Glioblastomas exploit truncated O-linked glycans for local and distant immune modulation via the macrophage galactose-type lectin.

Glioblastoma is the most aggressive brain malignancy, for which immunotherapy has failed to prolong survival. Glioblastoma-associated immune infiltrates are dominated by tumor-associated macrophages and microglia (TAMs), which are key mediators of immune suppression and resistance to immunotherapy. We and others demonstrated aberrant expression of glycans in different cancer types. These tumor-associated glycans trigger inhibitory signaling in TAMs through glycan-binding receptors. We investigated the glioblastoma glycocalyx as a tumor-intrinsic immune suppressor. We detected increased expression of both tumor-associated truncated O-linked glycans and their receptor, macrophage galactose-type lectin (MGL), on CD163+ TAMs in glioblastoma patient-derived tumor tissues. In an immunocompetent orthotopic glioma mouse model overexpressing truncated O-linked glycans (MGL ligands), high-dimensional mass cytometry revealed a wide heterogeneity of infiltrating myeloid cells with increased infiltration of PD-L1+ TAMs as well as distant alterations in the bone marrow (BM). Our results demonstrate that glioblastomas exploit cell surface O-linked glycans for local and distant immune modulation.

Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment.

BACKGROUND: Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells. METHODS: We present a workflow for ex vivo imaging of optically cleared brain tumor tissues and subsequent computational modeling. This workflow was used for quantification of the microvasculature in relation to nuclear or cellular density in healthy mouse brain tissues and in human orthotopic, infiltrative GBM8 and E98 glioblastoma models. RESULTS: Ex vivo cleared mouse brain tissues had a >10-fold imaging depth as compared to intravital imaging of mouse brain in vivo. Imaging of optically cleared brain tissue allowed quantification of the 3D microvascular characteristics in healthy mouse brains and in tissues with diffuse, infiltrative growing GBM8 brain tumors. Detailed 3D visualization revealed the organization of tumor cells relative to the vasculature, in both gray matter and white matter regions, and patterns of multicellular GBM networks collectively invading the brain parenchyma. CONCLUSIONS: Optical tissue clearing opens new avenues for combined quantitative and 3D microscopic analysis of the topographical relationship between GBM cells and their microenvironment.

The immunological landscape of peripheral blood in glioblastoma patients and immunological consequences of age and dexamethasone treatment.

Background: Glioblastomas manipulate the immune system both locally and systemically, yet, glioblastoma-associated changes in peripheral blood immune composition are poorly studied. Age and dexamethasone administration in glioblastoma patients have been hypothesized to limit the effectiveness of immunotherapy, but their effects remain unclear. We compared peripheral blood immune composition in patients with different types of brain tumor to determine the influence of age, dexamethasone treatment, and tumor volume. Methods: High-dimensional mass cytometry was used to characterise peripheral blood mononuclear cells of 169 patients with glioblastoma, lower grade astrocytoma, metastases and meningioma. We used blood from medically-refractory epilepsy patients and healthy controls as control groups. Immune phenotyping was performed using FlowSOM and t-SNE analysis in R followed by supervised annotation of the resulting clusters. We conducted multiple linear regression analysis between intracranial pathology and cell type abundance, corrected for clinical variables. We tested correlations between cell type abundance and survival with Cox-regression analyses. Results: Glioblastoma patients had significantly fewer naive CD4+ T cells, but higher percentages of mature NK cells than controls. Decreases of naive CD8+ T cells and alternative monocytes and an increase of memory B cells in glioblastoma patients were influenced by age and dexamethasone treatment, and only memory B cells by tumor volume. Progression free survival was associated with percentages of CD4+ regulatory T cells and double negative T cells. Conclusion: High-dimensional mass cytometry of peripheral blood in patients with different types of intracranial tumor provides insight into the relation between intracranial pathology and peripheral immune status. Wide immunosuppression associated with age and pre-operative dexamethasone treatment provide further evidence for their deleterious effects on treatment with immunotherapy.

Immune involvement of the contralateral hemisphere in a glioblastoma mouse model.

BACKGROUND: Glioblastoma (GBM) is the most common and deadliest form of brain cancer in adults. Standard treatment, consisting of surgery and radiochemotherapy, only provides a modest survival benefit and is incapable of combating infiltrating GBM cells in other parts of the brain. New therapies in clinical trials, such as anti-programmed cell death 1 immunotherapy, have so far shown limited success in GBM. Moreover, it is unclear how the growth of GBM suppresses the immune system locally at the site of the brain tumor or if distant sites of tumor cell migration are also involved. Invasive GBM cells in brain tissue beyond the primary tumor limit the use of surgery, thus immunotherapy could be beneficial if activated/suppressed immune cells are present in the contralateral hemisphere. METHODS: Here, we used a syngeneic orthotopic GL26 GBM mouse model and multiparameter fluorescence-activated cell sorting analysis to study the phenotype of resident and infiltrating immune cells in both the brain tumor hemisphere and contralateral hemisphere. RESULTS: We show that lymphoid cells, including tumor antigen-specific CD8+ tumor-infiltrating lymphocytes (TILs) are present in the tumor and are characterized by a tolerogenic phenotype based on high immune checkpoint expression. Massive infiltration of myeloid cells is observed, expressing immune checkpoint ligands, suggesting an immune-dependent coinhibitory axis limiting TIL responses. Surprisingly, these phenotypes are paralleled in the contralateral hemisphere, showing that infiltrating immune cells are also present at distant sites, expressing key immune checkpoints and immune checkpoint ligands. CONCLUSION: Whole-brain analysis indicates active immune involvement throughout the brain, both at the site of the primary tumor and in the contralateral hemisphere. Using the right combination and timing, immune checkpoint blockade could have the potential to activate immune cells at the site of the brain tumor and at distant sites, thereby also targeting diffusely infiltrating GBM cells.

Glycan modification of glioblastoma-derived extracellular vesicles enhances receptor-mediated targeting of dendritic cells.

Glioblastoma is the most prevalent and aggressive primary brain tumour for which total tumour lysate-pulsed dendritic cell vaccination is currently under clinical evaluation. Glioblastoma extracellular vesicles (EVs) may represent an enriched cell-free source of tumour-associated (neo-) antigens to pulse dendritic cells (DCs) for the initiation of an anti-tumour immune response. Capture and uptake of EVs by DCs could occur in a receptor-mediated and presumably glycan-dependent way, yet the glycan composition of glioblastoma EVs is unknown. Here, we set out to characterize the glycocalyx composition of glioblastoma EVs by lectin-binding ELISA and comprehensive immunogold transmission electron microscopy (immuno-TEM). The surface glycan profile of human glioblastoma cell line-derived EVs (50-200 nm) was dominated by α-2,3- and α-2,6 linked sialic acid-capped complex N-glycans and bi-antennary N-glycans. Since sialic acids can trigger immune inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, we screened for Siglec ligands on the EVs. Glioblastoma EVs showed significant binding to Siglec-9, which is highly expressed on DCs. Surprisingly, however, glioblastoma EVs lack glycans that could bind Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), a receptor that mediates uptake and induction of CD4+ and CD8+ T cell activation. Therefore, we explored whether modification of the EV glycan surface could reduce immune inhibitory Siglec binding, while enhancing EV internalization by DCs in a DC-SIGN dependent manner. Desialylation with a pan-sialic acid hydrolase led to reduction of sialic acid expression on EVs. Moreover, insertion of a high-affinity ligand (LewisY) for DC-SIGN resulted in a four-fold increase of uptake by monocyte-derived DCs. In conclusion, we show that the glycocalyx composition of EVs is a key factor of efficient DC targeting and that modification of the EV glycocalyx potentiates EVs as anti-cancer vaccine.

Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21.

Gliomas are primary, diffusely infiltrating brain tumors. Microglia are innate immune cells in the CNS and make up a substantial portion of the tumor mass. Glioma cells shape their microenvironment, communicating with and reprogramming surrounding cells, resulting in enhanced angiogenesis, immune suppression, and remodeling of the extracellular matrix. Glioma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Mouse glioma cells stably expressing a palmitoylated GFP to label EVs were implanted intracranially into syngeneic miR-21-null mice. Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs. These findings attest to EV-dependent microRNA delivery as studied in an in vivo-based model and provide insight into the reprograming of microglial cells by tumor cells to create a favorable microenvironment for cancer progression.

Glycan-Modified Apoptotic Melanoma-Derived Extracellular Vesicles as Antigen Source for Anti-Tumor Vaccination

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated tumor-derived apoptotic extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer.

Extracellular RNAs: A New Awareness of Old Perspectives.

Extracellular RNA (exRNA) has recently expanded as a highly important area of study in biomarker discovery and cancer therapeutics. exRNA consists of diverse RNA subpopulations that are normally protected from degradation by incorporation into membranous vesicles or by lipid/protein association. They are found circulating in biofluids, and have proven highly promising for minimally invasive diagnostic and prognostic purposes, particularly in oncology. Recent work has made progress in our understanding of exRNAs-from their biogenesis, compartmentalization, and vesicle packaging to their various applications as biomarkers and therapeutics, as well as the new challenges that arise in isolation and purification for accurate and reproducible analysis. Here we review the most recent advancements in exRNA research.

The importance of trabecular hypertrophy in right ventricular adaptation to chronic pressure overload.

To assess the contribution of right ventricular (RV) trabeculae and papillary muscles (TPM) to RV mass and volumes in controls and patients with pulmonary arterial hypertension (PAH). Furthermore, to evaluate whether TPM shows a similar response as the RV free wall (RVFW) to changes in pulmonary artery pressure (PAP) during follow-up. 50 patients underwent cardiac magnetic resonance (CMR) and right heart catheterization at baseline and after one-year follow-up. Furthermore 20 controls underwent CMR. RV masses were assessed with and without TPM. TPM constituted a larger proportion of total RV mass and RV end-diastolic volume (RVEDV) in PAH than in controls (Mass: 35 ± 7 vs. 25 ± 5 %; p < 0.001; RVEDV: 17 ± 6 vs. 12 ± 6 %; p = 0.003). TPM mass was related to the RVFW mass in patients (baseline: R = 0.65; p < 0.001; follow-up: R = 0.80; p < 0.001) and controls (R = 0.76; p < 0.001). In PAH and controls, exclusion of TPM from the assessment resulted in altered RV mass, volumes and function than when included (all p < 0.01). Changes in RV TPM mass (ß = 0.44; p = 0.004) but not the changes in RVFW mass (p = 0.095) were independently related to changes in PAP during follow-up. RV TPM showed a larger contribution to total RV mass in PAH (~35 %) compared to controls (~25 %). Inclusion of TPM in the analyses significantly influenced the magnitude of the RV volumes and mass. Furthermore, TPM mass was stronger related to changes in PAP than RVFW mass. Our results implicate that TPM are important contributors to RV adaptation during pressure overload and cannot be neglected from the RV assessment.