Role of haematopoietic cells and endothelial progenitors in tumour angiogenesis

Bone marrow-derived cells include haematopoietic cell lineages and the recently described endothelial progenitor cells (EPCs). It has been recently emphasised that these marrow-derived cells contribute to tumour angiogenesis, and different mechanisms have been proposed that account for this activity. Whereas haematopoietic cells may promote tumour angiogenesis through the release of proangiogenic factors or by creating permissive conditions in the tumour microenvironment that favour the growth of locally derived blood vessels ("paracrine" role), endothelial progenitors are thought to directly incorporate into nascent blood vessels as bona fide endothelial cells ("building block" role). The relative contribution of these distinct pathways to tumour angiogenesis is the subject of intense investigation and debate.     

The mathematical modelling of tumour angiogenesis and invasion

In order to accomplish the transition from avascular to vascular growth, solid tumours secrete a diffusible substance known as tumour angiogenesis factor (TAF) into the surrounding tissue. Endothelial cells which form the lining of neighbouring blood vessels respond to this chemotactic stimulus in a well-ordered sequence of events comprising, at minimum, of a degradation of their basement membrane, migration and proliferation. Capillary sprouts are formed which migrate towards the tumour eventually penetrating it and permitting vascular growth to take place. It is during this stage of growth that the insidious process of invasion of surrounding tissues can and does take place. A model mechanism for angiogenesis is presented which includes the diffusion of the TAF into the surrounding host tissue and the response of the endothelial cells to the chemotactic stimulus. Numerical simulations of the model are shown to compare very well with experimental observations. The subsequent vascular growth of the tumour is discussed with regard to a classical reaction-diffusion pre-pattern model.     

Low Dose Geldanamycin Inhibits Hepatocyte Growth Factor- and Hypoxia-Stimulated Invasion of Cancer Cells

Hepatocyte growth factor (HGF) receptor Met and hypoxia-inducible factor-1 (HIF-1) signaling pathways are commonly activated in aggressive tumors and promote progression. Since both Met and HIF-1α proteins are heat shock protein (Hsp) 90 clients, Hsp90 inhibitors might be expected to positively impact tumor progression. Here, we systematically evaluated the inhibitory effects of the prototypical Hsp90 inhibitor geldanamycin (GA) on cellular processes involved in invasion and angiogenesis in T24 bladder cancer cells stimulated with HGF and chemical hypoxia. First, we demonstrated the positive feedback loop between Met and HIF-1 pathways, which serves to sustain and amplifies their signaling in T24 cells. GA down-regulated Met by inhibiting new protein maturation, thereby dampening HGF signaling. HGF and chemical hypoxia with CoCl2 cooperatively promoted in vitro invasion and vascular endothelial growth factor (VEGF) secretion, while CoCl2 but not HGF activated urokinase-type plasminogen activator and matrix metalloproteinase 2, both of which promote invasion and angiogenesis. Low dose GA (100 nmol/L) inhibited these processes by suppressing both HGF and HIF-1 pathways. Notably, brief GA pretreatment inhibited in vitro invasion and VEGF secretion induced by HGF as effectively as did continuous treatment. Moreover, we found that GA inhibited activation of focal adhesion kinase, focal adhesion assembly, and actin reorganization induced by HGF and integrin engagement by extracellular matrix. Thus, GA widely suppresses extrinsic stimuli-induced signaling that contribute to tumor invasion and angiogenesis in this bladder carcinoma model, suggesting the utility of Hsp90 inhibitors in preventing tumor progression and metastasis.     

Cooperative effect of roscovitine and irradiation targets angiogenesis and induces vascular destabilization in human breast carcinoma

Angiogenesis is considered as an essential process for tumour development and invasion. Previously, we demonstrated that cyclin-dependent kinase inhibition by roscovitine induces a radiosensitization and a synergistic antitumoral effect in human carcinoma but its effect on the microenvironment and tumour angiogenesis remains unknown. Here, we investigated the effect of the combination roscovitine and ionizing radiation (IR) on normal cells in vitro and on tumour angiogenesis in MDA-MB 231 tumour xenografts. We observed that the combination roscovitine and IR induced a marked reduction of angiogenic hot spot and microvascular density in comparison with IR or roscovitine treatments alone. The Ang-2/Tie-2 ratio was increased in presence of reduced vascular endothelial growth factor level suggesting vessel destabilization. In vitro , no radiosensitization effect of roscovitine was found in endothelial, fibroblast, and keratinocyte cells. IR potentiated the antiproliferative effect of roscovitine without inducing apoptosis in endothelial cells. Roscovitine decreased IR-stimulated vascular endothelial growth factor secretion of MDA-MB 231 and endothelial cells. A reduction in the endothelial cells invasion and the capillary-like tube formation in Matrigel were observed following the combination roscovitine and IR. This combined treatment targets angiogenesis resulting in microvessel destabilization without inducing normal cell toxicity.     

Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis

In contrast to the aberrant control of proliferation, apoptosis, angiogenesis and lifespan, the cellular mechanisms that cause local invasion and metastasis of tumour cells are still poorly understood. New experimental approaches have identified different types of epithelial-plasticity changes in tumour cells towards fibroblastoid phenotypes as crucial events that occur during metastasis, and many molecules and signalling pathways cooperate to trigger these processes.     

Targeting key steps in metastatic tumour progression

The key determinants of tumour progression and discriminators of benign and malignant lesions include neoangiogenesis (the induction of a new blood supply) and the capacity of malignant cells to invade and metastasise. It is now recognized that these processes can be co-ordinately regulated by the activity of specific genes -- often distinct from those involved in early oncogenesis -- and involve common signalling pathways. Cell motility and chemotaxis (the ability to respond to gradients of chemoattractants) are implicated in both tumour-cell invasion and response of activated endothelial cells to angiogenic cytokines, and provide interesting and novel points for therapeutic intervention.     

Modulators of endothelial cell filopodia

Filopodia are an important feature of actively motile cells, probing the pericellular environment for chemotactic factors and other molecular cues that enable and direct the movement of the cell. They also act as points of attachment to the extracellular matrix for the cell, generating tension that may act to pull the cell forward and/or stabilize the cell as it moves. Endothelial cell motility is a critical aspect of angiogenesis, but only a limited number of molecules have been identified as specific regulators of endothelial cell filopodia. Recent reports, however, provide evidence for the involvement of PECAM-1, an endothelial cell adhesion and signaling molecule, in the formation of endothelial cell filopodia. This commentary will focus on these studies and their suggestion that at least two PECAM-1-regulated pathways are involved in the processes that enable filopodial protrusions by endothelial cells. Developing a more complete understanding of the role of PECAM-1 in mediating various endothelial cell activities, such as the extension of filopodia, will be essential for exploiting the therapeutic potential of targeting PECAM-1.     

Modulation of VEGF signalling output by the Notch pathway

The formation of blood vessels within the vascular system entails a variety of cellular processes, including proliferation, migration and differentiation. In many cases, these diverse processes need to be finely coordinated among neighbouring endothelial cells in order to establish a functional vascular network. For instance, during angiogenic sprouting specialized endothelial tip cells follow guidance cues and migrate extensively into avascular tissues while trailing stalk cells must stay connected to the patent blood vessel. The vascular endothelial growth factor (VEGF) and Notch signalling pathways have emerged as the major players in governing these different cellular behaviours. In particular, recent work indicates an important role for Notch signalling in determining how an endothelial cell responds to VEGF. In this review, we provide an overview of these biochemically distinct pathways and discuss how they may interact during endothelial cell differentiation and angiogenesis.     

In Vitro Inhibition of Angiogenesis by Antibodies Directed against the 37kDa/67kDa Laminin Receptor

The 37kDa/67kDa laminin receptor (LRP/LR) is a central receptor mediating interactions between tumour cells and the basement membrane and is thereby a key player in adhesion and invasion, essential processes in metastatic cancer. To affect continued tumour growth, tumours induce angiogenesis for the constant delivery of nutrients and oxygen. This study aims to determine the blocking effect of the anti-LRP/LR specific antibody, W3 on the angiogenic potential of HUVE (human umbilical vein endothelial) cells. Flow cytometric analysis revealed that 97% of HUVE cells display cell surface LRP/LR. An angiogenesis assay was conducted employing HUVE cells seeded on the basement membrane reconstituent Matrigel™ supplemented with the pro-angiogenic factor vascular endothelial growth factor (VEGF). Post 18h incubation at 37°C tubular structures, namely tube lengths were assessed. Treatment of established tubular structures with 100 µg/ml anti-LRP/LR specific antibody completely blocked angiogenesis. Our findings suggest a central role of the 37kDa/67kDa LRP/LR in tube formation and recommends anti-LRP/LR specific antibodies as potential therapeutic tools for treatment of tumour angiogenesis.     

Vascular endothelial growth factor A is a potential prognostic biomarker and correlates with immune cell infiltration in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths among cancer patients. Vascular endothelial growth factor A (VEGFA) is involved in regulating biological processes, such as angiogenesis and vascular permeability, and is very closely related to the pathogenesis of various tumours, especially vascular-rich, solid tumours. Clinical data of patients with HCC and other tumours were analysed through public databases, such as the TCGA database, Gene Expression Omnibus database, Human Protein Atlas database, STRING, Tumour Immune Estimation Resource and Kaplan–Meier Plotter. The tumour tissues and adjacent normal tissues of patients with HCC from Hunan Provincial People's Hospital were collected to verify the expression of VEGFA by immunohistochemistry, immunofluorescence, Western blotting and qPCR. VEGFA expression is elevated in multiple tumour types and correlates with the prognosis of tumour patients. VEGFA is involved in regulating the tumour microenvironment and immune cell function in tumour development. Inhibition of VEGFA reduces proliferation, invasion, and migration and promotes apoptosis in HCC cells. VEGFA is a potential predictive biomarker for the diagnosis and prognosis of HCC.     

Integrin signalling during tumour progression

During progression from tumour growth to metastasis, specific integrin signals enable cancer cells to detach from neighbouring cells, re-orientate their polarity during migration, and survive and proliferate in foreign microenvironments. There is increasing evidence that certain integrins associate with receptor tyrosine kinases (RTKs) to activate signalling pathways that are necessary for tumour invasion and metastasis. The effect of these integrins might be especially important in cancer cells that have activating mutations, or amplifications, of the genes that encode these RTKs.     

Tumour-secreted miR-9 promotes endothelial cell migration and angiogenesis by activating the JAK-STAT pathway

Angiogenesis plays a crucial role during tumorigenesis and much progress has been recently made in elucidating the role of VEGF and other growth factors in the regulation of angiogenesis. Recently, microRNAs (miRNAs) have been shown to modulate a variety of physiogical and pathological processes. We identified a set of differentially expressed miRNAs in microvascular endothelial cells co-cultured with tumour cells. Unexpectedly, most miRNAs were derived from tumour cells, packaged into microvesicles (MVs), and then directly delivered to endothelial cells. Among these miRNAs, we focused on miR-9 due to the strong morphological changes induced in cultured endothelial cells. We found that exogenous miR-9 effectively reduced SOCS5 levels, leading to activated JAK-STAT pathway. This signalling cascade promoted endothelial cell migration and tumour angiogenesis. Remarkably, administration of anti-miR-9 or JAK inhibitors suppressed MV-induced cell migration in vitro and decreased tumour burden in vivo. Collectively, these observations suggest that tumour-secreted miRNAs participate in intercellular communication and function as a novel pro-angiogenic mechanism.     

Coupled mathematical model of tumorigenesis and angiogenesis in vascular tumours

Objectives: Mathematical models are useful for studying vascular and avascular tumours, because these allow for more logical experimental design and provide valuable insights into the underlying mechanisms of their growth and development. The processes of avascular tumour growth and the development of capillary networks through tumour‐induced angiogenesis have already been extensively investigated, albeit separately. Despite the clinical significance of vascular tumours, few studies have combined these approaches to develop a single comprehensive growth and development model. Materials and methods: We develop a continuum‐based mathematical model of vascular tumour growth. In the model, angiogenesis is initiated through the release of angiogenic growth factors (AGFs) by cells in the hypoxic regions of the tumour. The nutrient concentration within the tumour reflects the influence of capillary growth and invasion induced by AGF. Results and conclusions: Parametric and sensitivity studies were performed to evaluate the influence of different model parameters on tumour growth and to identify the parameters with the most influence, which include the rates of proliferation, apoptosis and necrosis, as well as the diffusion of sprout tips and the size of the region affected by angiogenesis. An optimization was performed for values of the model parameters that resulted in the best agreement with published experimental data. The resulting model solution matched the experimental data with a high degree of correlation (r = 0.85).     

PV1 down‐regulation via shRNA inhibits the growth of pancreatic adenocarcinoma xenografts

PV1 is an endothelial‐specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour‐bearing mice by single‐dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down‐regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC‐1 and BxPC‐3). The effect observed is because of down‐regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down‐regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis.     

Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development

An increased rate of lipid synthesis in cancerous tissues has long been recognised as an important aspect of the rewired metabolism of transformed cells. However, the contribution of lipids to cellular transformation, tumour development and tumour progression, as well as their potential role in facilitating the spread of cancerous cells to secondary sites, are not yet fully understood. In this article, we review the recent findings that support the importance of lipid synthesis and metabolism in tumorigenesis. Specifically, we explore the role of aberrant lipid biosynthesis in cancer cell migration and invasion, and in the induction of tumour angiogenesis. These processes are crucial for the dissemination of tumour cells and formation of metastases, which constitute the main cause of cancer mortality.     

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.