embedded tissue to identify phenotypic, functional, and spatial biomarkers correlating with patient outcome. C-GBM tumors were enriched in STAT3-driven CD32+CD44+HLA-DR+ monocyte-derived macrophages (MDM) compared to NC-GBM (19 ± 8% vs. 6 ± 2%; p< 0.001) and depleted in lymphocytes (2.9 ± 1% vs. 7.6 ± 2%; p< 0.001) and tissue-resident microglia (1.8 ± 0.3% vs. 7 ± 3%; p< 0.001). Exhausted T cells in C-GBM co-expressed checkpoint receptors PD-1 and TIGIT. K-means clustering identified 10 immunological niches in GBM. Macrophage-tumor niches were most common in C-GBM (17.93% of niches), followed by T cell-microglia-tumor niches (17.72%). Within NC-GBM niches, T cell-T cell interactions were more prevalent (log odds ratio = 0.90) and correlated with improved outcome.These findings suggest that factors within the periventricular space negatively influence the immune microenvironment within GBM tumors. Clinically targetable immune biomarkers were identified in C-GBM. Notably, radiologic assessment of lateral ventricle contact may guide clinical trial design for immunotherapies in neuro-oncology based on tumor proximity to the ventricle wall.
Abstract
Proton therapy is an innovative radiation therapy modality that has been shown to induce higher cytotoxicity to tumor cells compared to standard x-ray therapy while sparing normal tissue. ...However, the impact of proton radiotherapy on the growth and progression of glioblastoma (GBM) has not been defined. We hypothesize that understanding tumor evolution and micro environmental responses to radiotherapy is critical for developing and targeting interventions. In this study, we investigate the effect of proton versus standard photon radiotherapy on tumor growth and the microenvironment in an orthotopic syngeneic GBM model over the course of treatment. We characterized tumor tissues at early, progressing, and terminal phases post irradiation by single cell transcriptomics, bulk RNA sequencing, and histology. We find that proton irradiation led to a drastic reduction in tumor volume at early stages before eventual recurrence. Proton treatment exhibits equivalent if not superior, tumor control when compared to normal photon radiation. Single cell transcriptomics analyses show a dramatic increase in immune cell populations including T cell and macrophage populations which peak shortly before tumor recurrence. In addition, we identify an increase in exhausted T cell markers such as PD1, Tim-3, and Lag-3 in T cells post irradiation. Treatment with therapeutic agents that reprogram microglia/macrophages to an anti-tumor phenotype and activate T cell effector functions effectively eradicate GBM recurrence post irradiation. Thus, our study demonstrates a powerful combined treatment strategy with radiation against GBM and provides valuable information about how tumor growth and microenvironment respond to proton therapies, as well as potential mechanisms of tumor response and recurrence in GBM.
Abstract
The glioblastoma microenvironment is an immunosuppressive barrier to therapeutic innovation. We hypothesized intratumor convection enhanced delivery (CED) of gene therapy vectors could ...reprogram the glioblastoma immune microenvironment and elucidate therapeutic vulnerabilities. To test this, SB28 or GL261 glioblastoma allografts were implanted into immunocompetent mice and treated with CED of attenuated adeno-associated virus 9 vectors (AAV9) encoding experimental cytokines (Il1b, Ccl4, or Apoa1) underlying infiltration or activation of anti-tumor immune cells in other intracranial tumors. Serial intracranial bioluminescence was used to assess glioblastoma growth and animals were monitored for survival. The impact of gene therapy perturbations on the glioblastoma immune microenvironment was assessed using histology, immunohistochemistry, single-cell mass cytometry (CyTOF), and multiplexed cytokine assays. Serial body weight and systemic cytokine measurements showed no evidence of treatment toxicity. AAV9-APOA1 or AAV9-IL1B gene therapy CED treatments attenuated SB28 growth and prolonged survival, decreasing immunosuppressive macrophage infiltration and increasing CD8 T cell and microglia infiltration of the glioblastoma microenvironment compared to control AAV9 vectors. Gene therapy CED treatments did not attenuate GL261 growth or prolong survival, but CyTOF of human glioblastomas (n=6) in comparison to preclinical models revealed untreated GL261 glioblastomas were endogenously enriched in CD8 T cells and other lymphoid lineages compared to untreated SB28 or human glioblastomas. Multiplexed cytokine assays demonstrated suppression of intratumor IL6 is a conserved mechanism of action underlying glioblastoma gene therapy responses. Single-cell RNA sequencing analysis of 32,877 cells from human glioblastomas (n=11) showed IL6 is predominantly produced by radial glial like cancer stem cells or endothelial cells in the tumor microenvironment. In support of these findings, survival from intracranial SB28 glioblastomas was prolonged in Il6 knockout C57BL/6J mice compared to wildtype mice. In summary, we report a novel strategy using gene therapy and CED to reprogram the glioblastoma immune microenvironment, revealing IL6 drives glioblastoma immunosuppression and growth.
Abstract
Tissue organization plays an important role in tumor development and evolution. Systematic investigation of the differences in the local composition of the tumor microenvironment (TME) in ...glioblastoma (GBM) in distinct regions of the tumor is needed for identification of traits driving tumor recurrence. Thus, we performed an analysis of three tumors, six spatially distant biopsies each, representing tumor surface, core, and deep margin. GBM cell fluorescence generated by the 5-aminolevulinic acid (5-ALA) metabolic labeling prior to surgery allowed us to enrich for the tumor cell fractions and the cells of the microenvironment, respectively, from each biopsy. Using RNA-seq and scRNA-seq we compared the transcriptomic profiles of GBM and tumor microenvironment (TME) at distinct locations within the tumor. We observed a gradient of GBM subtypes across the tumor regions, with a molecular identity progressively less defined from the tumor core towards the margins. Interestingly, the single-cell based copy number analysis revealed a subset of GBM cells that appeared to evade the 5-ALA labelling. The high prevalence of these 5-ALA-negative GBM cells within the deep tumor margins points to a metabolically distinct subpopulation that could be responsible for local tumor recurrence. The phenotypic gradient of cancer cells from tumor core towards the margins was also mirrored by tumor microenvironment-enriched fractions. Deep margin samples from the profiled tumors were transcriptionally more divergent than biopsies from other regions. Moreover, they were depleted from major populations of TME cells. In situ analysis revealed that TERT promoter mutant cells are not equally distributed across the tumor indicating the coexistence of GBM sub-clones with distinct evolutionary potential. Altogether, our results provide a novel insight into co-evolution of tumor microenvironment and GBM clonal heterogeneity within the context of the tumor tissue.
Abstract
The effects of tumor-associated neutrophils (TANs) on glioblastoma biology remain poorly understood. Flow cytometric analysis of 5 newly diagnosed glioblastoma fresh tissue specimens ...surprisingly revealed a high fraction (21.0-34.1%) of TANs expressed MHCII, a marker of antigen-presenting cells not classically associated with neutrophils and not expressed in matched peripheral blood (PBNs). Transcriptomic profiling confirmed that patient TANs upregulated expression of MHCII subunits (HLA-DR), chaperones (HLA-DM), and costimulatory ligands (CD86/83). Ex vivo cocultures further demonstrated that TANs activated patient-matched naïve T cells in an MHCII-dependent manner, while PBNs did not (CD25 MFI fold-change: 1.1 vs 3.2, p< 0.001). The antitumoral relevance of this property was confirmed in a syngeneic mouse glioma model, wherein αLy6G-mediated neutrophil depletion in T-cell-competent mice (Balb/c, n= 13) yielded endpoint tumors that had reduced CD8+ T cell infiltration (p= 0.0024) and were 2.4-fold larger by BLI (p= 0.0383) than controls, but had no bearing on tumor burden in T-cell-deficient (athymic) mice. Given the absence of MHCII+ neutrophils in circulation, we interrogated the inducibility of this phenotype by tumor-conditioned media in murine marrow/blood, finding that only immature Ly6Glow bone marrow neutrophils had sufficient plasticity to express MHCII and process foreign antigen as measured by DQ-ovalbumin uptake/proteolysis. Because of the non-inducibility of PBNs, we investigated the hypothesis that MHCII+ TANs were recruited to the glioblastoma microenvironment from adjacent skull bone marrow by labeling this space with CFMDA in tumor-implanted mice; at 72h, skull marrow-derived neutrophils contributed disproportionately to MHCII+ TANs compared to all TANs (41.7% vs 9.3%). As confirmation, in the first-ever scRNA-seq of human TANs, we demonstrated via pseudotime analysis that MHCII+ neutrophils represent a developmental lineage seen in TANs but not matched PBNs. Given the immunostimulatory effects of this population, mechanisms to promote neutrophil egress and tumor infiltration from the skull marrow may have therapeutic value.
Abstract
Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy, all patients suffer recurrence of ...treatment-resistant tumors. Previous studies from our lab showed that astrocytes become reactive and generate tumor-supportive conditions for glioma cells, when exposed to hypoxia or irradiation. Reactive astrocytes are known to upregulate proteins of the complement system, especially complement component 3 (C3), in several neurological disorders. However, it remains unexplored how these complement proteins are expressed in stromal astrocytes in glioblastoma. Tissue sections from a glioma mouse model showed presence of C3 in the invasive front, and in hypoxic, and perivascular spaces of the tumors, where there is an abundance of astrocytes. C3 expression co-localized with Nestin and CD44, two markers of mesenchymal and/or stem-like glioma cells. Primary human astrocytes grown in hypoxic conditions upregulated C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. In general, a strong correlation between hypoxia (195 genes) and complement (200 genes) gene signatures were found in TCGA GBM dataset (R= 0.82, p-value= 0). Single-cell sequencing data from primary GBM tumors showed a subpopulation of highly C3-expressing astrocytes, which were enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, complement, hypoxia, and interferon signaling. Human glioma cell lines from proneural, classical and mesenchymal subtypes all showed increased CAIX and GLUT1 expression grown under lower oxygen tensions, while two out of three subtypes showed upregulation of stemness markers (OCT4 and NANOG1) as well as C3 and C3aR. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment, where a local expression of complement proteins could possibly lead to tumor promoting signaling, which will be further explored.
Abstract
INTRODUCTION
Glioblastoma (GBM) contains cell populations with distinct metabolic requirements, with fast-cycling cells harnessing aerobic glycolysis, and treatment-resistant slow-cycling ...cells (SCCs) preferentially engaging lipid metabolism. The interaction between immune and tumor cells, and how their metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood. Objectives: The primary objective of this project is to spatially and molecularly decode the GBM microenvironment heterogeneity with a specific focus on unraveling the metabolic links that underlie the interaction of SCCs with the immune compartment.
METHODS
Multiple murine glioma cell lines coupled with geospatial profiling were used to establish metabolic heterogeneity and communications, while various genetic and pharmacological approaches were applied to assess the effect of disrupting the metabolic interplay between SCCs and the immune system.
RESULTS
We determined that SCCs exhibit distinct metabolic dependencies, involving preferential lipid metabolism supported by enhanced fatty acid uptake. We also found that SCCs exhibit specific geospatial distribution and that tumor progression is regulated by their interactions with immune suppressive cells, which in turn work against tumor immune rejection by inhibiting T cell anti-tumor activity. The immune microenvironment shaped by SCCs is marked by specific metabolic features enhancing lipid exchange capacities that are exploited by SCCs to support their survival and functions. Importantly, disrupting lipid metabolic exchange sensitized tumors to chemotherapy.
CONCLUSION
Our results reveal that metabolic interactions between SCCs and tumor-associated immune suppressive cells within the GBM microenvironment play a critical role in the development of drug and immune resistant tumors. This study delineates these metabolic communications and assesses the potential therapeutic effect of disrupting these interactions to treat GBM. The insights generated from this project uncover fundamental principles of the emerging connections between the tumor microenvironment, cell metabolism, anti-tumor immunity, and associated therapeutic vulnerabilities.
