ADAM9 is present at endothelial cell - cell junctions and regulates monocyte - endothelial transmigration.

We have found that A Disintegrin And Metalloproteinase-9 (ADAM9) localises to cell-cell junctions with VE-Cadherin in confluent endothelial monolayers. Co-cultures of cells separately transfected with ADAM9-EGFP or ADAM9-HA showed expression is required in two adjacent cells for localisation to cell-cell junctions suggesting the ADAM9 ectodomain may self-associate. A direct interaction between ADAM9 ectodomains was confirmed using recombinant proteins and an ELISA based method. As the ADAM9 ectodomain can also exist as a soluble form physiologically, we examined if this could inhibit endothelial functions dependent on cell-cell junctions. The soluble ADAM9 ectodomain could not increase endothelial monolayer permeability or inhibit monocyte-endothelial adhesion, but could inhibit monocyte-endothelial transmigration. These novel findings point to ADAM9 playing an important role in endothelial cell biology that is distinct from the other ADAMs.

Fabrication of Biodegradable Synthetic Vascular Networks and Their Use as a Model of Angiogenesis.

One of the greatest challenges currently faced in tissue engineering is the incorporation of vascular networks within tissue-engineered constructs. The aim of this study was to develop a technique for producing a perfusable, 3-dimensional, cell-friendly model of vascular structures that could be used to study the factors affecting angiogenesis and vascular biology in engineered systems in more detail. Initially, biodegradable synthetic pseudovascular networks were produced via the combination of robocasting and electrospinning techniques. The internal surfaces of the vascular channels were then recellularized with human dermal microvascular endothelial cells (HDMECs) with and without the presence of human dermal fibroblasts (HDFs) on the outer surface of the scaffold. After 7 days in culture, channels that had been reseeded with HDMECs alone demonstrated irregular cell coverage. However, when using a co-culture of HDMECs inside and HDFs outside the vascular channels, coverage was found to be continuous throughout the internal channel. Using this cell combination, collagen gels loaded with vascular endothelial growth factor were deposited onto the outer surface of the scaffold and cultured for a further 7 days. After this, endothelial cell outgrowth from within the channels into the collagen gel was observed, showing that the engineered vasculature maintains its capacity for angiogenesis. Furthermore, the HDMECs appeared to have formed perfusable tubules within the gel. These results show promising steps towards the development of an in vitro platform for studying angiogenesis and vascular biology in a tissue engineering context.

The use of microphysiological systems to model metastatic cancer.

Cancer is one of the leading causes of death in the 21st century, with metastasis of cancer attributing to 90% of cancer-related deaths. Therefore, to improve patient outcomes there is a need for better preclinical models to increase the success of translating oncological therapies into the clinic. Current traditional staticin vitromodels lack a perfusable network which is critical to overcome the diffusional mass transfer limit to provide a mechanism for the exchange of essential nutrients and waste removal, and increase their physiological relevance. Furthermore, these models typically lack cellular heterogeneity and key components of the immune system and tumour microenvironment. This review explores rapidly developing strategies utilising perfusable microphysiological systems (MPS) for investigating cancer cell metastasis. In this review we initially outline the mechanisms of cancer metastasis, highlighting key steps and identifying the current gaps in our understanding of the metastatic cascade, exploring MPS focused on investigating the individual steps of the metastatic cascade before detailing the latest MPS which can investigate multiple components of the cascade. This review then focuses on the factors which can affect the performance of an MPS designed for cancer applications with a final discussion summarising the challenges and future directions for the use of MPS for cancer models.

Development of PCL PolyHIPE Substrates for 3D Breast Cancer Cell Culture.

