Microglia are tissue-resident macrophages of the CNS that orchestrate local immune responses and contribute to several neurological and psychiatric diseases. Little is known about human microglia and how they orchestrate their highly plastic, context-specific adaptive responses during pathology. Here we combined two high-dimensional technologies, single-cell RNAsequencing and time-of-flight mass cytometry, to identify microglia states in the human brain during homeostasis and disease. This approach enabled us to identify and characterize a previously unappreciated spectrum of transcriptional states in human microglia. These transcriptional states are determined by their spatial distribution, and they further change with aging and brain tumor pathology. This description of multiple microglia phenotypes in the human CNS may open promising new avenues for subset-specific therapeutic interventions.

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The dynamics and phenotypes of intratumoral myeloid cells during tumor progression are poorly understood. Here we define myeloid cellular states in gliomas by longitudinal single-cell profiling and demonstrate their strict control by the tumor genotype: in isocitrate dehydrogenase (IDH)-mutant tumors, differentiation of infiltrating myeloid cells is blocked, resulting in an immature phenotype. In late-stage gliomas, monocyte-derived macrophages drive tolerogenic alignment of the microenvironment, thus preventing T cell response. We define the IDH-dependent tumor education of infiltrating macrophages to be causally related to a complex re-orchestration of tryptophan metabolism, resulting in activation of the aryl hydrocarbon receptor. We further show that the altered metabolism of IDH-mutant gliomas maintains this axis in bystander cells and that pharmacological inhibition of tryptophan metabolism can reverse immunosuppression. In conclusion, we provide evidence of a glioma genotype-dependent intratumoral network of resident and recruited myeloid cells and identify tryptophan metabolism as a target for immunotherapy of IDH-mutant tumors.

Lamszus (2019) Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours,

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.

Purpose Dramatic advances in our understanding of the molecular pathophysiology of cancer, along with a rapidly expanding portfolio of molecular targeted drugs, have led to a paradigm shift toward personalized, biomarker-driven cancer treatment. Here, we report the 2-year experience of the Comprehensive Cancer Center Freiburg Molecular Tumor Board (MTB), one of the first interdisciplinary molecular tumor conferences established in Europe. The role of the MTB is to recommend personalized therapy for patients with cancer beyond standard-of-care treatment. Methods This retrospective case series includes 198 patients discussed from March 2015 through February 2017. The MTB guided individual molecular diagnostics, assessed evidence of actionability of molecular alterations, and provided therapy recommendations, including approved and off-label treatments as well as available matched clinical trials. Results The majority of patients had metastatic solid tumors (73.7%), mostly progressive (77.3%) after a mean of 2.0 lines of standard treatment. Diagnostic recommendations resulted in 867 molecular diagnostic tests for 172 patients (five per case), including exome analysis in 36 cases (18.2%). With a median turnaround time of 28 days, treatment recommendations were given to 104 patients (52.5%). These included single-agent targeted therapies (42.3%), checkpoint inhibitors (37.5%), and combination therapies (18.3%). Treatment recommendations were implemented in 33 of 104 patients (31.7%), of whom 19 (57.6%) showed stable disease or partial response, including 14 patients (7.1% of the entire population) receiving off-label treatments. Conclusion Personalized extended molecular-guided patient care is effective for a small but clinically meaningful proportion of patients in challenging clinical situations. Limited access to targeted drugs, lack of trials, and submission at late disease stage prevents broader applicability, whereas genome-wide analyses are not a strict requirement for predictive molecular testing.

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle ( ACTA2 ). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

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