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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).

Elderly patients constitute an expanding part of our society. Due to a continuously increasing life expectancy, an optimal quality of life is expected even into advanced age. Glioblastoma (GBM) is more common in older patients, but they are still often withheld from efficient treatment due to worry of worse tolerance and have a significantly worse prognosis compared to younger patients. Our retrospective observational study aimed to investigate the therapeutic benefit from a second resection in recurrent glioblastoma of elderly patients. We included a cohort of 39 elderly patients (> 65 years) with a second resection as treatment option in the case of a tumor recurrence. A causal inference model was built by multiple non- and semiparametric models, which was used to identify matched patients from our elderly GBM database which comprises 538 patients. The matched cohorts were analyzed by a Cox-regression model adjusted by time-dependent covariates. The Cox-regression analysis showed a significant survival benefit (Hazard Ratio: 0.6, 95% CI 0.36–0.9, p-value = 0.0427) for the re-resected group (18.0 months, 95% CI 13.97–23.2 months) compared to the group without re-resection (10.1 months, 95% CI 8.09–20.9 months). No differences in the co-morbidities or hemato-oncological side effects during chemotherapy could be detected. Anesthetic- and surgical complications were rare and comparable to the complication rate of patients undergoing the first-line resection. Taken together, in elderly patients, re-resection is an acceptable treatment option in the recurrent state of a glioblastoma. The individual evaluation of the patients′ medical status as well as the chances of withstanding general anesthesia needs to be done in close interdisciplinary consultation. If these requirements are met, elderly patients benefit from a re-resection.

Glioblastomas are highly malignant tumors of the central nervous system. Evidence suggests that these tumors display large intra- and inter-patient heterogeneity hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies1–3. However, the source for dynamic reorganization of cellular states within their spatial context remains elusive. Here, we in-depth characterized glioblastomas by spatially resolved transcriptomics, metabolomics and proteomics. By deciphering exclusive and shared transcriptional programs across patients, we inferred that glioblastomas develop along defined neural lineages and adapt to inflammatory or metabolic stimuli reminiscent of reactive transformation in mature astrocytes. Metabolic profiling and imaging mass cytometry supported the assumption that tumor heterogeneity is dictated by microenvironmental alterations. Analysis of copy number variation (CNV) revealed a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. Deconvolution of age-dependent transcriptional programs in malignant and non-malignant specimens identified the aging environment as the major driver of inflammatory transformation in GBM, suggesting that tumor cells adopt transcriptional programs similar to inflammatory transformation in astrocytes. Glioblastoma stem cells implanted into human neocortical slices of varying age levels, independently confirmed that the ageing environment dynamically shapes the intratumoral heterogeneity towards reactive transcriptional programs. Our findings provide insights into the spatial architecture of glioblastoma, suggesting that both locally inherent tumor as well as global alterations of the tumor microenvironment shape its transcriptional heterogeneity. Global age-related inflammation in the human brain is driving distinct transcriptional transformation in glioblastomas, which requires an adjustment of the currently prevailing glioma models.

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.

Glioblastomas are highly malignant tumors of the central nervous system. Evidence suggests that these tumors display large intra- and inter-patient heterogeneity hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies. However, the source for dynamic reorganization of cellular states within their spatial context remains elusive. Here, we in-depth characterized glioblastomas by spatially resolved transcriptomics, metabolomics and proteomics. By deciphering exclusive and shared transcriptional programs across patients, we inferred that glioblastomas develop along defined neural lineages and adapt to inflammatory or metabolic stimuli reminiscent of reactive transformation in mature astrocytes. Metabolic profiling and imaging mass cytometry supported the assumption that tumor heterogeneity is dictated by microenvironmental alterations. Analysis of copy number variation (CNV) revealed a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. Deconvolution of age-dependent transcriptional programs in malignant and non-malignant specimens identified the aging environment as the major driver of inflammatory transformation in GBM, suggesting that tumor cells adopt transcriptional programs similar to inflammatory transformation in astrocytes. Glioblastoma stem cells implanted into human neocortical slices of varying age levels, independently confirmed that the ageing environment dynamically shapes the intratumoral heterogeneity towards reactive transcriptional programs. Our findings provide insights into the spatial architecture of glioblastoma, suggesting that both locally inherent tumor as well as global alterations of the tumor microenvironment shape its transcriptional heterogeneity. Global age-related inflammation in the human brain is driving distinct transcriptional transformation in glioblastomas, which requires an adjustment of the currently prevailing glioma models.

SummaryThe diversity of molecular states and cellular plasticity of immune cells in the glioblastoma environment is still poorly understood. Here, we performed scRNA sequencing of the immune compartment and mapped potential cellular interactions leading to an immunosuppressive microenvironment and dysfunction of T cells. Through inferring the dynamic adaptation during T cell activation, we identified three different terminal states with unique transcriptional programs. Modeling of driver genes for terminal T cell fate identified IL-10 signaling alterations in a subpopulation of HAVCR2(+) T cells. To explore in depth cellular interactions, we established an in-silico model by the integration of spatial transcriptomic and scRNA-sequencing, and identified a subset of HMOX1+ myeloid cells defined by IL10 release leading to T cell exhaustion. We found a spatial overlap between HMOX(+) myeloid and HAVCR2(+) T cells, suggesting that myeloid-lymphoid interaction causes immunosuppression present in tumor regions with enriched mesenchymal gene expression. Using human neocortical GBM model, coupled with patient-derived T cells, we confirmed that the functional interaction between myeloid and lymphoid cells, leads to a dysfunctional state of T cells. This IL-10 driven T cell exhaustion was found to be rescued by JAK/STAT inhibition. A comprehensive understanding of the cellular states and plasticity of lymphoid cells in GBM will aid towards successful immunotherapeutic approaches.