Cancer is a becoming a huge social and economic burden on society, becoming one of the most significant barriers to life expectancy in the 21st century. In particular, breast cancer is one of the leading causes of death for women. One of the most significant difficulties to finding efficient therapies for specific cancers, such as breast cancer, is the efficiency and ease of drug development and testing. Tissue-engineered (TE) in vitro models are rapidly developing as an alternative to animal testing for pharmaceuticals. Additionally, porosity included within these structures overcomes the diffusional mass transfer limit whilst enabling cell infiltration and integration with surrounding tissue. Within this study, we investigated the use of high-molecular-weight polycaprolactone methacrylate (PCL-M) polymerised high-internal-phase emulsions (polyHIPEs) as a scaffold to support 3D breast cancer (MDA-MB-231) cell culture. We assessed the porosity, interconnectivity, and morphology of the polyHIPEs when varying mixing speed during formation of the emulsion, successfully demonstrating the tunability of these polyHIPEs. An ex ovo chick chorioallantoic membrane assay identified the scaffolds as bioinert, with biocompatible properties within a vascularised tissue. Furthermore, in vitro assessment of cell attachment and proliferation showed promising potential for the use of PCL polyHIPEs to support cell growth. Our results demonstrate that PCL polyHIPEs are a promising material to support cancer cell growth with tuneable porosity and interconnectivity for the fabrication of perfusable 3D cancer models.

Gelatin-containing porous polycaprolactone PolyHIPEs as substrates for 3D breast cancer cell culture and vascular infiltration.

Tumour survival and growth are reliant on angiogenesis, the formation of new blood vessels, to facilitate nutrient and waste exchange and, importantly, provide a route for metastasis from a primary to a secondary site. Whilst current models can ensure the transport and exchange of nutrients and waste via diffusion over distances greater than 200 µm, many lack sufficient vasculature capable of recapitulating the tumour microenvironment and, thus, metastasis. In this study, we utilise gelatin-containing polymerised high internal phase emulsion (polyHIPE) templated polycaprolactone-methacrylate (PCL-M) scaffolds to fabricate a composite material to support the 3D culture of MDA-MB-231 breast cancer cells and vascular ingrowth. Firstly, we investigated the effect of gelatin within the scaffolds on the mechanical and chemical properties using compression testing and FTIR spectroscopy, respectively. Initial in vitro assessment of cell metabolic activity and vascular endothelial growth factor expression demonstrated that gelatin-containing PCL-M polyHIPEs are capable of supporting 3D breast cancer cell growth. We then utilised the chick chorioallantoic membrane (CAM) assay to assess the angiogenic potential of cell-seeded gelatin-containing PCL-M polyHIPEs, and vascular ingrowth within cell-seeded, surfactant and gelatin-containing scaffolds was investigated via histological staining. Overall, our study proposes a promising composite material to fabricate a substrate to support the 3D culture of cancer cells and vascular ingrowth.

MT1-MMP regulates VEGF-A expression through a complex with VEGFR-2 and Src.

Membrane-type-1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent type-I transmembrane metalloproteinase involved in pericellular proteolysis, migration and invasion, with elevated levels correlating with a poor prognosis in cancer. MT1-MMP-mediated transcriptional regulation of genes in cancer cells can contribute to tumour growth, although this is poorly understood at a mechanistic level. In this study, we investigated the mechanism by which MT1-MMP regulates the expression of VEGF-A in breast cancer cells. We discovered that MT1-MMP regulates VEGFR-2 cell surface localisation and forms a complex with VEGFR-2 and Src that is dependent on the MT1-MMP hemopexin domain and independent of its catalytic activity. Although the localisation of VEGFR-2 was independent of the catalytic and intracellular domain of MT1-MMP, intracellular signalling dependent on VEGFR-2 activity leading to VEGF-A transcription still required the MT1-MMP catalytic and intracellular domain, including residues Y573, C574 and DKV582. However, there was redundancy in the function of the catalytic activity of MT1-MMP, as this could be substituted with MMP-2 or MMP-7 in cells expressing inactive MT1-MMP. The signalling cascade dependent on the MT1-MMP-VEGFR-2-Src complex activated Akt and mTOR, ultimately leading to increased VEGF-A transcription.

LPS activates ADAM9 dependent shedding of ACE from endothelial cells.

Angiotensin-I converting enzyme (ACE) is a zinc dependent peptidase with a major role in regulating vasoactive peptide metabolism. ACE, a transmembrane protein, undergoes proteolysis, or shedding, by an as yet unidentified proteinase to release a catalytically active soluble form of the enzyme. Physiologically, soluble ACE in plasma is derived primarily from endothelial cells. We demonstrate that ACE shedding from confluent endothelial cells is increased in response to bacterial lipopolysaccharide, but not phorbol esters. Characterisation of lipopolysaccharide stimulated shedding showed that there is a lag phase before soluble ACE can be detected which is sensitive to inhibitors of translation, NF-κB, TNFα and TNFR-I/II. The shedding phase is less sensitive to these inhibitors, but is ablated by BB-94, a Matrix Metalloproteinase (MMP)/A Disintegrin and Metalloproteinase (ADAM) inhibitor. Tissue Inhibitor of Metalloproteinase (TIMP) profiling suggested a requirement for ADAM9 in lipopolysaccharide induced ACE shedding, which was confirmed by depletion with siRNA. Transient transfection of ADAM9 and ACE cDNAs into HEK293 cells demonstrated that ADAM9 requires both membrane anchorage and its catalytic domain to shed ACE.

Overexpression of the oncostatin M receptor in cervical squamous cell carcinoma cells is associated with a pro-angiogenic phenotype and increased cell motility and invasiveness.

Oncostatin M receptor (OSMR) shows frequent copy number gain and overexpression in advanced cervical squamous cell carcinoma (SCC). We used cell-based in vitro assays, RNA interference, and integrative gene expression profiling to investigate the functional significance of this observation. CaSki and SW756 were selected as representative cervical SCC cells that overexpressed OSMR, and ME180 and MS751 as cells that did not. The STAT-dependent pro-angiogenic factors VEGF-A and ID1 were rapidly induced by OSM in CaSki/SW756 but not in ME180/MS751. However, rapid induction did occur in MS751 following forced OSMR overexpression, while depleting OSMR in CaSki abrogated VEGF-A expression. Conditioned medium from both CaSki and SW756 stimulated endothelial tube formation in vitro, effects that were inhibited by depleting OSMR in the SCC cells. For both CaSki and SW756, migration in a wound healing assay and invasion through Matrigel were stimulated by OSM and consistently inhibited by OSMR depletion. The phenotype was rescued by transfection with OSMR containing a silent mutation that provided specific siRNA resistance. Overall, there was a positive correlation between OSMR levels and invasiveness. We used gene expression profiling to identify genes induced by OSM in CaSki/SW756 but not in ME180/MS751. The most prominent gene ontology category groups for the differentially expressed genes were cell motility/invasion, angiogenesis, signal transduction, and apoptosis. We also profiled 23 cervical SCC samples, identifying genes that were differentially expressed in cases with OSMR overexpression versus those without. Integration of the datasets identified 15 genes that showed consistent differential expression in association with OSMR levels in vitro and in vivo. We conclude that OSMR overexpression in cervical SCC cells provides increased sensitivity to OSM, which induces pro-malignant changes. OSMR is a potential prognostic and therapeutic target in cervical SCC. The genes that mediate OSM:OSMR effects will be valuable indicators of the effectiveness of antibody blockade in pre-clinical systems.

Modulation of integrin α4ß1 by ADAM28 promotes lymphocyte adhesion and transendothelial migration.

ADAMs (a disintegrin and metalloproteinase) are a family of type I transmembrane glycoproteins related to snake venom metalloproteases and disintegrins. They are regulatory proteins that modulate intercellular adhesion and the bioavailability of growth factors, and have been implicated in many disease states, including cancer, immunity and inflammation. One member of the ADAM family, ADAM28, has been reported to bind to the integrin α4ß1 in humans; however, the distribution of ADAM28 and the biological consequences of ADAM28-α4ß1 interactions are yet to be fully elucidated. The expression of ADAM28 in human and murine tissues was examined by multiple Affymetrix microarray analyses, real-time RT-PCR (reverse transcription-PCR) and immunohistochemical staining. We found that ADAM28 has a relatively restricted expression pattern in mouse and human and is highly expressed in the B-lymphocyte lineage, including chronic lymphocytic leukaemic B-cells. The murine B-lymphoma line L1-2 and recombinant soluble murine ADAM28 were used to investigate ADAM28-α4ß1 interactions. Our data reveal that ADAM28 binding to α4ß1 is typical of integrin-ligand interactions, since it is attenuated by anti-functional integrin antibodies, and is enhanced by Mn2+ and the integrin mAb (monoclonal antibody) 9EG7. However, a key finding was that soluble ADAM28 unexpectedly enhanced α4ß1-dependent cell adhesion to VCAM-1 (vascular cell adhesion molecule-1). In so doing ADAM28 was able to influence lymphocyte adhesion to, and migration through, endothelial monolayers, suggesting a physiological role for ADAM28 in regulating the specific spatial and temporal transendothelial migration of lymphocytes.

