Pulmonary MicroRNA Changes Alter Angiogenesis in Chronic Obstructive Pulmonary Disease and Lung Cancer.

The pulmonary endothelium is dysfunctional in chronic obstructive pulmonary disease (COPD), a known risk factor for lung cancer. The pulmonary endothelium is altered in emphysema, which is disproportionately affected by cancers. Gene and microRNA expression differs between COPD and non-COPD lung. We hypothesised that the alteration in microRNA expression in the pulmonary endothelium contributes to its dysfunction. A total of 28 patients undergoing pulmonary resection were recruited and endothelial cells were isolated from healthy lung and tumour. MicroRNA expression was compared between COPD and non-COPD patients. Positive findings were confirmed by quantitative polymerase chain reaction (qPCR). Assays assessing angiogenesis and cellular migration were conducted in Human Umbilical Vein Endothelial Cells (n = 3-4) transfected with microRNA mimics and compared to cells transfected with negative control RNA. Expression of miR-181b-3p, miR-429 and miR-23c (all p < 0.05) was increased in COPD. Over-expression of miR-181b-3p was associated with reduced endothelial sprouting (p < 0.05). miR-429 was overexpressed in lung cancer as well and exhibited a reduction in tubular formation. MicroRNA-driven changes in the pulmonary endothelium thus represent a novel mechanism driving emphysema. These processes warrant further study to determine if they may be therapeutic targets in COPD and lung cancer.

CAR T cells targeting tumor endothelial marker CLEC14A inhibit tumor growth.

Engineering T cells to express chimeric antigen receptors (CARs) specific for antigens on hematological cancers has yielded remarkable clinical responses, but with solid tumors, benefit has been more limited. This may reflect lack of suitable target antigens, immune evasion mechanisms in malignant cells, and/or lack of T cell infiltration into tumors. An alternative approach, to circumvent these problems, is targeting the tumor vasculature rather than the malignant cells directly. CLEC14A is a glycoprotein selectively overexpressed on the vasculature of many solid human cancers and is, therefore, of considerable interest as a target antigen. Here, we generated CARs from 2 CLEC14A-specific antibodies and expressed them in T cells. In vitro studies demonstrated that, when exposed to their target antigen, these engineered T cells proliferate, release IFN-γ, and mediate cytotoxicity. Infusing CAR engineered T cells into healthy mice showed no signs of toxicity, yet these T cells targeted tumor tissue and significantly inhibited tumor growth in 3 mouse models of cancer (Rip-Tag2, mPDAC, and Lewis lung carcinoma). Reduced tumor burden also correlated with significant loss of CLEC14A expression and reduced vascular density within malignant tissues. These data suggest the tumor vasculature can be safely and effectively targeted with CLEC14A-specific CAR T cells, offering a potent and widely applicable therapy for cancer.

An evaluation of the tumour endothelial marker CLEC14A as a therapeutic target in solid tumours.

Earlier studies identified the transmembrane cell surface C-type lectin CLEC14A as a putative tumour endothelial marker. For CLEC14A to progress as a vascular target in solid tumours an in-depth analysis of CLEC14A expression in human healthy and tumour tissue is needed. It is here shown that an analysis of 5332 RNA expression profiles in the public domain confirmed high expression of CLEC14A in tumour compared to healthy human tissue. It is further shown by immunohistochemistry that CLEC14A protein is absent, or expressed at a very low level, in healthy human and primate tissue. In contrast, CLEC14A is expressed on the vasculature of a range of human solid tumours, with particularly high expression in more than half of renal cell carcinomas. Elevated levels of CLEC14A transcripts were identified in some non-cancer pathologies; such comorbidities may need to be excluded from trials of therapies targeting this marker. It is further shown that, as CLEC14A expression can be induced by the absence of shear stress, it is imperative that freshly collected as opposed to aged or post-mortem tissue be analysed. We conclude that CLEC14A is a promising target to enable development of novel anti-cancer therapies for solid tumours.

Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin-binding protein.

Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.

C-type lectin domain group 14 proteins in vascular biology, cancer and inflammation.

The C-type lectin domain (CTLD) group 14 family of transmembrane glycoproteins consist of thrombomodulin, CD93, CLEC14A and CD248 (endosialin or tumour endothelial marker-1). These cell surface proteins exhibit similar ectodomain architecture and yet mediate a diverse range of cellular functions, including but not restricted to angiogenesis, inflammation and cell adhesion. Thrombomodulin, CD93 and CLEC14A can be expressed by endothelial cells, whereas CD248 is expressed by vasculature associated pericytes, activated fibroblasts and tumour cells among other cell types. In this article, we review the current literature of these family members including their expression profiles, interacting partners, as well as established and speculated functions. We focus primarily on their roles in the vasculature and inflammation as well as their contributions to tumour immunology. The CTLD group 14 family shares several characteristic features including their ability to be proteolytically cleaved and engagement of some shared extracellular matrix ligands. Each family member has strong links to tumour development and in particular CD93, CLEC14A and CD248 have been proposed as attractive candidate targets for cancer therapy.

Sphingosine-1-phosphate signalling drives an angiogenic transcriptional programme in diffuse large B cell lymphoma.

Although the over-expression of angiogenic factors is reported in diffuse large B-cell lymphoma (DLBCL), the poor response to anti-VEGF drugs observed in clinical trials suggests that angiogenesis in these tumours might be driven by VEGF-independent pathways. We show that sphingosine kinase-1 (SPHK1), which generates the potent bioactive sphingolipid sphingosine-1-phosphate (S1P), is over-expressed in DLBCL. A meta-analysis of over 2000 cases revealed that genes correlated with SPHK1 mRNA expression in DLBCL were significantly enriched for tumour angiogenesis meta-signature genes; an effect evident in both major cell of origin (COO) and stromal subtypes. Moreover, we found that S1P induces angiogenic signalling and a gene expression programme that is present within the tumour vasculature of SPHK1-expressing DLBCL. Importantly, S1PR1 functional antagonists, including Siponimod, and the S1P neutralising antibody, Sphingomab, inhibited S1P signalling in DLBCL cells in vitro. Furthermore, Siponimod, also reduced angiogenesis and tumour growth in an S1P-producing mouse model of angiogenic DLBCL. Our data define a potential role for S1P signalling in driving an angiogenic gene expression programme in the tumour vasculature of DLBCL and suggest novel opportunities to target S1P-mediated angiogenesis in patients with DLBCL.

Iridium Nanoparticles for Multichannel Luminescence Lifetime Imaging, Mapping Localization in Live Cancer Cells.

The development of long-lived luminescent nanoparticles for lifetime imaging is of wide interest as luminescence lifetime is environmentally sensitive detection independent of probe concentration. We report novel iridium-coated gold nanoparticles as probes for multiphoton lifetime imaging with characteristic long luminescent lifetimes based on iridium luminescence in the range of hundreds of nanoseconds and a short signal on the scale of picoseconds based on gold allowing multichannel detection. The tailor-made IrC6 complex forms stable, water-soluble gold nanoparticles (AuNPs) of 13, 25, and 100 nm, bearing 1400, 3200, and 22 000 IrC6 complexes per AuNP, respectively. The sensitivity of the iridium signal on the environment of the cell is evidenced with an observed variation of lifetimes. Clusters of iridium nanoparticles show lifetimes from 450 to 590 ns while lifetimes of 660 and 740 ns are an average of different points in the cytoplasm and nucleus. Independent luminescence lifetime studies of the nanoparticles in different media and under aggregation conditions postulate that the unusual long lifetimes observed can be attributed to interaction with proteins rather than nanoparticle aggregation. Total internal reflection fluorescence microscopy (TIRF), confocal microscopy studies and 3D luminescence lifetime stacks confirm the presence of bright, nonaggregated nanoparticles inside the cell. Inductively coupled plasma mass spectrometry (ICPMS) analysis further supports the presence of the nanoparticles in cells. The iridium-coated nanoparticles provide new nanoprobes for lifetime detection with dual channel monitoring. The combination of the sensitivity of the iridium signal to the cell environment together with the nanoscaffold to guide delivery offer opportunities for iridium nanoparticles for targeting and tracking in in vivo models.

Sunitinib Treatment Enhances Metastasis of Innately Drug-Resistant Breast Tumors.

Antiangiogenic therapies have failed to confer survival benefits in patients with metastatic breast cancer (mBC). However, to date, there has not been an inquiry into the roles for acquired versus innate drug resistance in this setting. In this study, we report roles for these distinct phenotypes in determining therapeutic response in a murine model of mBC resistance to the antiangiogenic tyrosine kinase inhibitor sunitinib. Using tumor measurement and vascular patterning approaches, we differentiated tumors displaying innate versus acquired resistance. Bioluminescent imaging of tumor metastases to the liver, lungs, and spleen revealed that sunitinib administration enhances metastasis, but only in tumors displaying innate resistance to therapy. Transcriptomic analysis of tumors displaying acquired versus innate resistance allowed the identification of specific biomarkers, many of which have a role in angiogenesis. In particular, aquaporin-1 upregulation occurred in acquired resistance, mTOR in innate resistance, and pleiotrophin in both settings, suggesting their utility as candidate diagnostics to predict drug response or to design tactics to circumvent resistance. Our results unravel specific features of antiangiogenic resistance, with potential therapeutic implications. Cancer Res; 77(4); 1008-20. ©2016 AACR.

Tube-Forming Assays.

Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

Development of Antibody-Based Vaccines Targeting the Tumor Vasculature.

A functional vasculature is essential for tumor progression and malignant cell metastasis. Endothelial cells lining blood vessels in the tumor are exposed to a unique microenvironment, which in turn induces expression of specific proteins designated as tumor endothelial markers (TEMs). TEMs either localized at the plasma membrane or secreted into the extracellular matrix are accessible for antibody targeting, which can be either infused or generated de novo via vaccination. Recent studies have demonstrated vaccines against several TEMs can induce a strong antibody response accompanied by a potent antitumor effect in animal models. These findings present an exciting field for novel anticancer therapy development. As most of the TEMs are self-antigens, breaking tolerance is necessary for a successful vaccine. This chapter describes approaches to efficiently induce a robust antibody response against the tumor vasculature.

A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks in a wide spectrum of biological systems.

Glutamate dependent NMDA receptor 2D is a novel angiogenic tumour endothelial marker in colorectal cancer.

Current vascular-targeted therapies in colorectal cancer (CRC) have shown limited benefit. The lack of novel, specific treatment in CRC has been hampered by a dearth of specific endothelial markers. Microarray comparison of endothelial gene expression in patient-matched CRC and normal colon identified a panel of putative colorectal tumour endothelial markers. Of these the glutamate dependent NMDA receptor GRIN2D emerged as the most interesting target. GRIN2D expression was shown to be specific to colorectal cancer vessels by RTqPCR and IHC analysis. Its expression was additionally shown be predictive of improved survival in CRC. Targeted knockdown studies in vitro demonstrated a role for GRIN2D in endothelial function and angiogenesis. This effect was also shown in vivo as vaccination against the extracellular region of GRIN2D resulted in reduced vascularisation in the subcutaneous sponge angiogenesis assay. The utility of immunologically targeting GRIN2D in CRC was demonstrated by the vaccination approach inhibiting murine CRC tumour growth and vascularisation. GRIN2D represents a promising target for the future treatment of CRC.

Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2).

C-type lectin family 14, member A (CLEC14A), is a single-pass transmembrane glycoprotein that is overexpressed in tumor endothelial cells, and it promotes sprouting angiogenesis and modulates endothelial function via interactions with extracellular matrix proteins. Here, we show that CLEC14A is cleaved by rhomboid-like protein 2 (RHBDL2), one of 3 catalytic mammalian rhomboid-like (RHBDL) proteases, but that it is not cleaved by RHBDL1 or -3. Site-directed mutagenesis identified the precise site at which RHBDL2 cleaves CLEC14A, and targeted, small interfering RNAs that knockdown endogenous CLEC14A and RHBDL2 in human endothelial cells validated the specificity of CLEC14A shedding by RHBDL2. Loss of endogenous cleaved CLEC14A increased endothelial migration 2-fold, whereas that addition of recombinant cleaved CLEC14A inhibited the sprouting of human and murine endothelial cells 3-fold in several in vitro models. We assessed the in vivo role of cleaved CLEC14A in angiogenesis by using the rodent subcutaneous sponge implant model, and we found that CLEC14A protein inhibited vascular density by >50%. Finally, we show that cleaved CLEC14A binds to sprouting endothelial tip cells. Our data show that the ectodomain of CLEC14A regulates sprouting angiogenesis and suggests a role for RHBDL2 in endothelial function.-Noy, P. J., Swain, R. K., Khan, K., Lodhia, P., Bicknell, R. Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2).

MCAM and LAMA4 Are Highly Enriched in Tumor Blood Vessels of Renal Cell Carcinoma and Predict Patient Outcome.

