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Nature Communications 13, 925 (2022). doi:10.1038/s41467-022-28523-1, Published by Nature Publishing Group UK, [London]

Glioblastomas are malignant tumors of the central nervous system hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies. The source of the dynamic reorganization within the spatial context of these tumors remains elusive. Here, we characterized glioblastomas in-depth by spatially resolved transcriptomics, metabolomics and proteomics. By deciphering regional shared transcriptional programs across patients, we infer that glioblastomas are organized by spatial segregation of lineage states and adapt to inflammatory or metabolic stimuli reminiscent of reactive transformation in mature astrocytes. Integration of metabolic imaging and image 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. Funding: DHH is funded by the Else Kroner-Fresenius Foundation. The work is part of the MEPHISTO project (PI: DHH and DD), funded by BMBF (iGerman Ministry of Education and Research) (project number: 031L0260B). The Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) supports the work of AW (project number: SFB 824 C04). VR, KJ and UGH funded by BMBF (Bundes Ministerium fur Bildung und Forschung) (project number: FMT 13GW0230A), US was supported by the Fordergemeinschaft Kinderkrebszentrum Hamburg. We thank Dietmar Pfeifer for here helpful advice. We thank Biorender.com. We thank Stella Maria Carro for her support and the provision of her laboratory facilities and equipment. Declaration of Interest: No potential conflicts of interest were disclosed by the authors. Ethical Approval: The local ethics committee of the University of Freiburg approved the data evaluation, imaging procedures and experimental design (protocol 100020/09 and 472/15_160880).

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.

Despite recent advances in cancer immunotherapy, its efficacy in Glioblastoma (GBM) is limited due to poor understanding of molecular states and cellular plasticity of immune cells within the tumor microenvironment. Here, we combined spatial and single-cell transcriptomics of 47.284 immune cells, to map the potential cellular interactions leading to the immunosuppressive microenvironment and dysfunction of T cells. Computational approach identified a subset of IL10 releasing HMOX1+ myeloid cells which activates transcriptional programs towards a dysfunctional state in T cells, and was found to be localized within mesenchymal dominated subregions of the tumor. These findings were further validated by a human ex-vivo neocortical GBM model (n=6) coupled with patient derived peripheral T-cells. Finally, the dysfunctional transformation of T cells was shown to be rescued by JAK/STAT inhibition in both our model and in-vivo. We strongly believe that our findings would be the stepping stone towards successful development of immunotherapeutic approaches in GBM.