The Influence of VE-Cadherin on Adhesion and Incorporation of Breast Cancer Cells into Vascular Endothelium.

During metastasis, cancer cells that originate from the primary tumor circulate in the bloodstream, extravasate, and form micrometastases at distant locations. Several lines of evidence suggest that specific interactions between cancer cells and endothelial cells, in particular tumor cell adhesion to the endothelium and transendothelial migration, play a crucial role in extravasation. Here we have studied the role of vascular endothelial (VE)-cadherin which is expressed aberrantly by breast cancer cells and might promote such interactions. By comparing different human breast cancer cell lines, we observed that the number of cancer cells that adhered to endothelium correlated with VE-cadherin expression levels. VE-cadherin silencing experiments confirmed that VE-cadherin enhances cancer cell adhesion to endothelial cells. However, in contrast, the number of cancer cells that incorporated into the endothelium was not dependent on VE-cadherin. Thus, it appears that cancer cell adhesion and incorporation are distinct processes that are governed by different molecular mechanisms. When cancer cells incorporated into the endothelial monolayer, they formed VE-cadherin positive contacts with endothelial cells. On the other hand, we also observed tumor cells that had displaced endothelial cells, reflecting either different modes of incorporation, or a temporal sequence where cancer cells first form contact with endothelial cells and then displace them to facilitate transmigration. Taken together, these results show that VE-cadherin promotes the adhesion of breast cancer cells to the endothelium and is involved in the initial phase of incorporation, but not their transmigration. Thus, VE-cadherin might be of relevance for therapeutic strategies aiming at preventing the metastatic spread of breast cancer cells.

Hematopoietic hypoxia-inducible factor 2α deficiency ameliorates pathological retinal neovascularization via modulation of endothelial cell apoptosis.

A hallmark of proliferative retinopathies, such as retinopathy of prematurity (ROP), is a pathological neovascularization orchestrated by hypoxia and the resulting hypoxia-inducible factor (HIF)-dependent response. We studied the role of Hif2α in hematopoietic cells for pathological retina neovascularization in the murine model of ROP, the oxygen-induced retinopathy (OIR) model. Hematopoietic-specific deficiency of Hif2α ameliorated pathological neovascularization in the OIR model, which was accompanied by enhanced endothelial cell apoptosis. That latter finding was associated with up-regulation of the apoptosis-inducer FasL in Hif2α-deficient microglia. Consistently, pharmacological inhibition of the FasL reversed the reduced pathological neovascularization from hematopoietic-specific Hif2α deficiency. Our study found that the hematopoietic cell Hif2α contributes to pathological retina angiogenesis. Our findings not only provide novel insights regarding the complex interplay between immune cells and endothelial cells in hypoxia-driven retina neovascularization but also may have therapeutic implications for proliferative retinopathies.-Korovina, I., Neuwirth, A., Sprott, D., Weber, S., Sardar Pasha, S. P. B., Gercken, B., Breier, G., El-Armouche, A., Deussen, A., Karl, M. O., Wielockx, B., Chavakis, T., Klotzsche-von Ameln, A. Hematopoietic hypoxia-inducible factor 2α deficiency ameliorates pathological retinal neovascularization via modulation of endothelial cell apoptosis.

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased ß-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1ß production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identification of a clonally expanding haematopoietic compartment in bone marrow.

In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self-renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high-resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48- putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48- cells. Our results identify a previously unrecognized, vessel-associated BM compartment with a specific localization and properties distinct from the marrow cavity.

Receptor tyrosine kinase inhibitors: Are they real tumor killers?

Inhibiting tumor growth by targeting the tumor vasculature was first proposed by Judah Folkman almost 40 years ago. Since then, different approaches and numerous drugs and agents have been developed to achieve this goal, either with the aim of inhibiting tumor neoangiogenesis or normalizing the tumor vasculature. Among the most promising therapeutic targets are receptor tyrosine kinases (RTKs), some of which are predominantly expressed on tumor endothelial cells, although they are sometimes also present on tumor cells. The majority of RTK inhibitors investigated over the past two decades competes with ATP at the active site of the kinase and therefore block the phosphorylation of intracellular targets. Some of these drugs have been approved for therapy, whereas others are still in clinical trials. Here, we discuss the scientific basis, current status, problems and future prospects of RTK inhibition in anti-tumor therapy.

Mechanoinduction of lymph vessel expansion.

In the mammalian embryo, few mechanical signals have been identified to influence organ development and function. Here, we report that an increase in the volume of interstitial or extracellular fluid mechanically induces growth of an organ system, that is, the lymphatic vasculature. We first demonstrate that lymph vessel expansion in the developing mouse embryo correlates with a peak in interstitial fluid pressure and lymphatic endothelial cell (LEC) elongation. In 'loss-of-fluid' experiments, we then show that aspiration of interstitial fluid reduces the length of LECs, decreases tyrosine phosphorylation of vascular endothelial growth factor receptor-3 (VEGFR3), and inhibits LEC proliferation. Conversely, in 'gain-of-fluid' experiments, increasing the amount of interstitial fluid elongates the LECs, and increases both VEGFR3 phosphorylation and LEC proliferation. Finally, we provide genetic evidence that ß1 integrins are required for the proliferative response of LECs to both fluid accumulation and cell stretching and, therefore, are necessary for lymphatic vessel expansion and fluid drainage. Thus, we propose a new and physiologically relevant mode of VEGFR3 activation, which is based on mechanotransduction and is essential for normal development and fluid homeostasis in a mammalian embryo.

VEGFR2 Signaling Prevents Colorectal Cancer Cell Senescence to Promote Tumorigenesis in Mice With Colitis.

BACKGROUND & AIMS: Senescence prevents cellular transformation. We investigated whether vascular endothelial growth factor (VEGF) signaling via its receptor, VEGFR2, regulates senescence and proliferation of tumor cells in mice with colitis-associated cancer (CAC). METHODS: CAC was induced in VEGFR2(ΔIEC) mice, which do not express VEGFR2 in the intestinal epithelium, and VEGFR2(fl/fl) mice (controls) by administration of azoxymethane followed by dextran sodium sulfate. Tumor development and inflammation were determined by endoscopy. Colorectal tissues were collected for immunoblot, immunohistochemical, and quantitative polymerase chain reaction analyses. Findings from mouse tissues were confirmed in human HCT116 colorectal cancer cells. We analyzed colorectal tumor samples from patients before and after treatment with bevacizumab. RESULTS: After colitis induction, VEGFR2(ΔIEC) mice developed significantly fewer tumors than control mice. A greater number of intestinal tumor cells from VEGFR2(ΔIEC) mice were in senescence than tumor cells from control mice. We found VEGFR2 to activate phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT, resulting in inactivation of p21 in HCT116 cells. Inhibitors of VEGFR2 and AKT induced senescence in HCT116 cells. Tumor cell senescence promoted an anti-tumor immune response by CD8(+) T cells in mice. Patients whose tumor samples showed an increase in the proportion of senescent cells after treatment with bevacizumab had longer progression-free survival than patients in which the proportion of senescent tumor cells did not change before and after treatment. CONCLUSIONS: Inhibition of VEGFR2 signaling leads to senescence of human and mouse colorectal cancer cells. VEGFR2 interacts with phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT to inactivate p21. Colorectal tumor senescence and p21 level correlate with patient survival during treatment with bevacizumab.

HIF prolyl hydroxylase 2 (PHD2) is a critical regulator of hematopoietic stem cell maintenance during steady-state and stress.

Hypoxia is a prominent feature in the maintenance of hematopoietic stem cell (HSC) quiescence and multipotency. Hypoxia-inducible factor (HIF) prolyl hydroxylase domain proteins (PHDs) serve as oxygen sensors and may therefore regulate this system. Here, we describe a mouse line with conditional loss of HIF prolyl hydroxylase 2 (PHD2) in very early hematopoietic precursors that results in self-renewal of multipotent progenitors under steady-state conditions in a HIF1α- and SMAD7-dependent manner. Competitive bone marrow (BM) transplantations show decreased peripheral and central chimerism of PHD2-deficient cells but not of the most primitive progenitors. Conversely, in whole BM transfer, PHD2-deficient HSCs replenish the entire hematopoietic system and display an enhanced self-renewal capacity reliant on HIF1α. Taken together, our results demonstrate that loss of PHD2 controls the maintenance of the HSC compartment under physiological conditions and causes the outcompetition of PHD2-deficient hematopoietic cells by their wild-type counterparts during stress while promoting the self-renewal of very early hematopoietic progenitors.

HIF-1α is a protective factor in conditional PHD2-deficient mice suffering from severe HIF-2α-induced excessive erythropoiesis.

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2α. In contrast, HIF-1α served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1α resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2α-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2α-dependent erythrocytosis, whereas HIF-1α protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia.

Endothelial cadherins in cancer.

Cadherins are cell adhesion receptors that play important roles in embryogenesis and tissue homoeostasis. Endothelial cells express various members of the cadherin superfamily, in particular vascular endothelial (VE-) cadherin, which is the main adhesion receptor of endothelial adherens junctions and neural (N-) cadherin, which is normally localized outside the junctions and may mediate adhesion between endothelial cells and non-endothelial cells. Dysregulation of cadherin expression has been implicated in tumor progression, in particular the loss of epithelial (E-) cadherin expression or function and the gain of N-cadherin. Moreover, more recently, aberrant expression of VE-cadherin was observed in certain cancer types. In breast carcinoma, VE-cadherin was shown to promote tumor cell proliferation and invasion through enhancing TGF-ß signaling. Thus, in breast cancer, the cadherin switch involves another player, vascular endothelial cadherin, which is part of an intricate interplay of classical cadherins in breast cancer progression.

A role for prolyl hydroxylase domain proteins in hippocampal synaptic plasticity.

Hypoxia-inducible factors (HIFs) are key transcriptional regulators that play a major role in oxygen homeostasis. HIF activity is tightly regulated by oxygen-dependent hydroxylases, which additionally require iron and 2-oxoglutarate as cofactors. Inhibition of these enzymes has become a novel target to modulate the hypoxic response for therapeutic benefit. Inhibition of prolyl-4-hydroxylase domains (PHDs) have been shown to delay neuronal cell death and protect against ischemic injury in the hippocampus. In this study we have examined the effects of prolyl hydroxylase inhibition on synaptic transmission and plasticity in the hippocampus. Field excitatory postsynaptic potentials (fEPSPs) and excitatory postsynaptic currents (EPSCs) were elicited by stimulation of the Schaffer collateral pathway in the CA1 region of the hippocampus. Treatment of rat hippocampal slices with low concentrations (10 µM) of the iron chelator deferosoxamine (DFO) or the 2-oxoglutarate analogue dimethyloxalyl glycine (DMOG) had no effect on fEPSP. In contrast, application of 1 mM DMOG resulted in a significant decrease in fEPSP slope. Antagonism of the NMDA receptor attenuated the effects of DMOG on baseline synaptic signalling. In rat hippocampal slices pretreated with DMOG and DFO the induction of long-term potentiation (LTP) by tetanic stimulation was strongly impaired. Similarly, neuronal knockout of the single PHD family member PHD2 prevented murine hippocampal LTP. Preconditioning of PHD2 deficient hippocampi with either DMOG, DFO, or the PHD specific inhibitor JNJ-42041935, did not further decrease LTP suggesting that DMOG and DFO influences synaptic plasticity primarily by inhibiting PHDs rather than unspecific effects. These findings provide striking evidence for a modulatory role of PHD proteins on synaptic plasticity in the hippocampus.

Cardiomyocyte-specific prolyl-4-hydroxylase domain 2 knock out protects from acute myocardial ischemic injury.

Prolylhydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of the α-subunit of the transcription factor hypoxia inducible factor (HIF)-1. HIF-1 affects cardiac development as well as adaptation of the heart toward increased pressure overload or myocardial infarction. We have disrupted PHD2 in cardiomyocytes (cPhd (-/-)) using Phd2(flox/flox) mice in combination with MLCvCre mice, which resulted in HIF-1α stabilization and activation of HIF target genes in the heart. Although cPhd2(-/-) mice showed no gross abnormalities in cardiac filament structure or function, we observed a significant increased cardiac capillary area in those mice. cPhd2 (-/-) mice did not respond differently to increased mechanical load by transverse aortic constriction compared with their wild-type (wt) littermates. After ligation of the left anterior descending artery, however, the area at risk and area of necrosis were significantly smaller in the cPhd2(-/-) mice compared with Phd2 wt mice in line with the described pivotal role of HIF-1α for tissue protection in case of myocardial infarction. This correlated with a decreased number of apoptotic cells in the infarcted myocardium in the cPhd2(-/-) mice and significantly improved cardiac function 3 weeks after myocardial infarction.

Neuron-specific prolyl-4-hydroxylase domain 2 knockout reduces brain injury after transient cerebral ischemia.

BACKGROUND AND PURPOSE: Numerous factors involved in the adaptive response to hypoxia, including erythropoietin and vascular endothelial growth factor are transcriptionally regulated by hypoxia-inducible factors (HIFs). During normoxia, prolyl-4-hydroxylase domain (PHD) proteins hydroxylate HIF-α subunits, resulting in their degradation. We investigated the effect of neuronal deletion of PHD2, the most abundant isoform in brain, for stroke outcome. METHODS: We generated neuron-specific Phd2 knockout mice and subjected animals to systemic hypoxia or transient middle cerebral artery occlusion. Infarct volume and cell death were determined by histology. HIF-1α, HIF-2α, and HIF target genes were analyzed by immunoblotting and real-time polymerase chain reaction, respectively. RESULTS: Neuron-specific ablation of Phd2 significantly increased protein stability of HIF-1α and HIF-2α in the forebrain and enhanced expression of the neuroprotective HIF target genes erythropoietin and vascular endothelial growth factor as well as glucose transporter and glycolysis-related enzymes under hypoxic and ischemic conditions. Mice with Phd2-deficient neurons subjected to transient cerebral ischemia exhibited a strong reduction in infarct size, and cell death of hippocampal CA1 neurons located in the peri-infarct region was dramatically reduced in these mice. Vessel density in forebrain subregions, except for caudate-putamen, was not altered in Phd2-deficient animals. CONCLUSIONS: Our findings denote that the endogenous adaptive response on hypoxic-ischemic insults in the brain is at least partly dependent on the activity of HIFs and identify PHD2 as the key regulator for the protective hypoxia response. The results suggest that specific inhibition of PHD2 may provide a useful therapeutic strategy to protect brain tissue from ischemic injury.

Interplay between neural-cadherin and vascular endothelial-cadherin in breast cancer progression.