The structure and molecular signature of tumor-associated vasculature are distinct from those of the host tissue, offering an opportunity to selectively target the tumor blood vessels. To identify tumor-specific endothelial markers, we performed a microarray on tumor-associated and nonmalignant endothelium collected from patients with renal cell carcinoma (RCC), colorectal carcinoma, or colorectal liver metastasis. We identified a panel of genes consistently upregulated by tumor blood vessels, of which melanoma cell adhesion molecule (MCAM) and its extracellular matrix interaction partner laminin alpha 4 (LAMA4) emerged as the most consistently expressed genes. This result was subsequently confirmed by immunohistochemical analysis of MCAM and LAMA4 expression in RCC and colorectal carcinoma blood vessels. Strong MCAM and LAMA4 expression was also shown to predict poor survival in RCC, but not in colorectal carcinoma. Notably, MCAM and LAMA4 were enhanced in locally advanced tumors as well as both the primary tumor and secondary metastases. Expression analysis in 18 different cancers and matched healthy tissues revealed vascular MCAM as highly specific in RCC, where it was induced strongly by VEGF, which is highly abundant in this disease. Lastly, MCAM monoclonal antibodies specifically localized to vessels in a murine model of RCC, offering an opportunity for endothelial-specific targeting of anticancer agents. Overall, our findings highlight MCAM and LAMA4 as prime candidates for RCC prognosis and therapeutic targeting. Cancer Res; 76(8); 2314-26. ©2016 AACR.

Anti-angiogenic alternatives to VEGF blockade.

Angiogenesis is a major requirement for tumour formation and development. Anti-angiogenic treatments aim to starve the tumour of nutrients and oxygen and also guard against metastasis. The main anti-angiogenic agents to date have focused on blocking the pro-angiogenic vascular endothelial growth factors (VEGFs). While this approach has seen some success and has provided a proof of principle that such anti-angiogenic agents can be used as treatment, the overall outcome of VEGF blockade has been somewhat disappointing. There is a current need for new strategies in inhibiting tumour angiogenesis; this article will review current and historical examples in blocking various membrane receptors and components of the extracellular matrix important in angiogenesis. Targeting these newly discovered pro-angiogenic proteins could provide novel strategies for cancer therapy.

Inference of Low and High-Grade Glioma Gene Regulatory Networks Delineates the Role of Rnd3 in Establishing Multiple Hallmarks of Cancer.

Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.

FKBPL is a critical antiangiogenic regulator of developmental and pathological angiogenesis.

OBJECTIVE: The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models. APPROACH AND RESULTS: FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL's critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl(+/-) mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish. CONCLUSIONS: FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes.

Robo4 vaccines induce antibodies that retard tumor growth.

Tumor endothelial specific expression of Robo4 in adults identifies this plasma membrane protein as an anti-cancer target for immunotherapeutic approaches, such as vaccination. In this report, we describe how vaccination against Robo4 inhibits angiogenesis and tumor growth. To break tolerance to the auto-antigen Robo4, mice were immunised with the extracellular domain of mouse Robo4, fused to the Fc domain of human immunoglobulin within an adjuvant. Vaccinated mice show a strong antibody response to Robo4, with no objectively detectable adverse effects on health. Robo4 vaccinated mice showed impaired fibrovascular invasion and angiogenesis in a rodent sponge implantation assay, as well as a reduced growth of implanted syngeneic Lewis lung carcinoma. The anti-tumor effect of Robo4 vaccination was present in CD8 deficient mice but absent in B cell or IgG1 knockout mice, suggesting antibody dependent cell mediated cytotoxicity as the anti-vascular/anti-tumor mechanism. Finally, we show that an adjuvant free soluble Robo4-carrier conjugate can retard tumor growth in carrier primed mice. These results point to appropriate Robo4 conjugates as potential anti-angiogenic vaccines for cancer patients.

In vivo evidence for platelet-induced physiological angiogenesis by a COX driven mechanism.

We sought to determine a role for platelets in in vivo angiogenesis, quantified by changes in the capillary to fibre ratio (C:F) of mouse skeletal muscle, utilising two distinct forms of capillary growth to identify differential effects. Capillary sprouting was induced by muscle overload, and longitudinal splitting by chronic hyperaemia. Platelet depletion was achieved by anti-GPIbα antibody treatment. Sprouting induced a significant increase in C:F (1.42±0.02 vs. contralateral 1.29±0.02, P<0.001) that was abolished by platelet depletion, while the significant C:F increase caused by splitting (1.40±0.03 vs. control 1.28±0.03, P<0.01) was unaffected. Granulocyte/monocyte depletion showed this response was not immune-regulated. VEGF overexpression failed to rescue angiogenesis following platelet depletion, suggesting the mechanism is not simply reliant on growth factor release. Sprouting occurred normally following antibody-induced GPVI shedding, suggesting platelet activation via collagen is not involved. BrdU pulse-labelling showed no change in the proliferative potential of cells associated with capillaries after platelet depletion. Inhibition of platelet activation by acetylsalicylic acid abolished sprouting, but not splitting angiogenesis, paralleling the response to platelet depletion. We conclude that platelets differentially regulate mechanisms of angiogenesis in vivo, likely via COX signalling. Since endothelial proliferation is not impaired, we propose a link between COX1 and induction of endothelial migration.