Multi-omic approach identifies hypoxic tumor-associated myeloid cells that drive immunobiology of high-risk pediatric ependymoma.

Ependymoma (EPN) is a devastating childhood brain tumor. Single-cell analyses have illustrated the cellular heterogeneity of EPN tumors, identifying multiple neoplastic cell states including a mesenchymal-differentiated subpopulation which characterizes the PFA1 subtype. Here, we characterize the EPN immune environment, in the context of both tumor subtypes and tumor cell subpopulations using single-cell sequencing (scRNAseq, n = 27), deconvolution of bulk tumor gene expression (n = 299), spatial proteomics (n = 54), and single-cell cytokine release assays (n = 12). We identify eight distinct myeloid-derived subpopulations from which a group of cells, termed hypoxia myeloid cells, demonstrate features of myeloid-derived suppressor cells, including IL6/STAT3 pathway activation and wound healing ontologies. In PFA tumors, hypoxia myeloid cells colocalize with mesenchymal-differentiated cells in necrotic and perivascular niches and secrete IL-8, which we hypothesize amplifies the EPN immunosuppressive microenvironment. This myeloid cell-driven immunosuppression will need to be targeted for immunotherapy to be effective in this difficult-to-cure childhood brain tumor. [Abstract copyright: © 2023 The Author(s).

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Integration of single-nuclei RNA-sequencing, spatial transcriptomics and histochemistry defines the complex microenvironment of NF1-associated plexiform neurofibromas

Abstract Plexiform neurofibroma (PN) is a leading cause of morbidity in children with the genetic condition Neurofibromatosis Type 1 (NF1), often disfiguring or threatening vital structures. During formation of PN, a complex tumor microenvironment (TME) develops, with recruitment of neoplastic and non-neoplastic cell types being critical for growth and progression. Due to the cohesive cellularity of PN, single-cell RNA-sequencing is difficult and may result in a loss of detection of critical cellular subpopulations. To bypass this barrier, we performed single-nuclei RNA-sequencing (snRNA-seq) on 8 frozen PN samples, and integrated this with spatial transcriptomics (ST) in 4 PN samples and immunohistochemistry to provide morphological context to transcriptomic data. SnRNA-seq analysis definitively charted the heterogeneous cellular subpopulations in the PN TME, with the predominant fraction being fibroblast subtypes. PN showed a remarkable amount of inter-sample homogeneity regarding cellular subpopulation proportions despite being resected from a variety of anatomical locations. ST analysis identified distinct cellular subpopulations which were annotated using snRNA-seq data and correlated with histological features. Schwann cell/fibroblast interactions were identified by receptor/ligand interaction analysis demonstrating a high probability of Neurexin 1/Neuroligin 1 (NRXN1/NLGN1) receptor-ligand cross-talk predicted between fibroblasts and non-myelinated Schwann cells (NM-SC) and subtypes, respectively. We observed aberrant expression of NRXN1 and NLGN1 in our PN snRNA-seq data compared to a normal mouse sciatic nerve single-cell RNA-seq dataset. This pathway has never been described in PN and may indicate a clear and direct communication pathway between putative NM-SC cells of origin and surrounding fibroblasts, potentially driving disease progression. SnRNA-seq integrated with spatial transcriptomics advances our understanding of the complex cellular heterogeneity of PN TME and identify potential novel communication pathways that may drive disease progression, a finding that could provide translational therapy options for patients with these devastating tumors of childhood and early adulthood

Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism

Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons

Spatial transcriptomic analysis delineates epithelial and mesenchymal subpopulations and transition stages in childhood ependymoma

Background The diverse cellular constituents of childhood brain tumor ependymoma, recently revealed by single cell RNA-sequencing, may underly therapeutic resistance. Here we use spatial transcriptomics to further advance our understanding of the tumor microenvironment, mapping cellular subpopulations to the tumor architecture of ependymoma posterior fossa subgroup A (PFA), the commonest and most deadly childhood ependymoma variant. Methods Spatial transcriptomics data from intact PFA sections was deconvoluted to resolve the histological arrangement of neoplastic and non-neoplastic cell types. Key findings were validated using immunohistochemistry, in vitro functional assays and outcome analysis in clinically-annotated PFA bulk transcriptomic data. Results PFA are comprised of epithelial and mesenchymal histological zones containing a diversity of cellular states, each zone including co-existing and spatially distinct undifferentiated progenitor-like cells; a quiescent mesenchymal zone population, and a second highly mitotic progenitor population that is restricted to hypercellular epithelial zones and that is more abundant in progressive tumors. We show that myeloid cell interaction is the leading cause of mesenchymal transition in PFA, occurring in zones spatially distinct from hypoxia-induced mesenchymal transition, and these distinct EMT-initiating processes were replicated using in vitro models of PFA. Conclusions These insights demonstrate the utility of spatial transcriptomics to advance our understanding of ependymoma biology, revealing a clearer picture of the cellular constituents of PFA, their interactions and influence on tumor progression

Molecular Analyses Reveal Inflammatory Mediators in the Solid Component and Cyst Fluid of Human Adamantinomatous Craniopharyngioma

Pediatric adamantinomatous craniopharyngioma (ACP) is a highly solid and cystic tumor, often causing substantial damage to critical neuroendocrine structures such as the hypothalamus, pituitary gland, and optic apparatus. Paracrine signaling mechanisms driving tumor behavior have been hypothesized, with IL-6R overexpression identified as a potential therapeutic target. To identify potential novel therapies, we characterized inflammatory and immunomodulatory factors in ACP cyst fluid and solid tumor components. Cytometric bead analysis revealed a highly pro-inflammatory cytokine pattern in fluid from ACP compared to fluids from another cystic pediatric brain tumor, pilocytic astrocytoma. Cytokines and chemokines with particularly elevated concentrations in ACPs were IL-6, CXCL1 (GRO), CXCL8 (IL-8) and the immunosuppressive cytokine IL-10. These data were concordant with solid tumor compartment transcriptomic data from a larger cohort of ACPs, other pediatric brain tumors and normal brain. The majority of receptors for these cytokines and chemokines were also over-expressed in ACPs. In addition to IL-10, the established immunosuppressive factor IDO-1 was overexpressed by ACPs at the mRNA and protein levels. These data indicate that ACP cyst fluids and solid tumor components are characterized by an inflammatory cytokine and chemokine expression pattern. Further study regarding selective cytokine blockade may inform novel therapeutic interventions

Targeting integrated epigenetic and metabolic pathways in lethal childhood PFA ependymomas

Childhood posterior fossa group A ependymomas (PFAs) have limited treatment options and bear dismal prognoses compared to group B ependymomas (PFBs). PFAs overexpress the oncohistone-like protein EZHIP (enhancer of Zeste homologs inhibitory protein), causing global reduction of repressive histone H3 lysine 27 trimethylation (H3K27me3), similar to the oncohistone H3K27M. Integrated metabolic analyses in patient-derived cells and tumors, single-cell RNA sequencing of tumors, and noninvasive metabolic imaging in patients demonstrated enhanced glycolysis and tricarboxylic acid (TCA) cycle metabolism in PFAs. Furthermore, high glycolytic gene expression in PFAs was associated with a poor outcome. PFAs demonstrated high EZHIP expression associated with poor prognosis and elevated activating mark histone H3 lysine 27 acetylation (H3K27ac). Genomic H3K27ac was enriched in PFAs at key glycolytic and TCA cycle–related genes including hexokinase-2 and pyruvate dehydrogenase. Similarly, mouse neuronal stem cells (NSCs) expressing wild-type EZHIP (EZHIP-WT) versus catalytically attenuated EZHIP-M406K demonstrated H3K27ac enrichment at hexokinase-2 and pyruvate dehydrogenase, accompanied by enhanced glycolysis and TCA cycle metabolism. AMPKα-2, a key component of the metabolic regulator AMP-activated protein kinase (AMPK), also showed H3K27ac enrichment in PFAs and EZHIP-WT NSCs. The AMPK activator metformin lowered EZHIP protein concentrations, increased H3K27me3, suppressed TCA cycle metabolism, and showed therapeutic efficacy in vitro and in vivo in patient-derived PFA xenografts in mice. Our data indicate that PFAs and EZHIP-WT–expressing NSCs are characterized by enhanced glycolysis and TCA cycle metabolism. Repurposing the antidiabetic drug metformin lowered pathogenic EZHIP, increased H3K27me3, and suppressed tumor growth, suggesting that targeting integrated metabolic/epigenetic pathways is a potential therapeutic strategy for treating childhood ependymomas