Abstract
BACKGROUND
The Hedgehog pathway directs gene expression programs that are essential for glioblastoma stem cells in vitro, but a clinical trial suggested Hedgehog pathway inhibition does not ...improve glioblastoma outcomes (ABTC-0904). In pancreatic and bladder cancer, Hedgehog signaling through the tumor microenvironment induces stromal morphogens to inhibit cancer stem cell proliferation. Thus, we hypothesized Hedgehog signaling through the glioblastoma microenvironment may inhibit glioblastoma stem cell proliferation in vivo.
METHODS
Hedgehog signaling was studied using patient-derived, fluorescently-labeled IDH wild-type glioblastoma cells (GBM6, SF11956, SF11907, GPMP004, GPMP005) in vitro, in vivo after intracranial implantation in mice, or in 3D co-culture with iPSC-derived organoids comprised of human astrocytes or neurons. Genetic loss-of-function experiments were performed using CRISPR interference. Cell proliferation, stem cell marker expression, Sonic Hedgehog (SHH) expression, or primary cilia architecture were assessed using immunofluorescence, confocal microscopy, or live-cell imaging. Hedgehog signaling or morphogen expression were assessed using QPCR or single-cell RNA sequencing. Pharmacologic experiments were performed using vismodegib to inhibit the Hedgehog pathway, or FK506 to induce stromal morphogen expression.
RESULTS
Vismodegib increased glioblastoma cell proliferation and stem cell marker expression in vivo or in co-culture with astrocyte organoids. Single-cell RNA sequencing demonstrated glioblastoma cell co-culture with astrocyte organoids induced Hedgehog target genes and stem cell markers in glioblastoma cells, or stromal morphogens in astrocytes, which also expressed primary cilia. CRISPRi suppression of SHH in glioblastoma cells increased glioblastoma cell proliferation and stem cell marker expression in co-culture with astrocyte organoids. Neither vismodegib nor SHH suppression induced glioblastoma cell proliferation or stem cell marker expression in vitro or in co-culture with neuron organoids. FK506 induced astrocyte morphogen expression, inhibiting glioblastoma cell proliferation, stem cell marker expression, and the effects of vismodegib in co-culture with astrocyte organoids.
CONCLUSIONS
SHH from glioblastoma cells signals through astrocytes to inhibit cancer stem cell proliferation.
Abstract
Background
Significant intra- and inter-heterogeneity exists in gliomas. This provides clinical, radiological, diagnostic, and treatment challenges. To date, there have been few efforts to ...comprehensively catalog information obtained in the operating room that spatially links neuro-navigation localization to imaging characteristics, gross intraoperative visual findings, and pathological/molecular features. The value of this spatial localization is probably greatest in high grade gliomas, which have been demonstrated to have intra-tumoral histological and genetic/epigenetic heterogeneity.
METHODS
An IRB-approved institutional registry of patients undergoing clinically-indicated surgery with use of an image-guidance system (IGS) was launched in November 2019 and as of June 1, 2022 includes nearly 500 patients, of which 243 were diagnosed with gliomas. Intraoperatively, locations within the gross tumor or tumor-infiltrated brain were sampled at each surgeon’s discretion, and each sample was linked to their precise location with the IGS system (“geo-tagged”). The registry includes information regarding surgeon; anesthesia technique; use of intraoperative tumor fluorescence; tumor location and volume; pathologic diagnosis and molecular features, and sample number.
RESULTS
Of 243 gliomas, 26 were low grade and 217 were high grade with 174 being glioblastoma. For enhancing tumors, volume of enhancement ranged from 0.31 to 127.0 cm3 with an average of 22.9 cm3. Tumors were widely distributed throughout the cerebrum with 133 left-sided tumors, 110 right-sided and 32 spanning multiple lobes or deep subcortical structures including the brainstem. 51% of surgeries were under awake anesthesia, and 40% were performed using fluorescence guidance. The average number of navigation image-linked samples collected per tumor was 3.67; 3.48 in low grade gliomas, and 3.69 in high grade gliomas. Samples are archived in frozen and/or formalin-fixed, paraffin-embedded formats for future research.
CONCLUSION
This registry provides the foundation for correlation of imaging, intraoperative findings, and pathology in brain tumors, and it will support detailed laboratory/translational investigations addressing tumor heterogeneity.
