Isolation and characterization of human breast tumor-derived endothelial cells

Grange, Cristina; Bussolati, Benedetta; Bruno, Stefania; Fonsato, Valentina; Sapino, Anna; Camussi, Giovanni

doi:10.3892/or.15.2.381

Cited by 53 publications

(72 citation statements)

References 20 publications

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“…A similar surface expression profile was detected in mammary-derived TECs (mTECs) (data not shown). As extensively documented, TECs also differ from their normal counterpart for a rapid turnover rate in absence of growth factors (Bussolati et al, 2003;Grange et al, 2006). Indeed, monitoring rTEC and mTEC growth at low serum concentration (2%) for different time intervals (24, 48 and 72 h), an increase in cells in the S phase ( Figure 1b, upper panel) could be detected and this increased proliferative rate (Figure 1b, lower panel) was maintained for several passages (12 passages, data not shown).…”

Section: Tec Characterization and Functional Behaviormentioning

confidence: 98%

“…Thus, TECs, isolated from renal clear cell carcinomas and breast carcinomas by CD105-positive immunomagnetic sorting (Bussolati et al, 2003;Grange et al, 2006), were first analyzed for endothelial cell surface markers by cytofluorimetric analysis. As reported in Figure 1a, we found that renal TECs (rTECs) were positive for the expression of several endothelial cell markers, such as CD105, CD146 (c) Cell migration assay was performed in Boyden's chambers, as described in Materials and methods section.…”

Section: Tec Characterization and Functional Behaviormentioning

confidence: 99%

“…Human TECs derived from kidney and breast carcinomas (both cell lines were derived from primary tumors; breast carcinoma cells were derived from a woman 48 years old: T3bG2N0M0, while renal carcinoma cells were derived from a man 55 years old: T1bG2N0M0), kindly provided by Professor Camussi, were isolated and grown as previously described (Bussolati et al, 2003;Grange et al, 2006). rTECs and mTECs were maintained in endothelial basal medium-2 medium supplemented with 2% of fetal bovine serum until twelfth passage.…”

Section: Cell Culturesmentioning

confidence: 99%

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IL-3 is a novel target to interfere with tumor vasculature

et al. 2011

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Angiogenesis inhibiting agents are currently integral component of anticancer therapy. However, tumors, initially responsive to anti-angiogenic drugs or vascular targeting agents, can acquire resistance. The limited clinical efficacy might result from the heterogeneous nature of tumors or alternatively from the unique phenotype of tumor vascular cells, widely diverse from so-called 'normal' endothelium. Hence, defining the molecular mechanisms driving this diversity might provide a rational basis to design combinatory therapies that should be more effective in avoiding resistance. Herein, we demonstrated that tumor-derived endothelial cells (TECs) isolated from breast and kidney carcinomas retained an endothelial phenotype, but outspread independently of growth factors. Applying small interfering RNA approach, we demonstrated that interleukin (IL)-3, but not vascular endothelial growth factor, released by TECs, supports their autocrine growth and promotes in vivo vessel formation and tumor angiogenesis. Meanwhile, we found that the expression of the membrane-bound kit ligand (mbKitL) depends on IL-3, and it is crucial for adhesion of endothelial progenitor cells (EPCs) and inflammatory cells to TECs. These events required Akt activation. Finally, the finding that depletion of the mbKitL prevented EPC and inflammatory cell trafficking into vascular microenvironment, indicates that, as in bone marrow, the mbKitL can act as a membrane/adhesion molecule for c-Kit-expressing cells. These data provide evidences that an IL-3 autocrine loop can drive a tumor endothelial switch and that targeting IL-3 might confer a significant therapeutic advantage to hamper tumor angiogenesis.

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Section: Tec Characterization and Functional Behaviormentioning

confidence: 98%

Section: Tec Characterization and Functional Behaviormentioning

confidence: 99%

Section: Cell Culturesmentioning

confidence: 99%

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IL-3 is a novel target to interfere with tumor vasculature

et al. 2011

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“…ECs were isolated, using anti-CD105 antibody coupled to magnetic beads, by magnetic cell sorting using the MACS system (Miltenyi Biotec) and grown in complete EBM (Cambrex) supplemented with 10% FCS (Cambrex), 50 μg/mL gentamicin (Cambrex), and 2 mmol/L glutamine (Cambrex) as previously described (10). Cells were used at passages 3 to 15.…”

Section: Cell Culturesmentioning

confidence: 99%

“…Interestingly, TECs display a distinct and unique phenotype different from that of normal vascular ECs at molecular and functional levels (9)(10)(11). Recently, TECs obtained from breast carcinomas (B-TEC) have been established and characterized, showing an immature proangiogenic phenotype with enhanced proliferation, motility, and capillary-like tube formation compared with "normal" ECs (10,12).…”

Section: Introductionmentioning

confidence: 99%

Multiple Roles of Protein Kinase A in Arachidonic Acid–Mediated Ca2+ Entry and Tumor-Derived Human Endothelial Cell Migration

Fiorio

Genova

Pupo

et al. 2010

Molecular Cancer Research

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We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca 2+ entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca 2+ signaling in the same cells. B-TEC stimulation with 5 μmol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser 1177 , and NO release was measured with the fluorescent NO-sensitive probe DAR4M-AM. PKA inhibition by the use of the membrane-permeable PKA inhibitory peptide myristoylated PKI 14-22 completely prevented both AA-and NOinduced calcium entry and abolished B-TEC migration promoted by AA. AA-dependent calcium entry and cell migration were significantly affected by both the NOS inhibitor N G -nitro-L-arginine methyl ester and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, suggesting that NO release is functionally involved in the signaling dependent on AA. Moreover, pretreatment with carboxyamidotriazole, an antiangiogenic compound that interferes with agonist-activated calcium entry, prevented AA-dependent B-TEC motility. Interestingly, even in the absence of AA, enhancement of the cyclic AMP/PKA pathway with the adenylyl cyclase activator forskolin evoked a calcium entry dependent on NOS recruitment and NO release. The functional relevance of AA-induced calcium entry could be restricted to tumor-derived endothelial cells (EC) because AA evoked a smaller calcium entry in normal human microvascular ECs compared with B-TECs, and even more importantly, it was unable to promote cell motility in wound healing assay. This evidence opens an intriguing opportunity for differential pharmacologic treatment between normal and tumor-derived human ECs.

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The proangiogenic phenotype of human tumor-derived endothelial cells depends on thrombospondin-1 downregulation via phosphatidylinositol 3-kinase/Akt pathway

et al. 2006

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Tumor-derived endothelial cells (TEC) display increased survival and angiogenic properties in respect to normal endothelial cells. The aim of this study was to investigate the mechanism potentially involved in TEC proangiogenic phenotype. We found that thrombospondin-1 (TSP-1), a potent physiological inhibitor of angiogenesis, was significantly reduced in TEC in respect to normal endothelial cells. This reduction was confirmed by immunofluorescence in the intratumor vessels of clear cell renal carcinomas. As TEC were shown to display a basal upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we evaluated the possible regulation of TSP-1 by this pathway by using LY294002 and wortmannin, the PI3K inhibitors, and rapamycin, the mammalian target of rapamycin (mTOR) inhibitor. In addition, we developed negative dominant TEC for Akt. TSP-1 production by TEC was enhanced by the treatment with LY294002 and wortmannin and with rapamycin, suggesting a negative regulation of TSP-1 expression by the PI3K/Akt/mTOR pathway. In addition, downregulation of Akt activation in negative dominant Akt TEC enhanced TSP-1 expression and release. Administration of exogenous TSP-1 to TEC reduced their proangiogenic properties in vitro and in vivo. In parallel, blockade of TSP-1 with an anti-TSP-1 antibody in negative dominant Akt TEC restored their proangiogenic phenotype to levels similar to wild-type TEC. In conclusion, these results indicate that the upregulation of the PI3K/Akt/mTOR pathway is responsible for the inhibition of TSP-1 synthesis which is critical in determining the proangiogenic phenotype of TEC. Strategies aimed to inhibit the PI3K/Akt/mTOR pathway may restore a normal quiescent endothelial phenotype in TEC by promoting TSP-1 production.

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Isolation and characterization of human breast tumor-derived endothelial cells

Cited by 53 publications

References 20 publications

IL-3 is a novel target to interfere with tumor vasculature

IL-3 is a novel target to interfere with tumor vasculature

Multiple Roles of Protein Kinase A in Arachidonic Acid–Mediated Ca2+ Entry and Tumor-Derived Human Endothelial Cell Migration

The proangiogenic phenotype of human tumor-derived endothelial cells depends on thrombospondin-1 downregulation via phosphatidylinositol 3-kinase/Akt pathway

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