Expression Of Hypoxia Markers Research Articles

BIG enhances Arg/N-degron pathway-mediated protein degradation to regulate Arabidopsis hypoxia responses and suberin deposition.

BIG/DARK OVEREXPRESSION OF CAB1/TRANSPORT INHIBITOR RESPONSE3 is a 0.5-MDa protein associated with multiple functions in Arabidopsis (Arabidopsis thaliana) signalling and development. However, the biochemical functions of BIG are unknown. We investigated a role for BIG in the Arg/N-degron pathways, in which substrate protein fate is influenced by the N-terminal (Nt) residue. We crossed a big loss-of-function allele to two N-degron pathway E3 ligase mutants, proteolysis6 (prt6) and prt1, and examined the stability of protein substrates. Stability of model substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants and the abundance of the PRT6 physiological substrates, HYPOXIA-RESPONSIVE ERF2 (HRE2) and VERNALIZATION2 (VRN2) was similarly increased in prt6 big double mutants. Hypoxia marker expression was enhanced in prt6 big double mutants; this constitutive response required arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated increased expression of multiple hypoxia-responsive genes in the double mutant relative to prt6, but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and broader processes in Arabidopsis.

Cancer cells depend on environmental lipids for proliferation when electron acceptors are limited.

Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can be a proliferation bottleneck that is influenced by environmental conditions. However, a comprehensive quantitative understanding of metabolic processes that may be affected by NAD+ deficiency is currently missing. Here, we show that de novo lipid biosynthesis can impose a substantial NAD+ consumption cost in proliferating cancer cells. When electron acceptors are limited, environmental lipids become crucial for proliferation because NAD+ is required to generate precursors for fatty acid biosynthesis. We find that both oxidative and even net reductive pathways for lipogenic citrate synthesis are gated by reactions that depend on NAD+ availability. We also show that access to acetate can relieve lipid auxotrophy by bypassing the NAD+ consuming reactions. Gene expression analysis demonstrates that lipid biosynthesis strongly anti-correlates with expression of hypoxia markers across tumor types. Overall, our results define a requirement for oxidative metabolism to support biosynthetic reactions and provide a mechanistic explanation for cancer cell dependence on lipid uptake in electron acceptor-limited conditions, such as hypoxia.

Persistent biliary hypoxia and lack of regeneration are key mechanisms in the pathogenesis of posttransplant nonanastomotic strictures.

Background and AimsNonanastomotic biliary strictures (NAS) are a major cause of morbidity after orthotopic liver transplantation (OLT). Although ischemic injury of peribiliary glands (PBGs) and peribiliary vascular plexus during OLT has been associated with the later development of NAS, the exact underlying mechanisms remain unclear. We hypothesized that bile ducts of patients with NAS suffer from ongoing biliary hypoxia and lack of regeneration from PBG stem/progenitor cells.Approach and ResultsForty‐two patients, requiring retransplantation for either NAS (n = 18), hepatic artery thrombosis (HAT; n = 13), or nonbiliary graft failure (controls; n = 11), were included in this study. Histomorphological analysis of perihilar bile ducts was performed to assess differences in markers of cell proliferation and differentiation in PBGs, microvascular density (MVD), and hypoxia. In addition, isolated human biliary tree stem cells (hBTSCs) were used to examine exo‐metabolomics during in vitro differentiation toward mature cholangiocytes.Bile ducts of patients with NAS or HAT had significantly reduced indices of PBG mass, cellular proliferation and differentiation (mucus production, secretin receptor expression, and primary cilia), reduced MVD, and increased PBG apoptosis and hypoxia marker expression, compared to controls. Metabolomics of hBTSCs during in vitro differentiation toward cholangiocytes revealed a switch from a glycolytic to oxidative metabolism, indicating the need for oxygen.ConclusionsNAS are characterized by a microscopic phenotype of chronic biliary hypoxia attributed to loss of microvasculature, resulting in reduced proliferation and differentiation of PBG stem/progenitor cells into mature cholangiocytes. These findings suggest that persistent biliary hypoxia is a key mechanism underlying the development of NAS after OLT.

Abstract 3438: Hypoxia inhibits BMP4-induced astrocytic differentiation in glioma stem cells

Abstract The tumor microenvironment is an important contributor to malignancy in glioblastoma (GBM) by influencing stemness properties in glioma stem cells (GSCs) and their capacity for differentiation into various cell types. Tumor hypoxia is a common feature of the GBM microenvironment and can influence cell state through a variety of processes including the inhibition of oxygen-dependent lysine histone demethylases (KDMs) and subsequent epigenetic changes required for differentiation or maintenance of the stem cell state. However, the precise mechanisms underlying hypoxic restriction of differentiation to specific neural lineages is unclear. Here, we show that hypoxia suppresses astrocytic differentiation induced by bone morphogenetic protein 4 (BMP4) in patient-derived GSCs. In 8/10 patient derived GSC lines, BMP4 induced expression of astrocytic markers, such as glial fibrillary acidic protein, and this induction was repressed by hypoxia in 7/10 lines. Interestingly, significant anti-proliferative effects of BMP4 induced differentiation were only observed in 3/10 lines and was rescued in only one GSC line by hypoxia, suggesting that these processes are uncoupled. Although hypoxia may directly limit the activity of oxygen-dependent KDMs important in this process, levels of S-2-hydroxyglutarate (S-2HG), an endogenous KDM inhibitor, were also increased in 5/8 GSC lines under hypoxia. Exogenous treatment with S-2HG or a small molecule KDM6 family inhibitor, phenocopied the repressive effects of hypoxia on BMP4 induced differentiation. In patient tumors, hypoxic areas surrounding pseudopalisading necrosis showed high expression of hypoxia markers, and these hypoxia markers were anti-correlated with markers of astrocytic differentiation. Together, these results demonstrate that the hypoxic tumor microenvironment in GBM, through multiple mechanisms limiting oxygen-dependent KDM activity, promote maintenance of the malignant stemness state by inhibiting astrocytic differentiation. This study implicates tumour hypoxia as a key regulator of stemness and a therapeutic target to promote differentiation in GBM. Treatment strategies that target hypoxic cells are urgently needed to overcome these challenges. Citation Format: Ronald S. Wu, Elisabeth Liedtke, Sheila Mansouri, Samantha Brazier, Paul Guilhamon, Nicole I. Park, Eric Chen, Mathieu Lupien, Daniel D. De Carvalho, Peter G. Dirks, Gelareh Zadeh, Bradley G. Wouters. Hypoxia inhibits BMP4-induced astrocytic differentiation in glioma stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3438.

Elevated Intraocular Pressure Causes Abnormal Reactivity of Mouse Retinal Arterioles.

Objective Glaucoma is a leading cause of severe visual impairment and blindness. Although high intraocular pressure (IOP) is an established risk factor for the disease, the role of abnormal ocular vessel function in the pathophysiology of glaucoma gains more and more attention. We tested the hypothesis that elevated intraocular pressure (IOP) causes vascular dysfunction in the retina. Methods High IOP was induced in one group of mice by unilateral cauterization of three episcleral veins. The other group received sham surgery only. Two weeks later, retinal vascular preparations were studied by video microscopy in vitro. Reactive oxygen species (ROS) levels and expression of hypoxia markers and of prooxidant and antioxidant redox genes as well as of inflammatory cytokines were determined. Results Strikingly, responses of retinal arterioles to stepwise elevation of perfusion pressure were impaired in the high-IOP group. Moreover, vasodilation responses to the endothelium-dependent vasodilator, acetylcholine, were markedly reduced in mice with elevated IOP, while no differences were seen in response to the endothelium-independent nitric oxide donor, sodium nitroprusside. Remarkably, ROS levels were increased in the retinal ganglion cell layer including blood vessels. Expression of the NADPH oxidase isoform, NOX2, and of the inflammatory cytokine, TNF-α, was increased at the mRNA level in retinal explants. Expression of NOX2, but not of the hypoxic markers, HIF-1α and VEGF-A, was increased in the retinal ganglion cell layer and in retinal blood vessels at the protein level. Conclusion Our data provide first-time evidence that IOP elevation impairs autoregulation and induces endothelial dysfunction in mouse retinal arterioles. Oxidative stress and inflammation, but not hypoxia, appear to be involved in this process.

Aspartate is a limiting metabolite for cancer cell proliferation under hypoxia and in tumours.

SUMMARYAs oxygen is essential for many metabolic pathways, tumor hypoxia may impair cancer cell proliferation (1–4). However, the limiting metabolites for proliferation under hypoxia and in tumors are unknown. Here, we assessed proliferation of a collection of cancer cells upon inhibition of the mitochondrial electron transport chain (ETC), a major metabolic pathway requiring molecular oxygen (5). Sensitivity to ETC inhibition varied across cell lines, and subsequent metabolomic analysis uncovered aspartate availability as a major determinant of sensitivity. Cell lines least sensitive to ETC inhibition maintain aspartate levels by importing it through an aspartate/glutamate transporter, SLC1A3. Genetic or pharmacologic modulation of SLC1A3 activity markedly altered cancer cell sensitivity to ETC inhibitors. Interestingly, aspartate levels also decrease under low oxygen, and increasing aspartate import by SLC1A3 provides a competitive advantage to cancer cells at low oxygen levels and in tumor xenografts. Finally, aspartate levels in primary human tumors negatively correlate with the expression of hypoxia markers, suggesting that tumor hypoxia is sufficient to inhibit ETC and, consequently, aspartate synthesis in vivo. Therefore, aspartate may be a limiting metabolite for tumor growth and aspartate availability could be targeted for cancer therapy.

COL1A1, PRPF40A, and UCP2 correlate with hypoxia markers in non-small cell lung cancer

PurposeCollagen 1A1 (COL1A1), RNA-binding and pre-mRNA Processing Factor (PRPF40A), and Uncoupling Protein 2 (UCP2) were identified as downstream effectors of cytoglobin (CYGB), which was shown implicated in tumour biology. Although these three genes have been previously associated with cancer, little is known about their status in lung malignancies.MethodsHereby, we investigated the expression and promoter methylation of COL1A1, PRPF40A, and UCP2 in 156 non-small cell lung cancer (NSCLC) and adjacent normal tissues.ResultsWe demonstrate that COL1A1 and PRPF40A mRNAs are significantly overexpressed in NSCLC (p < 1 × 10−4), while UCP2 exhibits a trend of upregulation (p = 0.066). Only COL1A1 promoter revealed hypermethylation in NSCLCs (36%), which was particularly evident in squamous cell carcinomas (p = 0.024) and in the tumours with moderate-to-good differentiation (p = 0.01). Transcript level of COL1A1, as well as PRPF40A and UCP2, exhibited striking association (p ≤ 0.001) with the expression of hypoxia markers. In addition, we demonstrate in lung cancer cell lines exposed to hypoxia or oxidative stress that COL1A1 transcription significantly responds to oxygen depletion, while other genes showed the modest upregulation in stress conditions.ConclusionIn conclusion, our data revealed that COL1A1, UCP2, and PRPF40A are novel players implicated in the complex network of hypoxia response in NSCLC.

Genetic polymorphisms in key hypoxia-regulated downstream molecules and phenotypic correlation in prostate cancer

BackgroundIn this study we sought if, in their quest to handle hypoxia, prostate tumors express target hypoxia-associated molecules and their correlation with putative functional genetic polymorphisms.MethodsRepresentative areas of prostate carcinoma (n = 51) and of nodular prostate hyperplasia (n = 20) were analysed for hypoxia-inducible factor 1 alpha (HIF-1α), carbonic anhydrase IX (CAIX), lysyl oxidase (LOX) and vascular endothelial growth factor (VEGFR2) immunohistochemistry expression using a tissue microarray. DNA was isolated from peripheral blood and used to genotype functional polymorphisms at the corresponding genes (HIF1A +1772 C > T, rs11549465; CA9 + 201 A > G; rs2071676; LOX +473 G > A, rs1800449; KDR – 604 T > C, rs2071559).ResultsImmunohistochemistry analyses disclosed predominance of positive CAIX and VEGFR2 expression in epithelial cells of prostate carcinomas compared to nodular prostate hyperplasia (P = 0.043 and P = 0.035, respectively). In addition, the VEGFR2 expression score in prostate epithelial cells was higher in organ-confined and extra prostatic carcinoma compared to nodular prostate hyperplasia (P = 0.031 and P = 0.004, respectively). Notably, for LOX protein the immunoreactivity score was significantly higher in organ-confined carcinomas compared to nodular prostate hyperplasia (P = 0.015). The genotype-phenotype analyses showed higher LOX staining intensity for carriers of the homozygous LOX +473 G-allele (P = 0.011). Still, carriers of the KDR−604 T-allele were more prone to have higher VEGFR2 expression in prostate epithelial cells (P < 0.006).ConclusionsProtein expression of hypoxia markers (VEGFR2, CAIX and LOX) on prostate epithelial cells was different between malignant and benign prostate disease. Two genetic polymorphisms (LOX +473 G > A and KDR−604 T > C) were correlated with protein level, accounting for a potential gene-environment effect in the activation of hypoxia-driven pathways in prostate carcinoma. Further research in larger series is warranted to validate present findings.

PTH-110 Ten Year Follow Up Case Control Study of The Effect of Portal Vein Embolisation (PVE) On The Survival of Patients with Colorectal Liver Metastases (CRLM) and The Influence of Hypoxia Factors on Tumour Growth

Introduction PVE facilitates hepatectomy in patients with a small anticipated future liver remnant, but stimulates tumour growth. Hypoxia may mediate this increased tumour growth as Hypoxia-Inducible-Factor-1α (HIF-1α) increases angiogenesis and invasion. A tendency to reduced survival following liver resection after PVE was noted in a previous study when compared with a case matched non PVE-group. We have performed a longer term follow up of patients who had a PVE prior to resection of CRLM and compared the outcome with a matched controlled group with measurement of tumour hypoxia. Methods Twenty-six patients who had PVE were compared with 25 case matched controls. Immunostaining was performed on serial formalin-fixed-paraffin-embedded CRLM sections for hypoxia regulated factors, HIF-1α and CA-9, vascular endothelial growth factor (VEGF) and a blood vessel marker, CD31. Disease progression, liver specific recurrence and actuarial survival were recorded. Results The clinicopathological characteristics of the cancers were comparable between the groups. The median follow-up was 115 months (range 106–124). Overall, 5 year, local hepatic recurrence-free survival and progression-free periods were reduced in those patients undergoing PVE (p = 0.026, p = 0.060, p = 0.001 and p = 0.008 respectively). The expression of hypoxia markers between the groups was similar. Conclusion This is the first long-term (>5 year) case matched series on outcome of patients with CRLM resected following PVE. Whilst, PVE facilitates potentially curative resection of CRLM, prognosis is less than those patients not requiring PVE. The mechanism behind this survival difference has not been established. Disclosure of Interest None Declared

Activated Schwann cells in pancreatic cancer are linked to analgesia via suppression of spinal astroglia and microglia

ObjectiveThe impact of glia cells during GI carcinogenesis and in cancer pain is unknown. Here, we demonstrate a novel mechanism how Schwann cells (SCs) become activated in the pancreatic cancer...

Abstract 2077: Snail modulates stemness properties in hypoxic Glioblastoma

Abstract Introduction: Glioblastoma (GBM) is the most aggressive form of glioma characterized by hypoxic regions with surrounding areas of necrosis. Hypoxia is responsible for inducing cancer invasiveness via epithelial-mesenchymal transition (EMT), thereby promoting tumor aggressiveness and therapeutic resistance. HIF-1α is known to upregulate Snail in various cancers. Snail over expression correlates with cancer stem cell-like properties in cancers. The association of EMT and stemness properties plays a vital role in tumors progression and might helps in therapeutic targeting. We have, therefore, investigated the correlation of EMT and Stem cell markers in hypoxic GBM. Method: mRNA expression of 5 EMT, 3 stemness and 5 hypoxia markers were analyzed in the 31 GBM samples and U373MG glioma cell line maintained under normoxia (20% O2) and severe hypoxia (0.2% O2) conditions for 24, 48 and 72 hrs. Correlation and cluster analysis were applied using SPSS 11.5, Cluster 3.0 software etc and R-software. Result: In tumors, spearman correlation was found to be significant (p≤0.05) between EMT markers and Hypoxia markers. Of all EMT markers, Snail correlated significantly with the maximum hypoxia markers, including HIF-1α. In addition, Snail was significantly correlated with Stem cell markers SOX2 (p&amp;lt;0.01) and Nestin (p&amp;lt;0.05). Gene Cluster analysis in 31 GBM arranged in decreasing order of Snail expression showed significant upregulation of SOX2 and Nestin in the high Snail expressing tertile. This correlation of gene expression in tumors was validated by in-vitro expression analysis of the molecules in U373MG cells, where we assessed significant increase in the expression of hypoxia markers (HIF-1α, VEGF and CA9), EMT (Snail and LOX) and SC markers (SOX2 and OCT4) were significantly increased under severe hypoxic conditions. Interpretation &amp; conclusions: Our finding for the first time suggests the role of Snail in provoking stemness of cancer cells in hypoxic GBM, thereby promoting aggressiveness of GBM. Note: This abstract was not presented at the meeting. Citation Format: Chitrangda srivastava, Khushboo Irshad, Arpit Sahu, Parthaprasad Chattopadhyay, Chitra Sarkar, Deepak kumar Gupta, Subrata Sinha, Kunzang Chosdol. Snail modulates stemness properties in hypoxic Glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2077. doi:10.1158/1538-7445.AM2014-2077

Immunohistochemical evaluation of hypoxia markers in the myocardium

Some proteins, termed hypoxia markers, are more highly expressed in hypoxic cells including ischemic cells. We evaluated the timing of the expression of various hypoxia markers and their specificity of expression in the myocardium in various causes of death. We selected two cases for each cause of death from the forensic autopsies performed by our department and evaluated the diffuse expression of hypoxia markers in the myocardium. We also evaluated the focal expression of these markers in the ventricles of five sudden cardiac death cases. We found that the expression of hypoxia markers in the myocardium varied depending on the cause of death and length of the dying process. This information may help in determining the cause of death in forensic medicine. In particular, we identified RBM3 and CCC9 as markers for ischemic heart disease. Immunohistological staining of myocardium with hypoxia markers could help to find the cause leading to death.

Prospective study assessing hypoxia-related proteins as markers for the outcome of treatment with sunitinib in advanced clear-cell renal cell carcinoma

BackgroundPrevious studies suggest that expression of hypoxia markers may be associated with response to antiangiogenic drugs. Thus, we aimed to identify predictors of sunitinib outcome in clear-cell renal cell carcinoma (ccRCC). Patients and methodsThe expression of eight key proteins related to hypoxia (CAIX, HIF1A, HIF2A, VEGFA, VEGFR1, VEGFR2, VEGFR3 and PDGFRB) and P-glycoprotein were assessed by immunohistochemistry in 67 primary ccRCC samples from prospectively recruited patients treated with first-line sunitinib. The proteins expression, VHL inactivation and EGLN3 mRNA content were compared with the patients' response to sunitinib. ResultsHigh expression of HIF2A and PDGFRB was associated with better sunitinib RECIST objective response (P = 0.024 and P = 0.026; respectively) and increased VEGFR3 expression was associated with longer progression-free survival (P = 0.012). VEGFR3 overexpression showed a negative correlation with VEGFR3 polymorphism rs307826 (P = 0.002), a sunitinib resistance predictor. With respect to overall survival (OS), high VEGFA was associated with short (P = 0.009) and HIF2A with long (P = 0.048) survival times. High EGLN3 mRNA content was associated with shorter OS (P = 0.023). ConclusionsWe found an association between several proteins involved in hypoxia and sunitinib efficacy. In addition, low VEGFR3 expression was associated with worse outcome and with VEGFR3 rs307826 variant allele, reinforcing VEGFR3 as a marker of sunitinib resistance.

Immunohistochemical expression of hypoxia markers in oral squamous cells carcinoma of the tongue

Immunohistochemical expression of hypoxia markers in oral squamous cells carcinoma of the tongue

Stromal Expression of Hypoxia Regulated Proteins Is an Adverse Prognostic Factor in Colorectal Carcinomas

Background: Hypoxia modifies the phenotype of tumors in a way that promotes tumor aggressiveness and resistance towards chemotherapy and radiotherapy. However, the expression and influence of hypoxia-regulated proteins on tumor biology are not well characterized in colorectal tumors. We studied the role of protein expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, carbonic anhydrase 9 (CA9) and glucose transporter 1 (GLUT1) in patients with colorectal adenocarcinomas. Methods: Expression of HIF-1α, HIF-2α, CA9 and GLUT1 was quantified by immunohistochemistry in 133 colorectal adenocarcinomas. The expression of hypoxia markers was correlated with clinicopathological variables and overall patient survival. Results: Expression of these hypoxia markers was detected in the epithelial compartment of the tumor cells as well as in tumor-associated stromal cells. Although tumor cells frequently showed expression of one or more of the investigated hypoxia markers, no correlation among these markers or with clinical response was found. However, within the tumor stroma, positive correlations between the hypoxia markers HIF-2α, CA9 and GLUT1 were observed. Furthermore expression of HIF-2α and CA9 in tumor-associated stroma were both associated with a significantly reduced overall survival. In the Cox proportional hazard model, stromal HIF-2α expression was an independent prognostic factor for survival. Conclusion: These observations show, that expression of hypoxia regulated proteins in tumor-associated stromal cells, as opposed to their expression in epithelial tumor cells, is associated with poor outcome in colorectal cancer. This study suggests that tumor hypoxia may influence tumor-associated stromal cells in a way that ultimately contributes to patient prognosis.

Angiogenesis and hypoxia in lymph node metastases is predicted by the angiogenesis and hypoxia in the primary tumour in patients with breast cancer

Hypoxia and angiogenesis are important factors in breast cancer progression. Little is known of hypoxia and angiogenesis in lymph node metastases of breast cancer. The aim of this study was to quantify hypoxia, by hypoxia-induced marker expression levels, and angiogenesis, by endothelial cell proliferation, comparing primary breast tumours and axillary lymph node metastases. Tissue sections of the primary tumour and a lymph node metastasis of 60 patients with breast cancer were immunohistochemically stained for the hypoxia-markers carbonic anhydrase 9 (CA9), hypoxia-inducible factor-1α (Hif-1α) and DEC-1 and for CD34/Ki-67. Endothelial cell proliferation fraction (ECP%) and tumour cell proliferation fraction (TCP%) were assessed. On haematoxylin–eosin stain, the growth pattern and the presence of a fibrotic focus were assessed. Hypoxia-marker expression, ECP% and TCP% in primary tumours and in lymph node metastases were correlated to each other and to clinico-pathological variables. Median ECP% and TCP% in primary tumours and lymph node metastases were comparable (primary tumours: ECP%=4.02, TCP%=19.54; lymph node metastases: ECP%=5.47, TCP%=21.26). ECP% correlated with TCP% (primary tumours: r=0.63, P<0.001; lymph node metastases: r=0.76, P<0.001). CA9 and Hif-1α expression were correlated (primary tumours P=0.005; lymph node metastases P<0.001). In primary tumours, CA9 and Hif-1α expression were correlated with DEC-1 expression (P=0.05), presence of a fibrotic focus (P<0.007) and mixed/expansive growth pattern (P<0.001). Primary tumours and lymph node metastases with CA9 or Hif-1α expression had a higher ECP% and TCP% (P<0.003); in primary tumours, mixed/expansive growth pattern and fibrotic focus were characterised by higher ECP% (P=0.03). Furthermore, between primary tumours and lymph node metastases a correlation was found for ECP%, TCP%, CA9 and Hif-1α expression (ECP% r=0.51, P<0.001; TCP r=0.77, P<0.001; CA9 and Hif-1α P<0.001). Our data demonstrate that the growth of breast cancer lymph node metastases is angiogenesis dependent and that angiogenesis and hypoxia in the primary tumour predict angiogenesis and hypoxia in the lymph node metastases. Together with previous findings in breast cancer liver metastases, which grow in 96% of cases angiogenesis independently, these data suggest that both the intrinsic growth characteristics and angiogenic potential of breast cancer cells and the site-specific tumour microenvironment determine angiogenesis and hypoxia in breast cancer.