Spatially resolved multi-omics deciphers bidirectional tumor-host interdependence in glioblastoma.

Glioblastomas are malignant tumors of the central nervous system hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies. The source of dynamic reorganization within the spatial context of these tumors remains elusive. Here, we characterized glioblastomas by spatially resolved transcriptomics, metabolomics, and proteomics. By deciphering regionally shared transcriptional programs across patients, we infer that glioblastoma is organized by spatial segregation of lineage states and adapts to inflammatory and/or metabolic stimuli, reminiscent of the reactive transformation in mature astrocytes. Integration of metabolic imaging and imaging mass cytometry uncovered locoregional tumor-host interdependence, resulting in spatially exclusive adaptive transcriptional programs. Inferring copy-number alterations emphasizes a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. A model of glioblastoma stem cells implanted into human and rodent neocortical tissue mimicking various environments confirmed that transcriptional states originate from dynamic adaptation to various environments.

T-cell dysfunction in the glioblastoma microenvironment is mediated by myeloid cells releasing interleukin-10.

Despite recent advances in cancer immunotherapy, certain tumor types, such as Glioblastomas, are highly resistant due to their tumor microenvironment disabling the anti-tumor immune response. Here we show, by applying an in-silico multidimensional model integrating spatially resolved and single-cell gene expression data of 45,615 immune cells from 12 tumor samples, that a subset of Interleukin-10-releasing HMOX1+ myeloid cells, spatially localizing to mesenchymal-like tumor regions, drive T-cell exhaustion and thus contribute to the immunosuppressive tumor microenvironment. These findings are validated using a human ex-vivo neocortical glioblastoma model inoculated with patient derived peripheral T-cells to simulate the immune compartment. This model recapitulates the dysfunctional transformation of tumor infiltrating T-cells. Inhibition of the JAK/STAT pathway rescues T-cell functionality both in our model and in-vivo, providing further evidence of IL-10 release being an important driving force of tumor immune escape. Our results thus show that integrative modelling of single cell and spatial transcriptomics data is a valuable tool to interrogate the tumor immune microenvironment and might contribute to the development of successful immunotherapies.

Inhibition of metabotropic glutamate receptor III facilitates sensitization to alkylating chemotherapeutics in glioblastoma.

Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.

Cyclooxygenase (COX) Inhibition by Acetyl Salicylic Acid (ASA) Enhances Antitumor Effects of Nitric Oxide in Glioblastoma In Vitro.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with a high recurrence rate and a median survival of about 16 months even with multimodal therapy. Novel experimental strategies against malignant gliomas include cyclooxygenase (COX) inhibition and nitric oxide (NO)-based therapies. Therapeutic doses of NO can be delivered to tumor cells by NO donors such as JS-K (O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) which releases NO upon enzymatic activation by glutathione S-transferase. COX-2 is frequently overexpressed in tumors and increases tumor invasiveness and angiogenesis. In this study, we show that pretreatment with acetyl salicylic acid (ASA) enhanced the cytotoxic antitumor effect of NO in vitro. Combination of low doses of JS-K and ASA revealed a dose-dependent synergistic increase of necrotic cell death under circumvention of classical apoptosis and alteration of the metabolic calcium level. These findings provide an opportunity to improve currently used therapeutic strategies in the treatment of gliomas with a well-established remedy.

ATF3 reduces migration capacity by regulation of matrix metalloproteinases via NFκB and STAT3 inhibition in glioblastoma.

Glioblastoma is associated with poor survival and a high recurrence rate in patients due to inevitable uncontrolled infiltrative tumor growth. The elucidation of the molecular mechanisms may offer opportunities to prevent relapses. In this study we investigated the role of the activating transcription factor 3 (ATF3) in migration of GBM cells in vitro. RNA microarray revealed that gene expression of ATF3 is induced by a variety of chemotherapeutics and experimental agents such as the nitric oxide donor JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate). We found NFκB and STAT3 to be downstream targets inhibited by overexpression of ATF3. We demonstrate that ATF3 is directly involved in the regulation of matrix metalloproteinase expression and activation. Overexpression of ATF3 therefore leads to a significantly reduced migration capacity and induction of tissue inhibitors of matrix metalloproteinases. Our study for the first time identifies ATF3 as a potential novel therapeutic target in glioblastoma.