INTRODUCTION: Deregulation of cadherin expression, in particular the loss of epithelial (E)-cadherin and gain of neural (N)-cadherin, has been implicated in carcinoma progression. We previously showed that endothelial cell-specific vascular endothelial (VE)-cadherin can be expressed aberrantly on tumor cells both in human breast cancer and in experimental mouse mammary carcinoma. Functional analyses revealed that VE-cadherin promotes tumor cell proliferation and invasion by stimulating transforming growth factor (TGF)-ß signaling. Here, we investigate the functional interplay between N-cadherin and VE-cadherin in breast cancer. METHODS: The expression of N-cadherin and VE-cadherin was evaluated by immunohistochemistry in a tissue microarray with 84 invasive human breast carcinomas. VE-cadherin and N-cadherin expression in mouse mammary carcinoma cells was manipulated by RNA interference or overexpression, and cells were then analyzed by immunofluorescence, reverse transcriptase-polymerase chain reaction, and western blot. Experimental tumors were generated by transplantation of the modified mouse mammary carcinoma cells into immunocompetent mice. Tumor growth was monitored, and tumor tissue was subjected to histological analysis. RESULTS: VE-cadherin and N-cadherin were largely co-expressed in invasive human breast cancers. Silencing of N-cadherin in mouse mammary carcinoma cells led to decreased VE-cadherin expression and induced changes indicative of mesenchymal-epithelial transition, as indicated by re-induction of E-cadherin, localization of ß-catenin at the cell membrane, decreased expression of vimentin and SIP1, and gain of epithelial morphology. Suppression of N-cadherin expression also inhibited tumor growth in vivo, even when VE-cadherin expression was forced. CONCLUSIONS: Our results highlight the critical role of N-cadherin in breast cancer progression and show that N-cadherin is involved in maintaining the malignant tumor cell phenotype. The presence of N-cadherin prevents the re-expression of E-cadherin and localization of ß-catenin at the plasma membrane of mesenchymal mammary carcinoma cells. N-cadherin is also required to maintain the expression of VE-cadherin in malignant tumor cells but not vice versa. Thus, N-cadherin acts in concert with VE-cadherin to promote tumor growth.

Overexpression of factor inhibiting HIF-1 enhances vessel maturation and tumor growth via platelet-derived growth factor-C.

Recent studies have revealed that the maturation state of vessels in tumors, in addition to vascularity, is a critical determinant of tumor growth. The role of oxygen-dependent signaling pathways in hypoxia-stimulated angiogenesis is well established, however, little is known about their impact on vessel maturation in tumors. Here, we have studied the function of the cellular oxygen sensor, factor inhibiting HIF-1 (FIH), which controls the activity of hypoxia-inducible factor-1. FIH silencing in mouse LM8 osteosarcoma stimulated angiogenesis but did not influence tumor growth. In contrast, FIH overexpression led to increased pericyte coverage of the tumor vasculature, reduced vessel leakiness and enhanced tumor growth. Vessel maturation was paralleled by up-regulation of platelet-derived growth factor (PDGF)-C in tumors and expression of PDGF receptor-α on pericytes. Ablation of PDGF-C in FIH-overexpressing tumor cells reduced pericyte coverage and tumor growth. Our data suggest that FIH-mediated PDGF-C induction in LM8 osteosarcoma stimulates the recruitment of PDGFR-α positive pericytes to the tumor vasculature, leading to vessel maturation and enhanced tumor growth.

Prognostic and predictive value of circulating tumor cell analysis in colorectal cancer patients.

OBJECTIVE: The aim of this study was to assess the prognostic and predictive values of circulating tumor cell (CTC) analysis in colorectal cancer patients. PATIENTS AND METHODS: Presence of CTCs was evaluated in 60 colorectal cancer patients before systemic therapy--from which 33 patients were also evaluable for CTC analysis during the first 3 months of treatment--through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 (targeting mucin 1 and EpCAM, respectively), followed by real-time RT-PCR analysis of the tumor-associated genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. RESULTS: Patients were stratified into groups according to CTC detection (CTC negative, when all marker genes were negative; and CTC positive when at least one of the marker genes was positive). Patients with CTC positivity at baseline had a significant shorter median progression-free survival (median PFS 181.0 days; 95% CI 146.9-215.1) compared with patients with no CTCs (median PFS 329.0 days; 95% CI 299.6-358.4; Log-rank P < .0001). Moreover, a statistically significant correlation was also founded between CTC detection during treatment and radiographic findings at the 6 month staging. This correlation applied to CTC results before therapy (odds ratio (OR), 6.22), 1 to 4 weeks after beginning of treatment (OR, 5.50), 5 to 8 weeks after beginning of treatment (OR, 7.94) 9 to 12 weeks after beginning of treatment (OR, 14.00) and overall CTC fluctuation during the course of treatment (OR, 20.57). CONCLUSION: The present study provides evidence of a strong correlation between CTC detection and radiographic disease progression in patients receiving chemotherapy for colorectal cancer. Our results suggest that in addition to the current prognostic factors, CTC analysis represent a potential complementary tool for prediction of colorectal cancer patients' outcome. Moreover, the present test allows for molecular characterization of CTCs, which may be of relevance to the creation of personalized therapies.

Multimarker gene analysis of circulating tumor cells in pancreatic cancer patients: a feasibility study.

OBJECTIVE: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. METHODS: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. RESULTS: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8-87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1-151.9) for CTC-negative patients (p = 0.01, log-rank test). CONCLUSION: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients.

Development of a molecular multimarker assay for the analysis of circulating tumor cells in adenocarcinoma patients.

BACKGROUND: The analysis of circulating tumor cells (CTCs) is emerging as a promising diagnostic tool in oncology. However, even if a variety of methods for CTC isolation have been already developed, their specificity and/or sensitivity still remain problematic. The aim of this study was to develop an immunomagnetic/real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the molecular detection of circulating tumor cells (CTCs) in peripheral blood (PB) of adenocarcinoma cancer patients. METHODS: The presence of CTCs was evaluated in 945 PB blood samples from 247 adenocarcinoma cancer patients and in 42 healthy controls by immunomagnetic enrichment using the antibodies BM7 and VU1D9 followed by real-time RT-PCR analysis of the marker genes KRT19, MUC1, EPCAM, CEACAM5, BIRCS, SCGB2A2, and ERBB2. RESULTS: The developed assay showed not only high specificity, as none of the healthy controls were found positive for the multimarker gene panel, but also great sensitivity as CTCs were detected in adenocarcinomas arising from 10 different organs. According to tumor primary origin, CTC positivity was detected in 33.3% of Ampulla of Vater adenocarcinomas, 69.6% of bile ducts adenocarcinomas, 61.3% of breast adenocarcinomas, 61.3% of cardia adenocarcinomas, 60.6% of colon adenocarcinomas, 66.7% of esophagus adenocarcinomas, 57.1% of pancreas adenocarcinomas, 66.7% of rectum adenocarcinomas, 33.3% of small intestine adenocarcinomas, and 62.2% of stomach adenocarcinomas. CONCLUSIONS: Our results suggest that the current developed technique can be used to detect CTCs in all major adenocarcinomas, with great sensitivity without compromising specificity.

Ablation of cholesterol biosynthesis in neural stem cells increases their VEGF expression and angiogenesis but causes neuron apoptosis.

Although sufficient cholesterol supply is known to be crucial for neurons in the developing mammalian brain, the cholesterol requirement of neural stem and progenitor cells in the embryonic central nervous system has not been addressed. Here we have conditionally ablated the activity of squalene synthase (SQS), a key enzyme for endogenous cholesterol production, in the neural stem and progenitor cells of the ventricular zone (VZ) of the embryonic mouse brain. Mutant embryos exhibited a reduced brain size due to the atrophy of the neuronal layers, and died at birth. Analyses of the E11.5-E15.5 dorsal telencephalon and diencephalon revealed that this atrophy was due to massive apoptosis of newborn neurons, implying that this progeny of the SQS-ablated neural stem and progenitor cells was dependent on endogenous cholesterol biosynthesis for survival. Interestingly, the neural stem and progenitor cells of the VZ, the primary target of SQS inactivation, did not undergo significant apoptosis. Instead, vascular endothelial growth factor (VEGF) expression in these cells was strongly upregulated via a hypoxia-inducible factor-1-independent pathway, and angiogenesis in the VZ was increased. Consistent with an increased supply of lipoproteins to these cells, the level of lipid droplets containing triacylglycerides with unsaturated fatty acyl chains was found to be elevated. Our study establishes a direct link between intracellular cholesterol levels, VEGF expression, and angiogenesis. Moreover, our data reveal a hitherto unknown compensatory process by which the neural stem and progenitor cells of the developing mammalian brain evade the detrimental consequences of impaired endogenous cholesterol biosynthesis.