Tumor-associated Astrocytes Research Articles

Medulloblastoma Spatial Transcriptomics Reveals Tumor Microenvironment Heterogeneity with High-Density Progenitor Cell Regions Correlating with High-Risk Disease.

The tumor microenvironment (TME) of medulloblastoma (MB) influences progression and therapy response, presenting a promising target for therapeutic advances. Prior single-cell analyses have characterized the cellular components of the TME but lack spatial context. To address this, we performed spatial transcriptomic sequencing on sixteen pediatric MB samples obtained at diagnosis, including two matched diagnosis-relapse pairs. Our analyses revealed inter- and intra-tumoral heterogeneity within the TME, comprised of tumor-associated astrocytes (TAAs), macrophages (TAMs), stromal components, and distinct subpopulations of MB cells at different stages of neuronal differentiation and cell cycle progression. We identified dense regions of quiescent progenitor-like MB cells enriched in patients with high-risk (HR) features and an increase in TAAs, TAMs, and dysregulated vascular endothelium following relapse. Our study presents novel insights into the spatial architecture and cellular landscape of the medulloblastoma TME, highlighting spatial patterns linked to HR features and relapse, which may serve as potential therapeutic targets.

MDB-106. MEDULLOBLASTOMA TUMOR MICROENVIRONMENTS SHOW INTRA- AND INTER-TUMORAL HETEROGENEITY ON SPATIAL TRANSCRIPTOMICS

Abstract BACKGROUND Medulloblastoma defines a heterogeneous set of neuroepithelial neoplasms of the posterior fossa. Four groups of medulloblastoma are recognized: wingless (WNT), sonic hedgehog (SHH), group 3, and group 4. The tumor microenvironment (TME) influences tumor progression and response to therapy and has emerged as a growing target for the study of novel therapeutic approaches. METHODS We show spatial gene expression architecture through 10X Visium Spatial Sequencing on 16 formalin-fixed paraffin-embedded (FFPE) samples representing all molecular groups of medulloblastoma. Spatial transcriptomics data is correlated with clinical parameters, including M-stage at diagnosis, tumor histological classification, and outcomes data including survival and relapse status. RESULTS Spatial sequencing demonstrates both intra- and inter-tumoral TME heterogeneity across molecular subgroups. Unsupervised clustering and uniform manifold approximate projection illustrate clusters of distinct spatial gene expression representing cell states from different components of the TME, including tumor-associated astrocytes (TAA), macrophages (TAM), and vascular endothelium. Medulloblastoma cells constitute the majority of cells and express marker genes representing different stages of neuronal differentiation and cell cycle. Pathway analysis reveals the enrichment of pathways associated with cell motility (RHO GTPase, MET, MAPK signaling), heat shock response, growth factor signaling, and adaptive immune response (TLR, MHC-II signaling). In addition, molecular pathways known in medulloblastoma, including NF-kB and TP53 regulatory pathways, were also enriched. Neighborhood-enrichment analysis reveals the co-localization of TAMs and TAAs in close spatial relationship with mitotic progenitor-like medulloblastoma cells. Additionally, these two cell types constitute a greater proportion of the TME in patients at relapse compared to initial diagnosis. CONCLUSION Spatial transcriptomics enables new insight into the heterogeneity of the medulloblastoma TME. Notably, our analysis elucidates the spatial association of TAMs and TAAs with proliferating tumor cells and an increased abundance of these cell types following relapse. These initial results support the role of TAMs and TAAs in medulloblastoma progression.

Single-cell transcriptomic sequencing identifies subcutaneous patient-derived xenograft recapitulated medulloblastoma.

Medulloblastoma (MB) is one of the most common malignant brain tumors that mainly affect children. Various approaches have been used to model MB to facilitate investigating tumorigenesis. This study aims to compare the recapitulation of MB between subcutaneous patient-derived xenograft (sPDX), intracranial patient-derived xenograft (iPDX), and genetically engineered mouse models (GEMM) at the single-cell level. We obtained primary human sonic hedgehog (SHH) and group 3 (G3) MB samples from six patients. For each patient specimen, we developed two sPDX and iPDX models, respectively. Three Patch+/- GEMM models were also included for sequencing. Single-cell RNA sequencing was performed to compare gene expression profiles, cellular composition, and functional pathway enrichment. Bulk RNA-seq deconvolution was performed to compare cellular composition across models and human samples. Our results showed that the sPDX tumor model demonstrated the highest correlation to the overall transcriptomic profiles of primary human tumors at the single-cell level within the SHH and G3 subgroups, followed by the GEMM model and iPDX. The GEMM tumor model was able to recapitulate all subpopulations of tumor microenvironment (TME) cells that can be clustered in human SHH tumors, including a higher proportion of tumor-associated astrocytes and immune cells, and an additional cluster of vascular endothelia when compared to human SHH tumors. This study was the first to compare experimental models for MB at the single-cell level, providing value insights into model selection for different research purposes. sPDX and iPDX are suitable for drug testing and personalized therapy screenings, whereas GEMM models are valuable for investigating the interaction between tumor and TME cells.

The Role of Astrocytes in Promoting Glioblastoma Pathogenicity

For this review, we study the ways that astrocytes lead to increased glioblastoma (GBM) cancer cell survival by interacting with the tumor microenvironment (TME). GBM cells exploit and alter surrounding somatic cells, such as astrocytes, to fuel their growth and metastasis. Astrocytes are the most abundant cells in the central nervous system (CNS) and occupy important roles in maintaining the blood-brain barrier and stabilizing synapses. Tumor-associated astrocytes (TAAs) help tumor progression by interacting with players of the TME. In summary, astrocytes support GBM pathogenicity by transferring mitochondria and cholesterol and directly promoting immunosuppression through the modulation of tumor-associated macrophages (TAMs). Targeting non-GBM cells such as astrocytes, which directly promotes its development, could become a new option for treating the lethal GBM disease. A further understanding of the interaction involved in astrocyte-driven GBM pathogenicity could identify promising molecular targets and effective strategies against GBM.

Glioma stem cell-derived exosomes induce the transformation of astrocytes via the miR-3065-5p/DLG2 signaling axis.

Tumor-associated astrocytes (TAAs) in the glioblastoma microenvironment play an important role in tumor development and malignant progression initiated by glioma stem cells (GSCs). In the current study, normal human astrocytes (NHAs) were cultured and continuously treated with GSC-derived exosomes (GSC-EXOs) induction to explore the mechanism by which GSCs affect astrocyte remodeling. This study revealed that GSC-EXOs can induce the transformation of NHAs into TAAs, with relatively swollen cell bodies and multiple extended processes. In addition, high proliferation, elevated resistance to temozolomide (TMZ), and increased expression of TAA-related markers (TGF-β, CD44, and tenascin-C) were observed in the TAAs. Furthermore, GSC-derived exosomal miR-3065-5p could be delivered to NHAs, and miR-3065-5p levels increased significantly in TAAs, as verified by miRNA expression profile sequencing and Reverse transcription polymerase chain reaction. Overexpression of miR-3065-5p also enhanced NHA proliferation, elevated resistance to TMZ, and increased the expression levels of TAA-related markers. In addition, both GSC-EXO-induced and miR-3065-5p-overexpressing NHAs promoted tumorigenesis of GSCs in vivo. Discs Large Homolog 2 (DLG2, downregulated in glioblastoma) is a direct downstream target of miR-3065-5p in TAAs, and DLG2 overexpression could partially reverse the transformation of NHAs into TAAs. Collectively, these data demonstrate that GSC-EXOs induce the transformation of NHAs into TAAs via the miR-3065-5p/DLG2 signaling axis and that TAAs can further promote the tumorigenesis of GSCs. Thus, precisely blocking the interactions between astrocytes and GSCs via exosomes may be a novel strategy to inhibit glioblastoma development, but more in-depth mechanistic studies are still needed.

Tumor-associated astrocytes promote tumor progression of Sonic Hedgehog medulloblastoma by secreting lipocalin-2.

Sonic Hedgehog (SHH) subgroup of medulloblastoma (MB) accounts for about 25% of all subgroups of MB. Tumor microenvironment (TME) may play a key role in the tumor progression and therapeutic resistance. Tumor-associated astrocytes (TAAs) are reshaped to drive tumor progression through multiple paracrine signals. However, the mechanism by which TAAs modulate MB cells remains elusive. Here, we illuminated that TAAs showed a specific and dynamic pattern during SHH-MB development. Most TAAs gathered to the tumor margin during the tumor progression, rather than evenly distributed in the early-stage tumors. We further demonstrated that lipocalin-2 (LCN2) secreted by TAAs could promote the tumor growth and was correlated with the poor prognosis of MB patients. Knocking down LCN2 in TAAs in vitro impeded the proliferation and migration abilities of MB cells. In addition, we identified that TAAs accelerated the tumor growth by secreting LCN2 via STAT3 signaling pathway. Accordingly, blockade of STAT3 signaling by its inhibitor WP1066 and AAV-Lcn2 shRNA, respectively, in TAAs abrogated the effects of LCN2 on tumor progression in vitro and in vivo. In summary, we for the first time clarified that LCN2, secreted by TAAs, could promote MB tumor progression via STAT3 pathway and has potential prognostic value. Our findings unveiled a new sight in reprogramming the TME of SHH-MB and provided a potential therapeutic strategy targeting TAAs.

OS10.7.A TUMOR-ASSOCIATED ASTROCYTES PROMOTE TUMOR PROGRESSION OF SONIC HEDGEHOG MEDULLOBLASTOMA BY SECRETING LCN2

Abstract BACKGROUND Sonic Hedgehog (SHH) subgroup of medulloblastoma (MB) accounts for about 25% of all subgroups of MB. Tumor microenvironment (TME) may play a key role in the tumor progression and therapeutic resistance. Tumor-associated astrocytes (TAAs) are reshaped to drive tumor progression through multiple paracrine signals. However, the mechanism by which TAAs modulate MB cells remains elusive. MATERIAL AND METHODS We explored the function of TAAs of SHH-MB in transgenic mouse model and co-culture system by a series of molecular biological techniques. To illuminate the TAAs distribution pattern during tumor progression of MB, we firstly detected MB transgenic mice bearing tumor at different time points by nuclear Magnetic resonance imaging (MRI). Histologic sections were then evaluated under microscope after H&E staining. The RNA-sequencing was also performed to evaluate differential expression between normal astrocytes (ASs) and tumor-associated astrocytes (TAAs). Bio-informatic analysis was followed to find the specific gene expressed in TAAs. Drug treatment of TAA inhibitor was finally applied in the animal model to clarify its effects on tumor progression. RESULTS We illuminated that TAAs showed a specific and dynamic pattern during SHH-MB development. Most TAAs gathered to the tumor margin during the tumor progression, rather than evenly distributed in the early-stage tumors and dissemination foci. We further demonstrated that LCN2 secreted by TAAs could promote the tumor growth and was correlated with the poor prognosis of MB patients. Knocking down LCN2 in TAAs in vitro impeded the proliferation and migration abilities of MB cells. In addition, we identified that TAAs accelerated the tumor growth by secreting LCN2 via STAT3 signaling pathway. Accordingly, blockade of STAT3 signaling by its inhibitor WP1066 in TAAs abrogated the effects of LCN2 on tumor progression in vitro and in vivo. CONCLUSION We for the first time clarified that LCN2, secreted by TAAs, could promote MB tumor progression via STAT3 pathway and has potential prognostic value. Our findings unveiled a new sight in reprogramming the TME of SHH-MB and provided a potential therapeutic strategy targeting TAAs.

CNSC-09. THE NEUROENDOCRINE FACTOR VGF IS NECESSARY FOR THE MAINTENANCE OF THE TUMOR STEM CELL POPULATION IN GLIOBLASTOMA

Abstract A major factor that contributes to the unchecked growth and recurrence of glioblastoma (GBM) tumors is the presence of a heterogeneous population of GBM stem-like cells (GSCs) that continuously replenish the tumor mass. Targeting strategies against this key cell population have rarely considered the trophic signals from peritumoral neural cells, which can protect the tumor cells during therapy and therefore promote recurrence. Here, we have focused on the little-known secreted factor VGF and its derived neuropeptides, which are largely restricted to the CNS and mediate paracrine communication between neural cells. We analyzed over 200 tissue specimens and cultured GBM cells, demonstrating that VGF is upregulated in malignant gliomas and expressed in GSCs of different subtypes, although it is markedly reduced in differentiated GBM cells. More importantly, we detected VGF expression in peritumoral astrocytes, even though this neuroendocrine factor has never been described in glial cells in normal brain. Co-culture experiments and scRNAseq data from clinical tumors confirmed that VGF is expressed in tumor-associated astrocytes, suggesting that these cells are a paracrine source of VGF in GBM. Accordingly, purified exogenous VGF increased the proliferation of GSCs and rescued GSC viability when the tumor cells were treated with the chemotherapeutic temozolomide. Moreover, purified VGF decreased the expression of inflammatory cytokines in activated macrophages, suggesting that this factor may also contribute to local immunosuppression in gliomas. Conversely, knockdown of VGF in GSCs was sufficient to decrease their viability, self-renewal, and expression of stemness-related genes. At the molecular level, VGF increased intracellular calcium and Akt/mTOR signaling in GSCs, suggesting a possible G protein coupled receptor-mediated mechanism of action. Taken together, our results explore for the first time the role of brain-derived VGF in GBM progression, highlighting the importance of disrupting the communication between the tumor and its microenvironment to achieve long-term anti-tumor efficacy.

P12.15.B Astrocyte immunometabolic regulation of the glioblastoma microenvironment drives tumor pathogenicity

Abstract Background Malignant brain tumors are the cause of a disproportionate level of morbidity and mortality among cancer patients, an unfortunate statistic that has remained constant for decades. Despite considerable advances in the molecular characterization of these tumors, targeting the cancer cells has yet to produce significant advances in treatment. An alternative strategy is to target cells in the glioblastoma microenvironment, such as tumor associated astrocytes. Astrocytes control multiple processes in health and disease, ranging from maintaining the brain's metabolic homeostasis, to modulating neuroinflammation. However, their role in glioblastoma pathogenicity is not well understood. Material and Methods Immunocompetent mice were implanted with murine glioma cell lines and the role of astrocyte in the tumor pathogenicity was analyzed, and further investigated using in-vitro co-cultures. Results Here we report that depletion of reactive astrocytes regresses glioblastoma and prolongs mouse survival. Analysis of the tumor-associated astrocyte translatome, revealed that astrocytes initiate transcriptional programs that shape the immune and metabolic compartments in the glioma microenvironment. Specifically, their expression of CCL2 and CSF1 governs the recruitment of tumor-associated macrophages and promotes a pro-tumorigenic macrophage phenotype. Concomitantly, we demonstrate that astrocyte-derived cholesterol is key to glioma cell survival, and that targeting astrocytic cholesterol efflux, via ABCA1, halts tumor progression. In summary, astrocytes control glioblastoma pathogenicity by reprogramming the immunological properties of the tumor microenvironment and supporting the non-oncogenic metabolic dependency of glioblastoma on cholesterol. Conclusion These findings suggest that targeting astrocyte immunometabolic signaling may help treat this uniformly lethal brain tumor.

Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α

BackgroundMedulloblastoma (MB) is the most common malignant brain tumor in children. Approximately one-third of MB patients remain incurable. Understanding the molecular mechanism of MB tumorigenesis is, therefore, critical for developing specific and effective treatment strategies. Our previous work demonstrated that astrocytes constitute the tumor microenvironment (TME) of MB and play an indispensable role in MB progression. However, the underlying mechanisms by which astrocytes are regulated and activated to promote MB remain elusive.MethodsBy taking advantage of Math1-Cre/Ptch1loxp/loxp mice, which spontaneously develop MB, primary MB cells and astrocytes were isolated and then subjected to administration and coculture in vitro. Immunohistochemistry was utilized to determine the presence of C3a in MB sections. MB cell proliferation was evaluated by immunofluorescent staining. GFAP and cytokine expression levels in C3a-stimulated astrocytes were assessed by immunofluorescent staining, western blotting, q-PCR and ELISA. C3a receptor and TNF-α receptor expression was determined by PCR and immunofluorescent staining. p38 MAPK pathway activation was detected by western blotting. Transplanted MB mice were treated with a C3a receptor antagonist or TNF-α receptor antagonist to investigate their role in MB progression in vivo.ResultsWe found that complement C3a, a fragment released from intact complement C3 following complement activation, was enriched in both human and murine MB tumor tissue, and its receptor was highly expressed on tumor-associated astrocytes (TAAs). We demonstrated that C3a activated astrocytes and promoted MB cell proliferation via the p38 MAPK pathway. Moreover, we discovered that C3a upregulated the production of proinflammatory cytokines, such as IL-6 and TNF-α in astrocytes. Application of the conditioned medium of C3a-stimulated astrocytes promoted MB cell proliferation, which was abolished by preincubation with a TNF-α receptor antagonist, indicating a TNF-α-dependent event. Indeed, we further demonstrated that administration of a selective C3a receptor or TNF-α receptor antagonist to mice subcutaneously transplanted with MB suppressed tumor progression in vivo.ConclusionsC3a was released during MB development. C3a triggered astrocyte activation and TNF-α production via the p38 pathway, which promoted MB cell proliferation. Our findings revealed the novel role of C3a-mediated TNF-α production by astrocytes in MB progression. These findings imply that targeting C3a and TNF-α may represent a potential novel therapeutic approach for human MB.

Abstract 2354: Astrocytes activated by tGLI1-expressing breast cancer brain metastases upregulate CNTF to activate astrocytes and promote breast cancer stem cells

Abstract Breast cancer is the most frequently diagnosed cancer, and second most common source of brain metastasis in women. Despite recent advances in detection and therapeutics, patients with breast cancer brain metastases (BCBM) survive only 6-18 months after diagnosis. HER2-enriched breast cancer and triple-negative breast cancer (TNBC) subtypes have the highest propensity to metastasize to the brain. Mechanisms that drive BCBM remain unclear, contributing to limited effective treatments and poor prognoses for HER2-enriched breast cancer and TNBC patients. Truncated glioma-associated oncogene homolog 1 (tGLI1), a gain-of-function GLI1 transcription factor discovered in our lab, promotes BCBM of circulating tumor cells in vivo. We previously reported that tGLI1 enriches the breast cancer stem cell (BCSC) subpopulation, and that tGLI1-positive BCSCs strongly activate astrocytes, the most abundant glial cell type in the brain. The mechanisms by which tGLI1-activated astrocytes promote BCBM have not been investigated. Since tumor-associated astrocytes have been reported to secrete specific cytokines, such as ciliary neurotrophic factor (CNTF), to activate astrocytes and cancer cells by binding to IL-6R and CNTF receptor-alpha (CNTFRα), we determined whether astrocytes activated by tGLI1-positive breast cancer cells secrete elevated levels of CNTF. Results of CNTF ELISA revealed that conditioned media from tGLI1-positive breast cancer cells increases the ability of astrocytes to secrete CNTF compared to GLI1-positive or control cells. Furthermore, we found that exogenous CNTF significantly activates astrocytes and promotes BCSCs, important mediators of tumor progression and metastasis in breast cancer. Analysis of patient datasets reveals CNTFRα is more highly expressed in brain metastases compared to breast cancer and normal breast tissues, suggesting that CNTF secreted by activated astrocytes can promote the growth of BCSCs in the brain. We further found that CNTF and CNTFRα gene signatures are significantly associated with worse brain metastasis-free survival, suggesting an important role for the tGLI1-CNTF-CNTFRα pathway in BCBM development. In summary, our study demonstrates for the first time that astrocytes activated by tGLI1-expressing BCBM secrete CNTF, CNTF further activates astrocytes and promotes BCSCs, and that this tumor-astrocyte interaction could be a novel mechanism for BCBM development and progression. Citation Format: Grace L. Wong, Sherona R. Sirkisoon, Noah R. Aguayo, Daniel L. Doheny, Dongqin Zhu, Angelina T. Regua, Austin Arrigo, Sara G. Manore, Calvin J. Wagner, Alexandra Thomas, Ravi Singh, Fei Xing, Guangxu Jin, Kounosuke Watabe, Hui-Wen Lo. Astrocytes activated by tGLI1-expressing breast cancer brain metastases upregulate CNTF to activate astrocytes and promote breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2354.

Tumor Microenvironment in Glioma Invasion.

A major malignant trait of gliomas is their remarkable infiltration capacity. When glioma develops, the tumor cells have already reached the distant part. Therefore, complete removal of the glioma is impossible. Recently, research on the involvement of the tumor microenvironment in glioma invasion has advanced. Local hypoxia triggers cell migration as an environmental factor. The transcription factor hypoxia-inducible factor (HIF) -1α, produced in tumor cells under hypoxia, promotes the transcription of various invasion related molecules. The extracellular matrix surrounding tumors is degraded by proteases secreted by tumor cells and simultaneously replaced by an extracellular matrix that promotes infiltration. Astrocytes and microglia become tumor-associated astrocytes and glioma-associated macrophages/microglia, respectively, in relation to tumor cells. These cells also promote glioma invasion. Interactions between glioma cells actively promote infiltration of each other. Surgery, chemotherapy, and radiation therapy transform the microenvironment, allowing glioma cells to invade. These findings indicate that the tumor microenvironment may be a target for glioma invasion. On the other hand, because the living body actively promotes tumor infiltration in response to the tumor, it is necessary to reconsider whether the invasion itself is friend or foe to the brain.

Abstract 3170: Targeting tumor-associated astrocyte dependence in glioblastoma treatment

Abstract Glioblastoma (GBM) is the most common malignant primary brain tumor and remains challenging to treat due to its aggressive nature with high rate of recurrence. Despite advances in therapy, prognosis is poor with median survival of 19 months with maximal therapy. The normal astrocytes within the GBM TME undergo a phenotypic shift, promoting alterations supportive of tumor growth. There is increasing attention on the role of tumor-associated astrocytes (TAA) in GBM progression and resistance to therapy. Previous studies assessing astrocyte support of GBM employ astrocytes derived from murine or fetal sources. We have isolated TAAs from patients with GBM as well as normal human astrocytes (NHA) from cancer-free patients undergoing surgical treatment of epilepsy. The cells are maintained at a low passage number (<10 passes). Our data emphasize the supportive role these cells in GBM progression and resistance to GBM therapy. Briefly, conditioned media (CM) from non-malignant, GFAP positive TAAs, demonstrate a propensity to induce invasion and migration of U87 GBM cells compared to the NHA CM. In addition, TAA CM significantly increased the IC50 dose of U87 cells treated with temozolomide from 536 μM to 1351 μM demonstrating treatment resistance (p<0.001). TAA CM induced temozolomide resistance of the GBM cells can be abolished with supra-therapeutic dosing. RNA sequencing (RNAseq) of two TAA and three NHA samples revealed 399 differentially expressed genes identified with 38 of those upregulated genes coding extracellular products. Expression data was verified by quantitative polymerase chain reaction (qPCR), thus validating the differential expression profile generated by RNAseq. The differential expression data was explored further using Ingenuity Pathway Analysis (IPA) and gene set enrichment analysis (GSEA). Attention was focused on genes with excreted factors as products or involved in the regulation of critical excreted products. We identified and validated high expression of IL1β in TAAs compared to NHAs. RNAseq data revealed multiple potential pathways for targeted therapies aimed at modulating the effect of TAAs in the tumor microenvironment. In ongoing studies, we will determine the efficacy of IL1β in attenuating the TAA-induced temozolomide resistance in GBM. These studies reveal for the first time a highly clinically relevant approach to attenuate TAA-facilitated GBM progression and therapy resistance. Citation Format: Joseph S. Domino, Anthony Alvarado, Levi Arnold, Sumedha Gunewardena, David Akhavan, Sufi M. Thomas. Targeting tumor-associated astrocyte dependence in glioblastoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3170.

Hypoxia-Induced Reactivity of Tumor-Associated Astrocytes Affects Glioma Cell Properties.

Glioblastoma is characterized by extensive necrotic areas with surrounding hypoxia. The cancer cell response to hypoxia in these areas is well-described; it involves a metabolic shift and an increase in stem cell-like characteristics. Less is known about the hypoxic response of tumor-associated astrocytes, a major component of the glioma tumor microenvironment. Here, we used primary human astrocytes and a genetically engineered glioma mouse model to investigate the response of this stromal cell type to hypoxia. We found that astrocytes became reactive in response to intermediate and severe hypoxia, similarly to irradiated and temozolomide-treated astrocytes. Hypoxic astrocytes displayed a potent hypoxia response that appeared to be driven primarily by hypoxia-inducible factor 2-alpha (HIF-2α). This response involved the activation of classical HIF target genes and the increased production of hypoxia-associated cytokines such as TGF-β1, IL-3, angiogenin, VEGF-A, and IL-1 alpha. In vivo, astrocytes were present in proximity to perinecrotic areas surrounding HIF-2α expressing cells, suggesting that hypoxic astrocytes contribute to the glioma microenvironment. Extracellular matrix derived from hypoxic astrocytes increased the proliferation and drug efflux capability of glioma cells. Together, our findings suggest that hypoxic astrocytes are implicated in tumor growth and potentially stemness maintenance by remodeling the tumor microenvironment.

STEM-30. NECROPTOTIC ASTROCYTES CONTRIBUTE TO MAINTAINING STEMNESS OF DISSEMINATED MEDULLOBLASTOMA THROUGH CCL2 SECRETION

Abstract Medulloblastoma (MB) with metastases at diagnosis and recurrence correlates with poor prognosis. Unfortunately, the molecular mechanism underlying metastases growth has received less attention than primary therapy-naïve MB. Though astrocytes have been frequently detected in brain tumors, their roles in regulating the stemness properties of MB stem-like cells (MBSCs) in disseminated lesions remain elusive. Effects of tumor-associated astrocytes (TAA)-secreted CCL2 on MBSCs self-renewal was determined by immunostaining analysis. Necroptosis of TAA was examined by measuring necrosome activity. Alterations in Notch signaling were examined after inhibition of CCL2. Progression of MBSCs-derived tumors was evaluated after pharmaceutical blockage of necroptosis. TAA, as the essential components of disseminated tumor, produced high level of CCL2 to shape inflammation microenvironment, which stimulated the enrichment of MBSCs in disseminated MB. In particular, CCL2 played a pivotal role in maintaining stem-like properties via JAK2/STAT3 mediated activation of Notch signaling. Loss of CCL2/CCR2 function repressed JAK2/STAT3-Notch pathway and impaired MBSCs proliferation, leading to a dramatic reduction of stemness, tumorigenicity and metastasizing capability. Furthermore, necroptosis-induced CCL2 release depended on RIP1/RIP3/MLKL activation in TAA, which promoted the oncogenic phenotype. Blockade of necroptosis resulted in CCL2 deprivation and compromised MBSCs self-proliferation, indicating MBSCs outsourced CCL2 from necroptotic TAA. Finally, CCL2 was upregulated in high-risk stages of MB, further supporting its value as a prognostic indicator. These findings highlighted the critical role of CCL2/CCR2 in Notch signaling activation in MBSCs, and revealed a necroptosis-associated glial cytokine microenvironment driving stemness maintenance in disseminations.

TAMI-53. THE ONCOMETABOLITE D-2HG INDUCE INFLAMMATORY ASTROGLIOSIS CAUSING NEUROTOXICITY AND SEIZURES IN IDH MUTATED GLIOMA

Abstract The role of tumor-associated astrocytes in the microenvironment of glioma has long been underestimated but is moving into the focus of current research. We explored the role of reactive astrocytes in IDH-mutated glioma using RNA-sequencing of purified astrocytes and microglia and single-nucleus RNA-sequencing of infiltrating tumor regions. Mapping of the transcriptional phenotype of astrocytes along developmental and reactive trajectories revealed an inflammatory transformation of IDH-mutated associated astrocytes. The major proportion of astrocytes is marked by complement-activation similar to findings in neuroinflammatory diseases. A human neocortical slices model with injected IDH-mutated patient-derived cells or D-2HG treatment (+/- microglia depletion) was used to map shared and unique transcriptional adaptation in astrocytes promoted by either tumor cells or metabolic alteration. High-dimensional electrophysiological profiling was used to investigate alterations in neural response to tumor-induced microenvironmental transformation. We showed that 2HG alone promote the inflammatory pattern of astrocytes, which causes neurotoxicity and seizures in our neocortical slice model. Depletion of microglia rescued the neurotoxicity suggesting that microglia predominantly drive inflammatory astrogliosis as a response to metabolic alteration the tumor environment. We showed that neurotoxic astrogliosis induced by the oncometabolite D-2HG via distinct microglia activation promote the evolution of frequently observed seizures in IDH-mutated glioma patients.

TAMI-05. THE IRRADIATED BRAIN MICROENVIRONMENT SUPPORTS GLIOMA STEMNESS AND SURVIVAL VIA ASTROCYTE-DERIVED TRANSGLUTAMINASE 2

Abstract The highest-grade gliomas invariably recur as incurable tumors following standard of care comprising surgery, radiotherapy, and chemotherapy. The majority of the recurrent tumors form within the area of the brain receiving high-dose irradiation during treatment of the primary tumor, indicating that the recurrent tumor forms in an irradiated microenvironment. The tumor microenvironment has been demonstrated to influence the therapeutic response and stemness characteristics of tumor cells, but the influence of radiation on the microenvironment and its subsequent consequences for tumor cells are incompletely understood. Here, we used genetically engineered glioma mouse models and human glioma samples to characterize the impact of standard of care radiotherapy on the brain tumor microenvironment. We found that tumor-associated astrocytes subjected to radiation in vitro could enhance tumor cell stemness and survival of co-cultured glioma cells. More aggressive gliomas formed in vivo when mouse brains were irradiated prior to tumor cell implantation, suggesting that the irradiated brain microenvironment supports tumor growth. We isolated the effect of irradiated astrocytes to extracellular matrix secreted by these cells, and specifically found that astrocyte-derived transglutaminase 2 (TGM2) is a stromal promoter of glioma stemness and radioresistance. TGM2 levels were increased after radiation in glioma mouse models. Recombinant TGM2 enhanced, and TGM2 inhibitors blocked, glioma cell stemness. In human GBM tissue, TGM2 levels were increased in recurrent vs. primary tumors. In summary, in addition to supporting TGM2 as a potential therapeutic target in glioma, our data indicate that radiotherapy results in a tumor-supportive microenvironment, the targeting of which may be necessary to overcome tumor cell therapeutic resistance and recurrence.

Niche-derived soluble DLK1 promotes glioma growth

Tumor cell behaviors associated with aggressive tumor growth such as proliferation, therapeutic resistance, and stem cell characteristics are regulated in part by soluble factors derived from the tumor microenvironment. Tumor-associated astrocytes represent a major component of the glioma tumor microenvironment, and astrocytes have an active role in maintenance of normal neural stem cells in the stem cell niche, in part via secretion of soluble delta-like noncanonical Notch ligand 1 (DLK1). We found that astrocytes, when exposed to stresses of the tumor microenvironment such as hypoxia or ionizing radiation, increased secretion of soluble DLK1. Tumor-associated astrocytes in a glioma mouse model expressed DLK1 in perinecrotic and perivascular tumor areas. Glioma cells exposed to recombinant DLK1 displayed increased proliferation, enhanced self-renewal and colony formation abilities, and increased levels of stem cell marker genes. Mechanistically, DLK1-mediated effects on glioma cells involved increased and prolonged stabilization of hypoxia-inducible factor 2alpha, and inhibition of hypoxia-inducible factor 2alpha activity abolished effects of DLK1 in hypoxia. Forced expression of soluble DLK1 resulted in more aggressive tumor growth and shortened survival in a genetically engineered mouse model of glioma. Together, our data support DLK1 as a soluble mediator of glioma aggressiveness derived from the tumor microenvironment.

Potassium and glutamate transport is impaired in scar-forming tumor-associated astrocytes

Unprovoked recurrent seizures are a serious comorbidity affecting most patients who suffer from glioma, a primary brain tumor composed of malignant glial cells. Cellular mechanisms contributing to the development of recurrent spontaneous seizures include the release of the excitatory neurotransmitter glutamate from glioma into extracellular space. Under physiological conditions, astrocytes express two high affinity glutamate transporters, Glt-1 and Glast, which are responsible for the removal of excess extracellular glutamate. In the context of neurological disease or brain injury, astrocytes become reactive which can negatively affect neuronal function, causing hyperexcitability and/or death. Using electrophysiology, immunohistochemistry, fluorescent in situ hybridization, and Western blot analysis in different orthotopic xenograft and allograft models of human and mouse gliomas, we find that peritumoral astrocytes exhibit astrocyte scar formation characterized by proliferation, cellular hypertrophy, process elongation, and increased GFAP and pSTAT3. Overall, peritumoral reactive astrocytes show a significant reduction in glutamate and potassium uptake, as well as decreased glutamine synthetase activity. A subset of peritumoral astrocytes displayed a depolarized resting membrane potential, further contributing to reduced potassium and glutamate homeostasis. These changes may contribute to the propagation of peritumoral neuronal hyperexcitability and excitotoxic death.

TMIC-52. REACTIVE ASTROCYTES AID THE EVOLUTION OF IMMUNOSUPPRESSIVE ENVIRONMENT AND DRIVE ONCOGENIC SIGNALING IN GLIOBLASTOMA

Abstract Reactive astrocytes are caused by multiple pathologies of the central nervous system, whereby they undergo distinct transcriptomic re-programming. Although the role of reactive astrocytes in some inflammatory diseases has been investigated, many central questions regarding the immunoregulatory functions of tumor-associated astrocytes and their crosstalk to microglia remain poorly understood. In our presented study, we purified astrocytes from various pathologies and different brain tumors to map the transcriptional landscape of reactive astrocytes. We identified the marker genes CHI3L1 and CD274 highly enriched in reactive astrocytes of the marginal astrogliosis scar at the tumor boarder. Human neocortical slices along with a microglia loss-of-function model were used to explore the crosstalk of microglia and reactive astrocytes within the tumor environment. Our results revealed that the reactive phenotype mutually arises from both, microglia and tumor cells. This interaction caused JAK/STAT signalling in reactive astrocytes along with a large release of anti-inflammatory cytokines such as TGFß and IL10. Additionally, inhibition of the JAK/STAT pathway recovered the release of anti-inflammatory cytokines and resulted in a pro-inflammatory environment. Besides the immunosuppressive properties, we found evidence that reactive astrocytes drove AKT and MAPK signaling in the tumor through astrocytic released CHI3L1 and consequential binding to IL13RA2. Our findings revealed increased malignant properties arising from astrocytic-tumor interaction, which were rescued by IL13RA2 inhibition. In a nutshell, reactive astrocytes have decisive regulatory tasks in the microenvironment of CNS tumors. Along with microglia, reactive astrocytes cause the evolution of an immunosuppressive environment and support malignant properties of the tumor.