Effects of tumor treating fields (TTFields) on human mesenchymal stromal cells.

PURPOSE: Mesenchymal stromal cells (MSCs) within the glioblastoma microenvironment have been shown to promote tumor progression. Tumor Treating Fields (TTFields) are alternating electric fields with low intensity and intermediate frequency that exhibit anti-tumorigenic effects. While the effects of TTFields on glioblastoma cells have been studied previously, nothing is known about the influence of TTFields on MSCs. METHODS: Single-cell RNA sequencing and immunofluorescence staining were employed to identify glioblastoma-associated MSCs in patient samples. Proliferation and clonogenic survival of human bone marrow-derived MSCs were assessed after TTFields in vitro. MSC' characteristic surface marker expression was determined using flow cytometry, while multi-lineage differentiation potential was examined with immunohistochemistry. Apoptosis was quantified based on caspase-3 and annexin-V/7-AAD levels in flow cytometry, and senescence was assessed with ß-galactosidase staining. MSCs' migratory potential was evaluated with Boyden chamber assays. RESULTS: Single-cell RNA sequencing and immunofluorescence showed the presence of glioblastoma-associated MSCs in patient samples. TTFields significantly reduced proliferation and clonogenic survival of human bone marrow-derived MSCs by up to 60% and 90%, respectively. While the characteristic surface marker expression and differentiation capacity were intact after TTFields, treatment resulted in increased apoptosis and senescence. Furthermore, TTFields significantly reduced MSCs' migratory capacity. CONCLUSION: We could demonstrate the presence of tumor-associated MSCs in glioblastoma patients, providing a rationale to study the impact of TTFields on MSCs. TTFields considerably increase apoptosis and senescence in MSCs, resulting in impaired survival and migration. The results provide a basis for further analyses on the role of MSCs in glioblastoma patients receiving TTFields.

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL.

ABSTRACT: The t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre-B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Multiomic spatial landscape of innate immune cells at human central nervous system borders.

The innate immune compartment of the human central nervous system (CNS) is highly diverse and includes several immune-cell populations such as macrophages that are frequent in the brain parenchyma (microglia) and less numerous at the brain interfaces as CNS-associated macrophages (CAMs). Due to their scantiness and particular location, little is known about the presence of temporally and spatially restricted CAM subclasses during development, health and perturbation. Here we combined single-cell RNA sequencing, time-of-flight mass cytometry and single-cell spatial transcriptomics with fate mapping and advanced immunohistochemistry to comprehensively characterize the immune system at human CNS interfaces with over 356,000 analyzed transcriptomes from 102 individuals. We also provide a comprehensive analysis of resident and engrafted myeloid cells in the brains of 15 individuals with peripheral blood stem cell transplantation, revealing compartment-specific engraftment rates across different CNS interfaces. Integrated multiomic and high-resolution spatial transcriptome analysis of anatomically dissected glioblastoma samples shows regionally distinct myeloid cell-type distributions driven by hypoxia. Notably, the glioblastoma-associated hypoxia response was distinct from the physiological hypoxia response in fetal microglia and CAMs. Our results highlight myeloid diversity at the interfaces of the human CNS with the periphery and provide insights into the complexities of the human brain's immune system.

Single-cell deconvolution reveals high lineage- and location-dependent heterogeneity in mesenchymal multivisceral stage 4 colorectal cancer.

Metastasized colorectal cancer (CRC) is associated with a poor prognosis and rapid disease progression. Besides hepatic metastasis, peritoneal carcinomatosis is the major cause of death in Union for International Cancer Control (UICC) stage IV CRC patients. Insights into differential site-specific reconstitution of tumor cells and the corresponding tumor microenvironment are still missing. Here, we analyzed the transcriptome of single cells derived from murine multivisceral CRC and delineated the intermetastatic cellular heterogeneity regarding tumor epithelium, stroma, and immune cells. Interestingly, we found an intercellular site-specific network of cancer-associated fibroblasts and tumor epithelium during peritoneal metastasis as well as an autologous feed-forward loop in cancer stem cells. We furthermore deciphered a metastatic dysfunctional adaptive immunity by a loss of B cell-dependent antigen presentation and consecutive effector T cell exhaustion. Furthermore, we demonstrated major similarities of this murine metastatic CRC model with human disease and - based on the results of our analysis - provided an auspicious site-specific immunomodulatory treatment approach for stage IV CRC by intraperitoneal checkpoint inhibition.

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.

The pre-existing T cell landscape determines the response to bispecific T cell engagers in multiple myeloma patients.

Bispecific T cell engagers (TCEs) have shown promise in the treatment of various cancers, but the immunological mechanism and molecular determinants of primary and acquired resistance to TCEs remain poorly understood. Here, we identify conserved behaviors of bone marrow-residing T cells in multiple myeloma patients undergoing BCMAxCD3 TCE therapy. We show that the immune repertoire reacts to TCE therapy with cell state-dependent clonal expansion and find evidence supporting the coupling of tumor recognition via major histocompatibility complex class I (MHC class I), exhaustion, and clinical response. We find the abundance of exhausted-like CD8+ T cell clones to be associated with clinical response failure, and we describe loss of target epitope and MHC class I as tumor-intrinsic adaptations to TCEs. These findings advance our understanding of the in vivo mechanism of TCE treatment in humans and provide the rationale for predictive immune-monitoring and conditioning of the immune repertoire to guide future immunotherapy in hematological malignancies.

Dysfunctional dendritic cells limit antigen-specific T cell response in glioma.

BACKGROUND: Dendritic cells (DC), the most potent professional antigen presenting cells capable of effective cross-presentation, have been demonstrated to license T helper cells to induce antitumor immunity in solid tumors. Specific DC subtypes are recruited to the injured brain by microglial chemokines, locally adapting to distinct transcriptional profiles. In isocitrate dehydrogenase (IDH) type 1 mutant gliomas, monocyte-derived macrophages have recently been shown to display an attenuated intratumoral antigen presentation capacity as consequence of the local accumulation of the oncometabolite R-2-hydroxyglutarate. The functionality and the contribution of DC to the IDH-mutant tumor microenvironment (TME) remains unclear. METHODS: Frequencies and intratumoral phenotypes of human DC in IDH-wildtype (IDHwt) and -mutant high-grade gliomas are comparatively assessed by transcriptomic and proteomic profiling. DC functionality is investigated in experimental murine glioblastomas expressing the model antigen ovalbumin. Single-cell sequencing-based pseudotime analyses and spectral flow cytometric analyses are used to profile DC states longitudinally. RESULTS: DC are present in primary and recurrent high-grade gliomas and interact with other immune cell types within the TME. In murine glioblastomas, we find an IDH-status-associated major histocompatibility class I-restricted cross-presentation of tumor antigens by DC specifically in the tumor but not in meninges or secondary lymphoid organs of tumor-bearing animals. In single-cell sequencing-based pseudotime and longitudinal spectral flow cytometric analyses, we demonstrate an IDH-status-dependent differential, exclusively microenvironmental education of DC. CONCLUSIONS: Glioma-associated DCs are relevantly abundant in human IDHwt and mutant tumors. Glioma IDH mutations result in specifically educated, dysfunctional DCs via paracrine reprogramming of infiltrating monocytes, providing the basis for combinatorial immunotherapy concepts against IDH mutant gliomas.

IGF1R expression by adult oligodendrocytes is not required in the steady-state but supports neuroinflammation.

In the central nervous system (CNS), insulin-like growth factor 1 (IGF-1) regulates myelination by oligodendrocyte (ODC) precursor cells and shows anti-apoptotic properties in neuronal cells in different in vitro and in vivo systems. Previous work also suggests that IGF-1 protects ODCs from cell death and enhances remyelination in models of toxin-induced and autoimmune demyelination. However, since evidence remains controversial, the therapeutic potential of IGF-1 in demyelinating CNS conditions is unclear. To finally shed light on the function of IGF1-signaling for ODCs, we deleted insulin-like growth factor 1 receptor (IGF1R) specifically in mature ODCs of the mouse. We found that ODC survival and myelin status were unaffected by the absence of IGF1R until 15 months of age, indicating that IGF-1 signaling does not play a major role in post-mitotic ODCs during homeostasis. Notably, the absence of IGF1R did neither affect ODC survival nor myelin status upon cuprizone intoxication or induction of experimental autoimmune encephalomyelitis (EAE), models for toxic and autoimmune demyelination, respectively. Surprisingly, however, the absence of IGF1R from ODCs protected against clinical neuroinflammation in the EAE model. Together, our data indicate that IGF-1 signaling is not required for the function and survival of mature ODCs in steady-state and disease.

Transcriptome Analysis Identifies Accumulation of Natural Killer Cells with Enhanced Lymphotoxin-ß Expression during Glioblastoma Progression.

Glioblastomas are the most common primary brain tumors. Despite extensive clinical and molecular insights into these tumors, the prognosis remains dismal. While targeted immunotherapies have shown remarkable success across different non-brain tumor entities, they failed to show efficacy in glioblastomas. These failures prompted the field to reassess the idiosyncrasies of the glioblastoma microenvironment. Several high-dimensional single-cell RNA sequencing studies generated remarkable findings about glioblastoma-associated immune cells. To build on the collective strength of these studies, we integrated several murine and human datasets that profiled glioblastoma-associated immune cells at different time points. We integrated these datasets and utilized state-of-the-art algorithms to investigate them in a hypothesis-free, purely exploratory approach. We identified a robust accumulation of a natural killer cell subset that was characterized by a downregulation of activation-associated genes with a concomitant upregulation of apoptosis genes. In both species, we found a robust upregulation of the Lymphotoxin-ß gene, a cytokine from the TNF superfamily and a key factor for the development of adaptive immunity. Further validation analyses uncovered a correlation of lymphotoxin signaling with mesenchymal-like glioblastoma regions in situ and in TCGA and CGGA glioblastoma cohorts. In summary, we identify lymphotoxin signaling as a potential therapeutic target in glioblastoma-associated natural killer cells.

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.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Neurocognitive and psychiatric post-coronavirus disease 2019 conditions: pathogenic insights of brain dysfunction following severe acute respiratory syndrome coronavirus 2 infection.

PURPOSE OF REVIEW: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), can trigger a myriad of neuropsychiatric manifestations. As a 2-year-old disease (at the writing of this manuscript), its long-term cognitive and neuropsychiatric implications, known as post-COVID-19 conditions, are incompletely recognized and mechanistically obscure. RECENT FINDINGS: Fatigue, anxiety, depression, posttraumatic stress disorder, and cognitive dysfunction are reported more frequently in COVID-19 survivors than in matching, non-COVID-19 population. Risk factors are unclear, including comorbidities, age at COVID-19 onset, or disease severity; women, however, have been reported to be at increased risk than men. Although the frequency of these symptoms decreases over time, at least one in five will have persistent cognitive and neuropsychiatric manifestations one year after recovering from COVID-19. SUMMARY: Neurocognitive and psychiatric post-COVID-19 long-term conditions are frequent and complex multifactorial sequelae. Several acute and chronic factors such as hypoxemia, cerebral thrombotic and inflammatory endothelial damage, and disruption of the blood-brain barrier (leading to parenchymal translocation of pro-inflammatory molecules, cytokines, and cytotoxic T lymphocytes) are involved, leading to microglial activation and astrogliosis. As an evolving topic, evidence derived from prospective studies will expand our understanding of post-COVID-19 these long-term outcomes.

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.

Evaluating microglial phenotypes using single-cell technologies.

Recent single-cell technologies have enabled researchers to simultaneously assess the transcriptomes and other modalities of thousands of cells within their spatial context. Here, we have summarized available single-cell methods for dissociated tissues and tissue slides with respect to the specifics of microglial biology. We have focused on next-generation-based technologies. We review the potential of these single-cell sequencing methods and newer multiomics approaches to extend the understanding of microglia function beyond the status quo.

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease.

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically.

GPCRomics of Homeostatic and Disease-Associated Human Microglia.

G-protein-coupled receptors (GPCRs) are critical sensors affecting the state of eukaryotic cells. To get systematic insight into the GPCRome of microglia, we analyzed publicly available RNA-sequencing data of bulk and single cells obtained from human and mouse brains. We identified 17 rhodopsin and adhesion family GPCRs robustly expressed in microglia from human brains, including the homeostasis-associated genes CX3CR1, GPR34, GPR183, P2RY12, P2RY13, and ADGRG1. Expression of these microglial core genes was lost upon culture of isolated cells ex vivo but could be acquired by human induced pluripotent stem cell (iPSC)-derived microglial precursors transplanted into mouse brains. CXCR4 and PTGER4 were higher expressed in subcortical white matter compared to cortical grey matter microglia, and ADGRG1 was downregulated in microglia obtained from normal-appearing white and grey matter tissue of multiple sclerosis (MS) brains. Single-cell RNA sequencing of microglia from active lesions, obtained early during MS, revealed downregulation of homeostasis-associated GPCR genes and upregulation of CXCR4 expression in a small subset of MS-associated lesional microglia. Functional presence of low levels of CXCR4 on human microglia was confirmed using flow cytometry and transwell migration towards SDF-1. Microglia abundantly expressed the GPCR down-stream signaling mediator genes GNAI2 (αi2), GNAS (αs), and GNA13 (α13), the latter particularly in white matter. Drugs against several microglia GPCRs are available to target microglia in brain diseases. In conclusion, transcriptome profiling allowed us to identify expression of GPCRs that may contribute to brain (patho)physiology and have diagnostic and therapeutic potential in human microglia.

IL-17 controls central nervous system autoimmunity through the intestinal microbiome.

Interleukin-17A- (IL-17A) and IL-17F-producing CD4+ T helper cells (TH17 cells) are implicated in the development of chronic inflammatory diseases, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). TH17 cells also orchestrate leukocyte invasion of the central nervous system (CNS) and subsequent tissue damage. However, the role of IL-17A and IL-17F as effector cytokines is still confused with the encephalitogenic function of the cells that produce these cytokines, namely, TH17 cells, fueling a long-standing debate in the neuroimmunology field. Here, we demonstrated that mice deficient for IL-17A/F lose their susceptibility to EAE, which correlated with an altered composition of their gut microbiota. However, loss of IL-17A/F in TH cells did not diminish their encephalitogenic capacity. Reconstitution of a wild-type-like intestinal microbiota or reintroduction of IL-17A specifically into the gut epithelium of IL-17A/F-deficient mice reestablished their susceptibility to EAE. Thus, our data demonstrated that IL-17A and IL-17F are not encephalitogenic mediators but rather modulators of intestinal homeostasis that indirectly alter CNS-directed autoimmunity.