Tumor vascularization occurs through several distinct biological processes, which not only vary between tumor type and anatomic location, but also occur simultaneously within the same cancer tissue. These processes are orchestrated by a range of secreted factors and signaling pathways and can involve participation of non-endothelial cells, such as progenitors or cancer stem cells. Anti-angiogenic therapies using either antibodies or tyrosine kinase inhibitors have been approved to treat several types of cancer. However, the benefit of treatment has so far been modest, some patients not responding at all and others acquiring resistance. It is becoming increasingly clear that blocking tumors from accessing the circulation is not an easy task to accomplish. Tumor vessel functionality and gene expression often differ vastly when comparing different cancer subtypes, and vessel phenotype can be markedly heterogeneous within a single tumor. Here, we summarize the current understanding of cellular and molecular mechanisms involved in tumor angiogenesis and discuss challenges and opportunities associated with vascular targeting.

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 upregulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates β1 integrin 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 proteolytic cleavage. The CD93-MMRN2 complex was required for activation of β1 integrin, 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 β1 integrin 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.

Glioblastoma is an aggressive brain tumor characterized by an abnormal blood vasculature that is hyperpermeable. Here, we report a novel role for CD93 in regulating angiogenesis in this setting by modulating cell-cell and cell-matrix adhesion of endothelial cells. Tissue microarray analysis demonstrated that vascular expression of CD93 was correlated with poor survival in a clinical cohort of patients with high-grade astrocytic glioma. Similarly, intracranial growth in the GL261 mouse model of glioma was delayed significantly in CD93 À/À hosts, resulting in improved survival compared with wild-type mice. This effect was associated with increased vascular permeability and decreased vascular perfusion of tumors, indicating reduced vessel functionality in the absence of CD93. RNAi-mediated attenuation of CD93 in endothelial cells diminished VEGFinduced tube formation in a three-dimensional collagen gel. CD93 was required for efficient endothelial cell migration and proper cell polarization in vitro. Further, in endothelial cells where CD93 was attenuated, decreased cell spreading led to a severe reduction in cell adhesion, a lack of proper cell contacts, a loss of VE-cadherin, and aberrant actin stress fiber formation. Our results identify CD93 as a key regulator of glioma angiogenesis and vascular function, acting via cytoskeletal rearrangements required for cell-cell and cell-matrix adhesion. Cancer Res; 75(21); 4504-16. Ó2015 AACR.

Glioblastoma is the most aggressive type of brain tumor with poor therapeutic response and prognosis. Passage of systemically delivered pharmacological agents into the brain is largely blocked by the blood-brain-barrier (BBB), an organotypic specialization of brain endothelial cells (EC). Tumor vessels in GBM are abnormal and more permeable, but the heterogeneity of BBB breakdown in different parts of the tumor vasculature and at the tumor's invasive front is largely unknown. Here, through single-cell RNA sequencing (scRNA-seq) of freshly isolated ECs from human glioblastoma and paired tumor peripheral tissues, we have constructed a molecular atlas of human brain ECs providing unprecedented molecular insight into the heterogeneity of the human BBB and its molecular alteration in glioblastoma. We identified 5 distinct EC phenotypes representing different states of EC activation and BBB impairment, and associated with different anatomical locations within and around the tumor. This unique data resource provides key information for designing rational therapeutic regimens and optimizing drug delivery.

The immature and dysfunctional vascular network within solid tumors poses a substantial obstacle to immunotherapy because it creates a hypoxic tumor microenvironment that actively limits immune cell infiltration. The molecular basis underpinning this vascular dysfunction is not fully understood. Using genome-scale receptor array technology, we showed here that insulin-like growth factor binding protein 7 (IGFBP7) interacts with its receptor CD93, and we subsequently demonstrated that this interaction contributes to abnormal tumor vasculature. Both CD93 and IGFBP7 were up-regulated in tumor-associated endothelial cells. IGFBP7 interacted with CD93 via a domain different from multimerin-2, the known ligand for CD93. In two mouse tumor models, blockade of the CD93/IGFBP7 interaction by monoclonal antibodies promoted vascular maturation to reduce leakage, leading to reduced tumor hypoxia and increased tumor perfusion. CD93 blockade in mice increased drug delivery, resulting in an improved antitumor response to gemcitabine or fluorouracil. Blockade of the CD93 pathway triggered a substantial increase in intratumoral effector T cells, thereby sensitizing mouse tumors to immune checkpoint therapy. Last, analysis of samples from patients with cancer under anti–programmed death 1/programmed death-ligand 1 treatment revealed that overexpression of the IGFBP7/CD93 pathway was associated with poor response to therapy. Thus, our study identified a molecular interaction involved in tumor vascular dysfunction and revealed an approach to promote a favorable tumor microenvironment for therapeutic intervention.

BackgroundVascular gene expression patterns in lower-grade gliomas (LGGs; diffuse World Health Organization [WHO] grades II–III gliomas) have not been thoroughly investigated. The aim of this study was to molecularly characterize LGG vessels and determine if tumor isocitrate dehydrogenase (IDH) mutation status affects vascular phenotype.MethodsGene expression was analyzed using an in-house dataset derived from microdissected vessels and total tumor samples from human glioma in combination with expression data from 289 LGG samples available in the database of The Cancer Genome Atlas. Vascular protein expression was examined by immunohistochemistry in human brain tumor tissue microarrays (TMAs) representing WHO grades II–IV gliomas and nonmalignant brain samples. Regulation of gene expression was examined in primary endothelial cells in vitro.ResultsGene expression analysis of WHO grade II glioma indicated an intermediate stage of vascular abnormality, less severe than that of glioblastoma vessels but distinct from normal vessels. Enhanced expression of laminin subunit alpha 4 (LAMA4) and angiopoietin 2 (ANGPT2) in WHO grade II glioma was confirmed by staining of human TMAs. IDH wild-type LGGs displayed a specific angiogenic gene expression signature, including upregulation of ANGPT2 and serpin family H (SERPINH1), connected to enhanced endothelial cell migration and matrix remodeling. Transcription factor analysis indicated increased transforming growth factor beta (TGFβ) and hypoxia signaling in IDH wild-type LGGs. A subset of genes specifically induced in IDH wild-type LGG vessels was upregulated by stimulation of endothelial cells with TGFβ2, vascular endothelial growth factor, or cobalt chloride in vitro.ConclusionIDH wild-type LGG vessels are molecularly distinct from the vasculature of IDH-mutated LGGs. TGFβ and hypoxia-related signaling pathways may be potential targets for anti-angiogenic therapy of IDH wild-type LGG.

Objective-High levels of circulating low-density lipoproteins (LDL) are a major atherosclerotic risk factor. The effects of intimal LDL on vascular smooth muscle cell (VSMC) phenotype and function during plaque remodeling and vascular repair are not fully understood. We have investigated whether exposure of VSMC to LDL induces changes on the proteomic profile of the heat shock protein (HSP) family-molecular chaperones involved in atherosclerosis. Methods & Results-2D electrophoresis demonstrates that LDL modifies the proteomic profile of HSP27 (HSPB1).Western blot analysis evidenced a significant HSP27 dephosphorylation after exposure of cells to native LDL (nLDL) and aggregated-LDL (agLDL) for 24 hours (PϽ0.05). Dephosphorylation of HSP27 was not related to changes in p38 MAPK phosphorylation. Both nLDL and agLDL induced relocalization of unphosphorylated HSP27 to the tip of actin stress fibers and focal adhesion structures in VSMC. During cell adhesion, phospho-HSP27 was located at the cell surface contact region in LDL-treated cells, whereas it remained cytosolic in control cells. Immunohistochemistry studies showed that phosphorylated HSP27 is rarely found in lipid-rich areas of atherosclerotic plaques in human coronary arteries. (oxidation, glycosylation, aggregation). Modified LDL have been associated with changes in endothelial function, 2 as well as with migration, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs). 3 Migration and proliferation of VSMCs, as well as extracellular matrix production and catabolism, are important events in the development of atherosclerosis and arterial remodeling. 4 VSMCs acquire or lose various functions to be able to fulfill the requirements for intimal remodeling. 5 Previous studies from our group demonstrated that LDL induce changes on VSMC gene expression and phenotype, leading to alterations in vascular function. 6 -8 We have recently demonstrated that aggregated LDL (agLDL) impair VSMC migration and wound repair after injury. 9 In response to stress, cells activate cytoprotective pathways. Heat shock proteins (HSP) are molecular chaperones that have the ability to protect proteins from damage induced by factors such as free radicals, heat, or ischemia. It has been demonstrated that HSP are highly expressed in atherosclerotic lesions of humans, rabbits, and apolipoprotein E-deficient mice. 10 Interestingly, HSP90 (HSP90AA1) expression is induced in VSMC by oxidative stress 11 and plays a role in the maintenance of endothelial barrier integrity by binding endothelial nitric oxide synthase and guanylyl cyclase (reviewed in Antonova et al 12 ). Overexpression of HSP70 (HSPA1A) in mouse heart increases resistance to ischemic injury. 13 Small HSP can also protect against ischemic injury in cardiomyocytes. 14 Among small HSP, HSP27 (HSPB1) acts as a molecular chaperone in vitro 15 and is also involved in F-actin assembly. 16,17 In smooth muscle, HSP27 is constitutively expressed and, when phosphorylated, colocalizes with contractile proteins. 18 In th...

CD40-stimulating immunotherapy can elicit potent anti-tumor responses by activating dendritic cells and enhancing T-cell priming. Tumor vessels orchestrate T-cell recruitment during immune response, but the effect of CD40-stimulating immunotherapy on tumor endothelial cells has not been evaluated. Here, we have investigated how tumor endothelial cells transcriptionally respond to CD40-stimulating immunotherapy by isolating tumor endothelial cells from agonistic CD40 mAb-or isotype-treated mice bearing B16-F10 melanoma, and performing RNA-sequencing. Gene set enrichment analysis revealed that agonistic CD40 mAb therapy increased interferon (IFN)-related responses in tumor endothelial cells, including up-regulation of the immunosuppressive enzyme Indoleamine 2, 3-Dioxygenase 1 (IDO1). IDO1 was predominantly expressed in endothelial cells within the tumor microenvironment, and its expression in tumor endothelium was positively correlated to T-cell infiltration and to increased intratumoral expression of IFNγ. In vitro, endothelial cells up-regulated IDO1 in response to T-cell-derived IFNγ, but not in response to CD40-stimulation. Combining agonistic CD40 mAb therapy with the IDO1 inhibitor epacadostat delayed tumor growth in B16-F10 melanoma, associated with increased activation of tumor-infiltrating T-cells. Hereby, we show that the tumor endothelial cells up-regulate IDO1 upon CD40-stimulating immunotherapy in response to increased IFNγ-secretion by T-cells, revealing a novel immunosuppressive feedback mechanism whereby tumor vessels limit T-cell activation.