Oligodendroglioma are defined by a distinct molecular phenotype marked by 1p19q co-deletion and simultaneous presence of an IDH1/2 mutation. These tumors showed a favorable clinical course and long-term survival of around 15 years. Due to the long course of the disease, prospective studies to determine the effectiveness of different therapeutic strategies are difficult, since the percentage of patients with multiple therapies is high. Here we report a computational approach to map the longitudinal growth pattern, to quantify the effect of therapies on tumor growth and to identify similarities and spatial heterogeneity of oligodendroglioma growth. In our study, we included a cohort of 44 histopathologically and molecularly stratified oligodendrogliomas WHO°II (n=23) and WHO°III (n=21). We started our investigation with the longitudinal tumor segmentation. All volumetric data were pinpointed to the times of tumor therapy within all patients. Next, we extracted first-order features of tumor growth and response to chemo- or radiotherapy as well as resection, resulting in a total number of 98 features. An unsupervised cluster was used to identify similarities between patients, which revealed 3 subgroups. The first subgroup contained patients with predominantly frontal oligodendrogliomas marked by increased response to radiotherapy. The second subgroup included temporal oligodendrogliomas with high response rate to PC/PCV chemotherapy and flagged by epilepsy. The third group was heterogeneous with varying growth behaviors. A survival analysis showed a better separation between low- and high-risk patients based on the growth pattern model, in contrast to the WHO grading system. Taken together, our analysis revealed a novel classification of oligodendroglioma based on the longitudinal growth pattern and therapeutical response. We also detected a spatial difference between frontally or temporally localized oligodendrogliomas. We plan to further investigate molecular data that explain these spatial differences, which also may uncover novel therapeutic strategies.

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.

Gap junctions have recently been shown to interconnect glioblastoma cells to a multicellular syncytial network, thereby allowing intercellular communication over long distances as well as enabling glioblastoma cells to form routes for brain microinvasion. Against this backdrop gap junction-targeted therapies might provide for an essential contribution to isolate cancer cells within the brain, thus increasing the tumor cells&rsquo, vulnerability to the standard chemotherapeutic agent temozolomide. By utilizing INI-0602&mdash, a novel gap junction inhibitor optimized for crossing the blood brain barrier&mdash, in an oncological setting, the present study was aimed at evaluating the potential of gap junction-targeted therapy on primary human glioblastoma cell populations. Pharmacological inhibition of gap junctions profoundly sensitized primary glioblastoma cells to temozolomide-mediated cell death. On the molecular level, gap junction inhibition was associated with elevated activity of the JNK signaling pathway. With the use of a novel gap junction inhibitor capable of crossing the blood&ndash, brain barrier&mdash, thus constituting an auspicious drug for clinical applicability&mdash, these results may constitute a promising new therapeutic strategy in the field of current translational glioblastoma research.

Therapeutically resected, adult brain segments were maintained and characterized for an extended period to study glioblastoma progression and treatment in its almost natural environment., When it comes to the human brain, models that closely mimic in vivo conditions are lacking. Living neuronal tissue is the closest representation of the in vivo human brain outside of a living person. Here, we present a method that can be used to maintain therapeutically resected healthy neuronal tissue for prolonged periods without any discernible changes in tissue vitality, evidenced by immunohistochemistry, genetic expression, and electrophysiology. This method was then used to assess glioblastoma (GBM) progression in its natural environment by microinjection of patient-derived tumor cells into cultured sections. The result closely resembles the pattern of de novo tumor growth and invasion, drug therapy response, and cytokine environment. Reactive transformation of astrocytes, as an example of the cellular nonmalignant tumor environment, can be accurately simulated with transcriptional differences similar to those of astrocytes isolated from acute GBM specimens. In a nutshell, we present a simple method to study GBM in its physiological environment, from which valuable insights can be gained. This technique can lead to further advancements in neuroscience, neuro-oncology, and pharmacotherapy.

Cancers 11(10), 1437 (2019). doi:10.3390/cancers11101437, Published by MDPI, Basel

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.

BACKGROUND Glioblastomas are referred to as immunologically “cold” tumors since their cellular environment promotes an anti-inflammatory environment leading to an unresolved barrier to immunotherapy. Although the role of reactive astrocytes in other inflammatory diseases has been investigated in several studies, the immunoregulatory functions of astrocytes in the tumor environment remains poorly understood. MATERIAL AND METHODS We purified reactive astrocytes from de-novo glioblastoma and non-infiltrated cortex specimens by immunoprecipitation and analyzed the transcriptional phenotype by RNA sequencing. In order to investigate the origin of astrocytic transformation, we used a microglia loss-of-function model in human organotypic slices. Microglia was depleted by clodronat stimulation. We injected tumor cells and analyzed gene expression of the astrocytes after 7d incubation by RNA sequencing. Environmental cytokines were analyzed by multi-ELISA. Immunostainings of slices were processed by confocal microscopy and 3D reconstruction. RESULTS Here we address the immunological impact of tumor-associated astrocytes, we were able to identify a novel reactive subtype marked by JAK/STAT pathway activation and CD274 expression. Our results show a distinct astrocytic transcriptional phenotype that mutually arises from both microglia and astrocytes of the tumor environment. This interaction leads to a large release of anti-inflammatory cytokines such as TGFß and IL10. The reactive subtype switch of astrocytes was recovered by inhibition of the JAK pathway, which caused an increase of pro-inflammatory environment. CONCLUSION Our results can form the basis for a novel therapeutic approach by directly targeting tumor-associated astrocytes in order to transformimmunologically “cold” into “warm” tumors.