Debunking the Myth of the Endogenous Antiangiogenic Vegfaxxxb Transcripts.

In this opinion article we critically assess evidence for the existence of a family of antiangiogenic vascular endothelial growth factor (Vegfaxxxb) transcripts, arising from the use of a phylogenetically conserved alternative distal splice site within exon 8 of the VEGFA gene. We explain that prior evidence for Vegfaxxxb transcripts in tissues rests heavily upon flawed RT-PCR methodologies, with the extensive use of 5'-tailing in primer design being the main issue. Furthermore, our analysis of large RNA-seq data sets (human and ovine) fails to identify a single Vegfaxxxb transcript. Therefore, we challenge the very existence of Vegfaxxxb transcripts, which further questions the physiological relevance of studies based on the use of 'anti-VEGFAxxxb' antibodies. Our analysis has implications for the proposed therapeutic use of isoform-specific anti-VEGFA strategies for treating cancer and retinopathies.

Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

Granulocyte-macrophage colony stimulating factor induces endothelial capillary formation through induction of membrane-type 1 matrix metalloproteinase expression in vitro.

In our study, we examined the mechanism by which granulocyte-macrophage colony stimulating factor (GM-CSF) regulates angiogenesis using in vitro models. GM-CSF significantly increased precapillary sprout-like formation from endothelial cell spheroids seeded in type-I collagen gels and tubule formation on coculture of endothelial cells with fibroblasts. In both cases, sprout and tubule formation was highly dependent on metalloproteinase activity. Tissue Inhibitor of metalloproteinase (TIMP) profiling in the spheroid and coculture models showed inhibition by TIMP-2 but not by TIMP-1, indicative of activity of membrane-type matrix metalloproteinases (MT-MMPs). GM-CSF induced sprout formation in spheroids was found to be potently inhibited by siRNA specific for MT1-MMP. Subsequent analysis showed that GM-CSF transiently increased MT1-MMP mRNA in endothelial cells in a MEK-dependent mechanism, which led to increased surface levels of MT1-MMP. This was accompanied by an increase in MT1-MMP-dependent degradation of DQ-collagen by lysates of GM-CSF stimulated endothelial cells. GM-CSF did not increase MT1-MMP levels in fibroblasts. The effect of GM-CSF on endothelial cell sprout formation could be mimicked by adenoviral transduction of intact spheroids with virus expressing MT1-MMP, but not by transduction of endothelial cells before spheroid formation, suggesting that upregulation of MT1-MMP must only occur in cells directly involved in tubule formation.

Membrane-type 4 matrix metalloproteinase promotes breast cancer growth and metastases.

Membrane-type matrix metalloproteinases (MT-MMP) constitute a subfamily of six distinct membrane-associated MMPs. Although the contribution of MT1-MMP during different steps of cancer progression has been well documented, the significance of other MT-MMPs is rather unknown. We have investigated the involvement of MT4-MMP, a glycosylphosphatidylinositol-anchored protease, in breast cancer progression. Interestingly, immunohistochemical analysis shows that MT4-MMP production at protein level is strongly increased in epithelial cancer cells of human breast carcinomas compared with normal epithelial cells. Positive staining for MT4-MMP is also detected in lymph node metastases. In contrast, quantitative reverse transcription-PCR analysis reveals similar MT4-MMP mRNA levels in human breast adenocarcinomas and normal breast tissues. Stable transfection of MT4-MMP cDNA in human breast adenocarcinoma MDA-MB-231 cells does not affect in vitro cell proliferation or invasion but strongly promotes primary tumor growth and associated metastases in RAG-1 immunodeficient mice. We provide for the first time evidence that MT4-MMP overproduction accelerates in vivo tumor growth, induces enlargement of i.t. blood vessels, and is associated with increased lung metastases. These results identify MT4-MMP as a new putative target to design anticancer strategies.

Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) induces FAS dependent apoptosis in human vascular smooth muscle cells.

Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain.

Differential Expression of VEGFA Isoforms Regulates Metastasis and Response to Anti-VEGFA Therapy in Sarcoma.

Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4.1.1 did not affect survival in the lung on intravenous injection. B20-4.1.1 inhibited subcutaneous tumor growth and decreased vascular density in both fs120 and fs188 tumors. However, migration of fs120, but not fs188 cells, in vitro was inhibited by B20-4.1.1. The greater survival of fs120 cells in the lung was associated with VEGFR1-dependent accumulation of CD11b-positive myeloid cells and higher expression of the VEGFR1 ligand, PlGF2, by the fs120 cells in vitro and in the plasma and lungs of fs120 tumor-bearing mice. We conclude that soluble VEGFA isoform expression increases fibrosarcoma metastasis through multiple mechanisms that vary in their sensitivity to anti-VEGF/VEGFR inhibition, with VEGFA-targeted therapy suppressing metastasis through effects on the primary tumor rather than the metastatic site. Cancer Res; 77(10); 2633-46. ©2017 AACR.

Investigating Neovascularization in Rat Decellularized Intestine: An In Vitro Platform for Studying Angiogenesis.

One of the main challenges currently faced by tissue engineers is the loss of tissues postimplantation due to delayed neovascularization. Several strategies are under investigation to create vascularized tissue, but none have yet overcome this problem. In this study, we produced a decellularized natural vascular scaffold from rat intestine to use as an in vitro platform for neovascularization studies for tissue-engineered constructs. Decellularization resulted in almost complete (97%) removal of nuclei and DNA, while collagen, glycosaminoglycan, and laminin content were preserved. Decellularization did, however, result in the loss of elastin and fibronectin. Some proangiogenic factors were retained, as fragments of decellularized intestine were able to stimulate angiogenesis in the chick chorioallantoic membrane assay. We demonstrated that decellularization left perfusable vascular channels intact, and these could be repopulated with human dermal microvascular endothelial cells. Optimization of reendothelialization of the vascular channels showed that this was improved by continuous perfusion of the vasculature and further improved by infusion of human dermal fibroblasts into the intestinal lumen, from where they invaded into the decellularized tissue. Finally we explored the ability of the perfused cells to form new vessels. In the absence of exogenous angiogenic stimuli, Dll4, a marker of endothelial capillary-tip cell activation during sprouting angiogenesis, was absent, indicating that the reformed vasculature was largely quiescent. However, after addition of vascular endothelial growth factor A, Dll4-positive endothelial cells could be detected, demonstrating that this engineered vascular construct maintained its capacity for neovascularization. In summary, we have demonstrated how a natural xenobiotic vasculature can be used as an in vitro model platform to study neovascularization and provide information on factors that are critical for efficient reendothelialization of decellularized tissue.

The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs.

The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane-bound precursors and involved in modulating cell-cell and cell-matrix interactions. ADAM8 (MS2, CD156) has been identified in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane-bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin-gamma, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membrane-associated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane-type metalloproteinases) which have been implicated in protein processing at the cell surface.

Metalloproteases cleave the urokinase-type plasminogen activator receptor in the D1-D2 linker region and expose epitopes not present in the intact soluble receptor.

Proteolytic cleavage of the urokinase plasminogen activator receptor (uPA(R)) prevents the binding of uPA and vitronectin while generating biologically active uPAR fragments. We have recently shown that matrix metalloproteinase-12 (MMP-12) releases cellular uPAR-antigen from stimulated human micro-vascular endothelial cells providing a novel feedback mechanism between the plasminogen activation and MMP systems. We now show that MMP-12 and other MMPs directly and efficiently cleave uPAR at the Thr86 paralal Tyr87 peptide bond located in the linker region connecting uPAR domains 1 and 2, releasing the major ligand binding domain 1 from the rest of the receptor. The possible biological importance of uPAR cleavage by MMPs is supported by the observation that also murine uPAR is cleaved by MMP-12 (at the Pro89 paralal Gln90 peptide bond), despite the limited sequence homology between the linker regions. Using an antibody raised against the human uPAR linker region we show that this region of uPAR, which contains the chemotactic SRSRY epitope, is exposed upon MMP cleavage.

Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics.

Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120) on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188) or wild type controls (fswt) were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively) were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine kinase receptor activation. VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results reveal novel roles of individual isoforms associated with cancer growth and metastasis and highlight the importance of understanding their diverse actions.

A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis.

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model--a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis.