Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231.

Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (-50 °C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism.

The relationship between semaphorin 3C and microvessel density in the progression of breast and oral neoplasia.

This study aimed to identify the expression of semaphorin 3C (SEMA3C) in the normal-metastatic spectrum of breast and oral cancers, and correlate expression with microvessel density (MVD, CD31), a surrogate marker of angiogenesis. Histological analysis revealed that SEMA3C expression was reduced in the development of oral cancer from normal oral tissue (P<0.0001) and expression was inversely correlated with MVD (r=-0.394, P=0.05). In contrast, SEMA3C expression increased in the transition from normal to invasive breast disease in epithelial/tumour cells (P=0.001) and endothelial cells (P=0.006), with both correlating weakly with MVD (r=0.35, p=0.03 and r=0.243, p=0.041 respectively). Furthermore, histological analysis of a breast cancer tissue microarray revealed a weak positive correlation with tumour grade (r=0.305, P=<0.001) and biological phenotype (r=0.237, p=0.004) with tumour cell expression of SEMA3C highest in triple negative and ER-, PR-, HER2+ subtypes. These data suggest that SEMA3C expression is differentially regulated in the development and progression of breast versus oral neoplasia, and that increased expression of SEMA3C may be modulating breast cancer progression and angiogenesis, and could represent a biomarker of metastatic disease.

Neuropilin-1 and neuropilin-2 expression in the adenoma-carcinoma sequence of colorectal cancer.

AIMS: Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are transmembrane glycoproteins which interact with vascular endothelial growth factor (VEGF) to prevent tumour cell apoptosis and regulate angiogenesis. However, the precise role of NRP1 and NRP2 in the adenoma-carcinoma sequence (ACS) of colorectal cancer remains unclear, and we aimed to determine this in surgical specimens comprising the ACS. METHODS AND RESULTS: Histological analysis demonstrated that epithelial NRP1 expression increased significantly across the ACS (P = 0.0007), and correlated with microvessel density (MVD; r = 0.505, P = 0.0003) and weakly with VEGF (r = 0.251, P = 0.001). In contrast, although NRP2 epithelial expression was increased significantly in all carcinomas (P < 0.002), there was no correlation with MVD, VEGF or NRP1. Furthermore, patients showing coexpression of NRP1 and NRP2 had a potentially worse prognosis than those expressing a single neuropilin or neither one. Although vascular expression of NRP1 increased significantly across the ACS (P = 0.0004) and correlated with MVD (r = 0.361, P = 0.0006), NRP2 vascular expression decreased significantly (P = 0.0001) and showed an inverse correlation with MVD (r=-0.506, P = 0.0001), suggesting differential roles for neuropilins in the angiogenic process during colorectal cancer development. CONCLUSIONS: These data suggest that an increase in NRP1 and NRP2 epithelial/tumour expression, as well as in NRP1 vascular expression, may be associated with disease progression in colorectal cancer.

Anti-angiogenic drug discovery: lessons from the past and thoughts for the future.

INTRODUCTION: Since the pioneering work of Judah Folkman, the discovery of bevacizumab has introduced the use of anti-angiogenic agents as a new modality for the treatment of cancer. Currently, hundreds of clinical trials involving anti-angiogenic agents, targeting different elements of the tumour angiogenesis pathway, are underway. However, thus far, the benefits of anti-angiogenic therapy in unselected patient populations are often marginal with harmful side effects. AREAS COVERED: This article presents a detailed discussion of the lessons learnt from the use of bevacizumab and other VEGF pathway inhibitors in the clinical setting. Specifically, this article provides a review of the literature on anti-VEGF agents and other angiogenesis inhibitors used in pre-clinical and clinical trials for cancer treatment. EXPERT OPINION: Future anti-angiogenic drug design centres on multiple protein targets and combinations including: growth factors, hypoxia-inducible factor and tumour endothelial cell markers unique to the tumour vasculature. Furthermore, treatment dosing, scheduling and combination with radiation and chemotherapy require further investigation, as does the potential of treating early disease, and the development of biomarkers which accurately predict response to therapy. These are essential for the future development of these drugs with individualised therapy likely to be the ultimate goal.

Neuropilins: expression and roles in the epithelium.

Initially found expressed in neuronal and then later in endothelial cells, it is well established that the transmembrane glycoproteins neuropilin-1 (NRP1) and neuropilin-2 (NRP2) play essential roles in axonal growth and guidance and in physiological and pathological angiogenesis. Neuropilin expression and function in epithelial cells has received little attention when compared with neuronal and endothelial cells. Overexpression of NRPs is shown to enhance growth, correlate with invasion and is associated with poor prognosis in various tumour types, especially those of epithelial origin. The contribution of NRP and its ligands to tumour growth and metastasis has spurred a strong interest in NRPs as novel chemotherapy drug targets. Given NRP's role as a multifunctional co-receptor with an ability to bind with disparate ligand families, this has sparked new areas of research implicating NRPs in diverse biological functions. Here, we review the growing body of research demonstrating NRP expression and role in the normal and neoplastic epithelium.

Anti-angiogenic properties of ADAMTS-4 in vitro.

Angiogenesis is an indispensable mechanism in development and in many pathologies, including cancer, synovitis and aberrant wound healing. Many angiogenic stimulators and inhibitors have been investigated, and some have progressed to the clinic. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a group of multifunctional proteinases. ADAMTS-1 and ADAMTS-8 have been reported to be anti-angiogenic. Here, we provide evidence that ADAMTS-4, like ADAMTS-1, is expressed by endothelial cells and binds to vascular endothelial groth factor (VEGF). Moreover, ADAMTS-4 inhibited human dermal microvascular endothelial cells (HuDMEC) VEGF-stimulated VEGF receptor (R) R2 phosphorylation, differentiation and migration, suggesting that ADAMTS-4 may be a novel anti-angiogenic molecule.

Transforming Growth Factor-ß and Endoglin Signaling Orchestrate Wound Healing.

Physiological wound healing is a complex process requiring the temporal and spatial co-ordination of various signaling networks, biomechanical forces, and biochemical signaling pathways in both hypoxic and non-hypoxic conditions. Although a plethora of factors are required for successful physiological tissue repair, transforming growth factor beta (TGF-ß) expression has been demonstrated throughout wound healing and shown to regulate many processes involved in tissue repair, including production of ECM, proteases, protease inhibitors, migration, chemotaxis, and proliferation of macrophages, fibroblasts of the granulation tissue, epithelial and capillary endothelial cells. TGF-ß mediates these effects by stimulating signaling pathways through a receptor complex which contains Endoglin. Endoglin is expressed in a broad spectrum of proliferating and stem cells with elevated expression during hypoxia, and regulates important cellular functions such as proliferation and adhesion via Smad signaling. This review focuses on how the TGF-ß family and Endoglin, regulate stem cell availability, and modulate cellular behavior within the wound microenvironment, includes current knowledge of the signaling pathways involved, and explores how this information may be applicable to inflammatory and/or angiogenic diseases such as fibrosis, rheumatoid arthritis and metastatic cancer.

Class 3 semaphorins and their receptors in physiological and pathological angiogenesis.

Class 3 semaphorins (Sema3) are a family of secreted proteins that were originally identified as axon guidance factors mediating their signal transduction by forming complexes with neuropilins and plexins. However, the wide expression pattern of Sema3 suggested additional functions other than those associated with the nervous system, and indeed many studies have now indicated that Sema3 proteins and their receptors play a role in angiogenesis. The present review specifically focuses on recent evidence for this role in both physiological and pathological angiogenesis.

Angiopoietins 1 and 2 and Tie-2 receptor expression in human ductal breast disease.

AIMS: This study aimed to identify the involvement of the angiopoietin/Tie-2 receptor system in breast cancer development, progression, metastasis and angiogenesis. METHODS AND RESULTS: We quantified and correlated angiopoietin-1 (Ang-1), Ang-2 and Tie-2 expression in sections of normal human breast, benign and premalignant hyperplastic tissue, pre-invasive and invasive cancer, and compared these findings with our previously published data on vascular endothelial growth factor (VEGF) and microvessel density (MVD) in the same samples. A breast cancer tissue microarray was used to evaluate the prognostic value of these factors. Histological analysis revealed a significant decrease in Ang-1 expression (P = 0.001) and an inverse correlation with MVD (r = -0.442, P = 0.008) and VEGF (r = -0.510, P = 0.002) in the non-invasive lesions. In contrast Ang-2 expression increased significantly (P = 0.0004) with increasing severity of lesion and correlated with MVD (r = 0.570; P = 0.0002), while Tie-2 expression remained relatively unchanged. Expression of all three factors was reduced in invasive breast cancer and did not correlate with oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), lymph node status or tumour grade. CONCLUSIONS: These data suggest that a change in the angiopoietin balance in favour of Ang-2 is associated with the angiogenic switch at the onset of hyperplasia in the breast. However, angiopoietins and the Tie-2 receptor are not related to known prognostic indicators in invasive breast cancer.

Expression of class 3 semaphorins and their receptors in human breast neoplasia.

AIMS: This study aimed to identify the involvement of class 3 semaphorins (Sema3) and receptors, neuropilins (Np1 and Np2) and plexins (A1-A4) in breast cancer development and angiogenesis. METHODS AND RESULTS: We quantified and correlated Sema3A, Sema3B, Sema3F and their known receptors and coreceptors Plexin-A1, Plexin-A3, Np1 and Np2 in sections of normal human breast, benign and pre-malignant hyperplastic tissue, pre-invasive and invasive cancer, and compared these findings with our previously published data on vascular endothelial growth factor (VEGF) and microvessel density (MVD) in the same samples. Histological analysis revealed that Sema3B was expressed more strongly and widely than Sema3A and 3F in normal breast tissue and all three semaphorins decreased with the transition from in situ to invasive cancer (P < 0.014). Plexin-A3 decreased significantly with progression towards invasive cancer (P < 0.045), whereas Plexin-A1 expression was only significantly reduced once invasion had occurred (P = 0.012). Np1 and Np2 were expressed in both endothelial and epithelial/tumour cells. Np2 expression did not change, but Np1 expression significantly increased in the spectrum from hyperplasia to ductal carcinoma in situ (P < 0.035), but decreased with invasive cancer. CONCLUSION: These data suggest that a decrease in class 3 semaphorin and their plexin receptors have some relationship with disease progression in ductal breast carcinoma.

Classification and functions of enteroendocrine cells of the lower gastrointestinal tract.

With over thirty different hormones identified as being produced in the gastrointestinal (GI) tract, the gut has been described as 'the largest endocrine organ in the body' (Ann. Oncol., 12, 2003, S63). The classification of these hormones and the cells that produce them, the enteroendocrine cells (EECs), has provided the foundation for digestive physiology. Furthermore, alterations in the composition and function of EEC may influence digestive physiology and thereby associate with GI pathologies. Whilst there is a rapidly increasing body of data on the role and function of EEC in the upper GI tract, there is a less clear-cut understanding of the function of EEC in the lower GI. Nonetheless, their presence and diversity are indicative of a role. This review focuses on the EECs of the lower GI where new evidence also suggests a possible relationship with the development and progression of primary adenocarcinoma.

Short-chain fatty acid level and field cancerization show opposing associations with enteroendocrine cell number and neuropilin expression in patients with colorectal adenoma.

BACKGROUND: Previous reports have suggested that the VEGF receptor neuropilin-1 (NRP-1) is expressed in a singly dispersed subpopulation of cells in the normal colonic epithelium, but that expression becomes dysregulated during colorectal carcinogenesis, with higher levels in tumour suggestive of a poor prognosis. We noted that the spatial distribution and morphology if NRP-1 expressing cells resembles that of enteroendocrine cells (EEC) which are altered in response to disease state including cancer and irritable bowel syndrome (IBS). We have shown that NRP-1 is down-regulated by butyrate in colon cancer cell lines in vitro and we hypothesized that butyrate produced in the lumen would have an analogous effect on the colon mucosa in vivo. Therefore we sought to investigate whether NRP-1 is expressed in EEC and how NRP-1 and EEC respond to butyrate and other short-chain fatty acids (SCFA - principally acetate and propionate). Additionally we sought to assess whether there is a field effect around adenomas. METHODOLOGY: Biopsies were collected at the mid-sigmoid, at the adenoma and at the contralateral wall (field) of 28 subjects during endoscopy. Samples were fixed for IHC and stained for either NRP-1 or for chromogranin A (CgA), a marker of EEC. Stool sampling was undertaken to assess individuals' butyrate, acetate and propionate levels. RESULT: NRP-1 expression was inversely related to SCFA concentration at the colon landmark (mid-sigmoid), but expression was lower and not related to SCFA concentration at the field. Likewise CgA+ cell number was also inversely related to SCFA at the landmark, but was lower and unresponsive at the field. Crypt cellularity was unaltered by field effect. A colocalisation analysis showed only a small subset of NRP-1 localised with CgA. Adenomas showed extensive, weaker staining for NRP-1 which contrastingly correlated positively with butyrate level. Field effects cause this relationship to be lost. Adenoma tissue shows dissociation of the co-regulation of NRP-1 and EEC. CONCLUSION: NRP-1 is inversely associated with levels of butyrate and other SCFA in vivo and is expressed in a subset of CgA expressing cells. EEC number is related to butyrate level in the same way.

Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation.

BACKGROUND: Neuropilin is a transmembrane receptor for vascular endothelial growth factor (VEGF) and is expressed in normal endothelial cells and upregulated in cancer cells. Neuropilin-1 (NRP-1) has been shown to promote tumour cell migration and survival in colon cancer in response to VEGF binding. The expression profiles of neuropilins, associated co-receptors and known ligands have been mapped in three colorectal cell lines: Caco-2, HCT116 & HT29. We have previously shown that butyrate, a naturally occurring histone deacetylase inhibitor (HDACi) produced by fermentation of fibre in the colon, causes apoptosis of colon cancer cell lines. RESULTS: Here we demonstrate that butyrate down-regulates NRP-1 and VEGF at the mRNA and protein level in colorectal cancer cell lines. NRP-1 is a known transcriptional target of Sp1, whose activity is regulated by acetylation. NRP-1 down-regulation by butyrate was associated with decreased binding affinity of Sp1 for canonical Sp-binding sites in the NRP-1 promoter. siRNA-mediated knock-down of Sp1 implied that Sp1 may have strong DNA binding activity but weak transactivation potential. CONCLUSION: The downregulation of the key apoptotic and angiogenesis regulator NRP-1 by butyrate suggests a novel contributory mechanism to the chemopreventive effect of dietary fibre.

A critical analysis of current in vitro and in vivo angiogenesis assays.

The study of angiogenesis has grown exponentially over the past 40 years with the recognition that angiogenesis is essential for numerous pathologies and, more recently, with the advent of successful drugs to inhibit angiogenesis in tumours. The main problem with angiogenesis research remains the choice of appropriate assays to evaluate the efficacy of potential new drugs and to identify potential targets within the angiogenic process. This selection is made more complex by the recognition that heterogeneity occurs, not only within the endothelial cells themselves, but also within the specific microenvironment to be studied. Thus, it is essential to choose the assay conditions and cell types that most closely resemble the angiogenic disease being studied. This is especially important when aiming to translate data from in vitro to in vivo and from preclinical to the clinic. Here we critically review and highlight recent advances in the principle assays in common use including those for endothelial cell proliferation, migration, differentiation and co-culture with fibroblasts and mural cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as chick chorioallantoic membrane (CAM), zebrafish, sponge implantation, corneal, dorsal air sac, chamber and tumour angiogenesis models. Finally, we briefly discuss the direction likely to be taken in future studies, which include the use of increasingly sophisticated imaging analysis systems for data acquisition.

Current status and future prospects for anti-angiogenic therapies in cancer.

BACKGROUND: Angiogenesis is essential for tumour growth and development and since the pioneering work of Judah Folkman several anti-angiogenic strategies have now been successfully employed. OBJECTIVE: This article aims to present a detailed review of current knowledge of the main pathways involved in angiogenesis, the strategies employed for inhibition and the current status of angiogenesis inhibitors in therapeutic use. METHODS: A systematic review of the literature was undertaken including angiogenesis in cancer and angiogenesis inhibitors in pre-clinical and clinical trials. CONCLUSION: While angiogenic inhibitors are now in clinical use, their limited benefits mean we must urgently develop strategies to improve the efficacy of this approach.

Tissue factor, angiogenesis and tumour progression.

Tissue factor, the primary initiator of the coagulation cascade, maintains vascular integrity in response to injury. It is now recognised that, in addition to the role as a procoagulant activator, tissue factor participates in many tumour-related processes that contribute to malignant disease progression. The present review details the recent evidence supporting a role for tissue factor in tumour haemostasis, angiogenesis, metastasis and malignant cell survival. Furthermore, future research directions are discussed that may enhance our understanding of the role and regulation of this protein, which could ultimately lead to the innovative design and development of new anticancer therapies.

The angiogenic switch occurs at the adenoma stage of the adenoma carcinoma sequence in colorectal cancer.

OBJECTIVE: The aim of this study was to examine the relationship between tissue factor (TF), vascular endothelial growth factor (VEGF) and the onset of angiogenesis in the adenoma-carcinoma sequence (ACS), the stepwise process encompassing colorectal cancer (CRC) disease progression. PATIENTS AND METHODS: 210 surgical specimens comprising the ACS were immunohistochemically stained for endothelial cells (CD31), VEGF and TF. Angiogenesis quantified using Chalkley grid analysis (microvascular density; MVD), and VEGF/TF expression were semiquantitatively graded and correlated with standard prognostic indicators including 5 year follow-up. VEGF and TF were measured by ELISA in tumour specimens and normal mucosa from an additional 90 CRC patients. RESULTS: There was a significant increase in MVD across the ACS (p < 0.0005) with significant correlations with Dukes' stage (p = 0.01) and lymph node involvement (p = 0.02). The greatest increase in MVD was related to the onset of dysplasia, with an associated significant increase in VEGF expression (p < 0.0005). There was a significant relationship between VEGF and TF expression in the initial phase of the ACS (k = 0.44, p < 0.005), although no correlation between VEGF or TF, and MVD, tumour size, Dukes' classification, lymph node involvement or survival was found. CONCLUSIONS: These findings are the first to suggest that the angiogenic switch occurs at the onset of dysplasia in the ACS, and provide further evidence of the close association between VEGF and TF in the early stages of CRC development.

Angiogenesis inhibitors found within the haemostasis pathway.

Angiogenesis, the development of new blood vessels from the existing vasculature, and haemostasis, the coagulation cascade leading to formation of a clot, are among the most consistent host responses associated with cancer. Importantly, these two pathways interrelate, with blood coagulation and fibrinolysis influencing tumor angiogenesis directly, thereby contributing to tumor growth. Moreover, many endogenous inhibitors of angiogenesis are found within platelets or harboured as cryptic fragments of haemostatic proteins. In this review we outline ways in which angiogenesis is coordinated and regulated by haemostasis in human cancer. Then we detail the experimental and pre-clinical evidence for the ability of many of these endogenous proteins to inhibit tumor angiogenesis and thus their potential to be anti-cancer agents, with particular reference to any clinical trials.

Current methods for assaying angiogenesis in vitro and in vivo.

Angiogenesis, the development of new blood vessels from an existing vasculature, is essential in normal developmental processes and in numerous pathologies, including diabetic retinopathy, psoriasis and tumour growth and metastases. One of the problems faced by angiogenesis researchers has been the difficulty of finding suitable methods for assessing the effects of regulators of the angiogenic response. The ideal assay would be reliable, technically straightforward, easily quantifiable and, most importantly, physiologically relevant. Here, we review the advantages and limitations of the principal assays in use, including those for the proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as sponge implantation, corneal, chamber, zebrafish, chick chorioallantoic membrane (CAM) and tumour angiogenesis models.

Alphastatin, a 24-amino acid fragment of human fibrinogen, is a potent new inhibitor of activated endothelial cells in vitro and in vivo.

Angiogenesis, the development of new blood vessels from existing vasculature, is crucial for the development and metastasis of solid tumors. Here, we show for the first time that a 24-amino acid peptide derived from the amino terminus of the alpha chain of human fibrinogen (termed "alphastatin") has potent antiangiogenic properties, inhibiting both the migration and tubule formation of human dermal microvascular endothelial cells in response to vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) in vitro. Moreover, alphastatin markedly inhibits the growth of tumors in a syngeneic murine model. Tumors from mice receiving daily injections of alphastatin for 12 days exhibited large areas of intravascular disruption and thrombosis with substantial cellular necrosis. Importantly, alphastatin administration had no detectable effect on vessels in such normal tissues as liver, lungs, and kidney. Taken together, these data indicate that alphastatin is a potent new antiangiogenic agent in vitro and antivascular agent in vivo.