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Background

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, characterized by aggressive growth and a dismal prognosis. Interleukin-33 (IL-33) and its receptor ST2 have emerged as regulators of glioma growth, but their exact function in tumorigenesis has not been deciphered. Indeed, previous studies on IL-33 in cancer have yielded somewhat opposing results as to whether it is pro- or anti-tumorigenic.

Methods

IL-33 expression was assessed in a GBM tissue microarray and public databases. As in vivo models we used orthotopic xenografts of patient-derived GBM cells, and syngenic models with grafted mouse glioma cells.

Results

We analyzed the role of IL-33 and its receptor ST2 in nonmalignant cells of the glioma microenvironment and found that IL-33 levels are increased in cells surrounding the tumor. Protein complexes of IL-33 and ST2 are mainly found outside of the tumor core. The IL-33-producing cells consist primarily of oligodendrocytes. To determine the function of IL-33 in the tumor microenvironment, we used mice lacking the ST2 receptor. When glioma cells were grafted to ST2-deficient mouse brains, the resulting tumors exhibited a more invasive growth pattern, and are associated with poorer survival, compared to wild-type mice. Tumors in ST2-deficient hosts are more invasive, with increased expression of extracellular matrix remodeling enzymes and enhanced tumor angiogenesis. Furthermore, the absence of ST2 leads to a more immunosuppressive environment.

Conclusions

Our findings reveal that glia-derived IL-33 and its receptor ST2 participate in modulating tumor invasiveness, tumor vasculature, and immunosuppression in glioma.

Compromised vascular integrity facilitates extravasation of cancer cells and promotes metastatic dissemination. CD93 has emerged as a target for antiangiogenic therapy, but its importance for vascular integrity in metastatic cancers has not been evaluated. Here, we demonstrate that CD93 participates in maintaining the endothelial barrier and reducing metastatic dissemination. Primary melanoma growth was hampered in CD93^–/– mice, but metastatic dissemination was increased and associated with disruption of adherens and tight junctions in tumor endothelial cells and elevated expression of matrix metalloprotease 9 at the metastatic site. CD93 directly interacted with vascular endothelial growth factor receptor 2 (VEGFR2) and its absence led to VEGF-induced hyperphosphorylation of VEGFR2 in endothelial cells. Antagonistic anti-VEGFR2 antibody therapy rescued endothelial barrier function and reduced the metastatic burden in CD93^–/– mice to wild-type levels. These findings reveal a key role of CD93 in maintaining vascular integrity, which has implications for pathological angiogenesis and endothelial barrier function in metastatic cancer.

Background: Glioblastoma multiforme (GBM) is the most common highly aggressive, primary malignant brain tumor in adults. Current experimental strategies include photodynamic therapy (PDT) and new drug delivery technologies such as nanoparticles, which could play a key role in the treatment, diagnosis, and imaging of brain tumors.

Objectives: The purpose of this study was to test the efficacy of PDT using AGuIX-TPP, a polysiloxane-based nanoparticle (AGuIX) that contains TPP (5,10,15,20-tetraphenyl-21H,23H-porphine), in biological models of glioblastoma multiforme and to investigate the vascular mechanisms of action at multiple complexity levels.

Methods: PDT effects were studied in monolayer and spheroid cell culture, as well as tumors in chicken chorioallantoic membranes (CAMs) and in mice were studied.

Results: Treatment was effective in both endothelial ECRF and glioma U87 cells, as well as in the inhibition of growth of the glioma spheroids. PDT using AGuIX-TPP inhibited U87 tumors growing in CAM and destroyed their vascularization. The U87 tumors were also grown in nude mice. Their vascular network, as well as oxygen partial pressure, were assessed using ultrasound and EPR oximetry. The treatment damaged tumor vessels and slightly decreased oxygen levels.

Conclusions: PDT with AGuIX-TPP was effective against glioma cells, spheroids, and tumors; however, in mice, its efficacy appeared to be strongly related to the presence of blood vessels in the tumor before the treatment.

Regulation of vascular permeability to plasma is essential for tissue and organ homeostasis and is mediated by endothelial cell-to-cell junctions that tightly regulate the trafficking of molecules between blood and tissue. The single-pass transmembrane glycoprotein CD93 is upregulated in endothelial cells during angiogenesis and controls cytoskeletal dynamics. However, its role in maintaining homeostasis by regulating endothelial barrier function has not been elucidated yet. Here, we demonstrate that CD93 interacts with vascular endothelial (VE)-cadherin and limits its phosphorylation and turnover. CD93 deficiency in vitro and in vivo induces phosphorylation of VE-cadherin under basal conditions, displacing it from endothelial cell–cell contacts. Consistent with this, endothelial junctions are defective in CD93^−/− mice, and the blood–brain barrier permeability is enhanced. Mechanistically, CD93 regulates VE-cadherin phosphorylation and turnover at endothelial junctions through the Rho/Rho kinase-dependent pathway. In conclusion, our results identify CD93 as a key regulator of VE-cadherin stability at endothelial junctions, opening up possibilities for therapeutic strategies directed to control vascular permeability.

Glioblastomas are aggressive brain tumors that are largely immunotherapy resistant. This is associated with immunosuppression and a dysfunctional tumor vasculature, which hinder T cell infiltration. LIGHT/TNFSF14 can induce high endothelial venules (HEVs) and tertiary lymphoid structures (TLS), suggesting that its therapeutic expression could promote T cell recruitment. Here, we use a brain endothelial cell-targeted ad-eno-associated viral (AAV) vector to express LIGHT in the glioma vasculature (AAV-LIGHT). We found that systemic AAV-LIGHT treatment induces tumor-associated HEVs and T cell-rich TLS, prolonging survival in aPD-1-resistant murine glioma. AAV-LIGHT treatment reduces T cell exhaustion and promotes TCF1+CD8+ stem-like T cells, which reside in TLS and intratumoral antigen-presenting niches. Tumor regres-sion upon AAV-LIGHT therapy correlates with tumor-specific cytotoxic/memory T cell responses. Our work reveals that altering vascular phenotype through vessel-targeted expression of LIGHT promotes efficient anti-tumor T cell responses and prolongs survival in glioma. These findings have broader implications for treatment of other immunotherapy-resistant cancers.

Determination of interactions between native proteins in cells is important for understanding function. Here the authors report MolBoolean as a method to detect interactions between endogenous proteins in subcellular compartments, using antibody-DNA conjugates for identification and signal amplification. Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.

To achieve successful vascular targeting in cancer, a better understanding of the molecular mechanisms that lead to tumour vascular abnormalities is required.  The transmembrane protein CD93 is highly expressed in the vasculature of several tumours including glioblastoma and has emerged as a potential anti-angiogenic target. This thesis work explores the mechanisms through which CD93 contributes to vascular function and facilitates tumour progression. In paper I, we identify that CD93 interacts with the extracellular matrix (ECM) glycoprotein multimerin-2, which stabilizes CD93 at the cell surface and anchors it to the ECM protein fibronectin. CD93 binds to integrinα5β1 and regulates the activity of integrinβ1 and fibronectin fibrillogenesis in vitro. Consistent with this, the tumor vessels of CD93^-/- mice bearing gliomas displayed an impaired integrinβ1 activity and fibronectin fibrillogenesis, suggesting that CD93-multimerin-2-fibronectin axis has an important role in tumour angiogenesis. In paper II, we explored the co-regulation of CD93 with other genes associated with glioblastoma vascular abnormalities. Using the publicly available Gliovis database for distinguished gene correlation analysis, we identified multimerin-2, fibronectin and angiopoietin-2 as candidate genes which are likely to be expressed with CD93 in glioblastoma vasculature. The expression of CD93, fibronectin and angiopoietin-2 was associated with high microvascular proliferation. Moreover, the presence of CD93 in a high proportion of the tumor vessels correlated with poor survival, suggesting that targeting CD93 can be beneficial for glioblastoma patients. In paper III, we explored the role of CD93 in the blood brain barrier (BBB) integrity. We demonstrate that CD93 regulates the activity of Rho-GTPases, thus stabilizing the endothelial junction molecules VE-cadherin and claudin-5 and preventing the internalization of VE-cadherin. Consistent with this, CD93^-/- mice displayed a compromised BBB and exhibited an increased vascular permeability. In paper IV, we further explored the consequences of endothelial barrier disruption upon CD93-deficiency in cancer. We demonstrate that CD93 binds to VEGFR2 and that the absence of CD93 enhances VEGF-induced VEGFR2 phosphorylation in vitro. Consistent with this, melanoma-bearing CD93^-/- mice displayed impaired vascular integrity and an enhanced MMP9 expression, leading to increased intravasation of tumour cells and increased metastatic spread. This phenotype was reversed to the wild-type level by inhibiting the VEGF-VEGFR2 signalling in CD93^-/- mice. Taken together, this thesis work reveals a key role of CD93 in regulating vascular maturation and stabilization in health and cancer, and unveils its contribution to tumour progression and metastasis.

BACKGROUND: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels, and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood.

METHODS: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1 -/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1-deletion on glioma immunity was determined by treating tumor bearing mice with PD-1-blocking antibodies.

RESULTS: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1-deletion, however, tumor vascular function was improved in ELTD1 -/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1 -/- mice. Consistent with an enhanced inflammatory response, ELTD1-deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade.

CONCLUSION: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggests that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.

Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is up-regulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates integrin-β1-signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytical cleavage. The CD93-MMRN2 complex was required for activation of integrin-β1, phosphorylation of focal adhesion kinase (FAK) and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of integrin-β1 and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.