High-sensitive spatially resolved T cell receptor sequencing with SPTCR-seq.

Spatial resolution of the T cell repertoire is essential for deciphering cancer-associated immune dysfunction. Current spatially resolved transcriptomic technologies are unable to directly annotate T cell receptors (TCR). We present spatially resolved T cell receptor sequencing (SPTCR-seq), which integrates optimized target enrichment and long-read sequencing for highly sensitive TCR sequencing. The SPTCR computational pipeline achieves yield and coverage per TCR comparable to alternative single-cell TCR technologies. Our comparison of PCR-based and SPTCR-seq methods underscores SPTCR-seq's superior ability to reconstruct the entire TCR architecture, including V, D, J regions and the complementarity-determining region 3 (CDR3). Employing SPTCR-seq, we assess local T cell diversity and clonal expansion across spatially discrete niches. Exploration of the reciprocal interaction of the tumor microenvironmental and T cells discloses the critical involvement of NK and B cells in T cell exhaustion. Integrating spatially resolved omics and TCR sequencing provides as a robust tool for exploring T cell dysfunction in cancers and beyond.

'Hippocampal innate inflammatory gliosis only' in pharmacoresistant temporal lobe epilepsy.

Drug-resistant mesial-temporal lobe epilepsy is a devastating disease with seizure onset in the hippocampal formation. A fraction of hippocampi samples from epilepsy-surgical procedures reveals a peculiar histological pattern referred to as 'gliosis only' with unresolved pathogenesis and enigmatic sequelae. Here, we hypothesize that 'gliosis only' represents a particular syndrome defined by distinct clinical and molecular characteristics. We curated an in-depth multiparameter integration of systematic clinical, neuropsychological as well as neuropathological analysis from a consecutive cohort of 627 patients, who underwent hippocampectomy for drug-resistant temporal lobe epilepsy. All patients underwent either classic anterior temporal lobectomy or selective amygdalohippocampectomy. On the basis of their neuropathological exam, patients with hippocampus sclerosis and 'gliosis only' were characterized and compared within the whole cohort and within a subset of matched pairs. Integrated transcriptional analysis was performed to address molecular differences between both groups. 'Gliosis only' revealed demographics, clinical and neuropsychological outcome fundamentally different from hippocampus sclerosis. 'Gliosis only' patients had a significantly later seizure onset (16.3 versus 12.2 years, P = 0.005) and worse neuropsychological outcome after surgery compared to patients with hippocampus sclerosis. Epilepsy was less amendable by surgery in 'gliosis only' patients, resulting in a significantly worse rate of seizure freedom after surgery in this subgroup (43% versus 68%, P = 0.0001, odds ratio = 2.8, confidence interval 1.7-4.7). This finding remained significant after multivariate and matched-pairs analysis. The 'gliosis only' group demonstrated pronounced astrogliosis and lack of significant neuronal degeneration in contrast to characteristic segmental neuron loss and fibrillary astrogliosis in hippocampus sclerosis. RNA-sequencing of gliosis only patients deciphered a distinct transcriptional programme that resembles an innate inflammatory response of reactive astrocytes. Our data indicate a new temporal lobe epilepsy syndrome for which we suggest the term 'Innate inflammatory gliosis only'. 'Innate inflammatory gliosis only' is characterized by a diffuse gliosis pattern lacking restricted hippocampal focality and is poorly controllable by surgery. Thus, 'innate inflammatory gliosis only' patients need to be clearly identified by presurgical examination paradigms of pharmacoresistant temporal lobe epilepsy patients; surgical treatment of this subgroup should be considered with great precaution. 'Innate inflammatory gliosis only' requires innovative pharmacotreatment strategies.

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.

Meclofenamate causes loss of cellular tethering and decoupling of functional networks in glioblastoma.

BACKGROUND: Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy. METHODS: In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis. RESULTS: TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity. CONCLUSION: The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach.