Understanding tumor endothelial cell abnormalities to develop ideal anti-angiogenic therapies

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels have been shown to differ from their normal counterparts, for example, by changes in morphology. An important concept in tumor angiogenesis is that tumor endothelial cells are assumed to be genetically normal, even though these endothelial cells are structurally and functionally abnormal. To date, many anti-angiogenic drugs have been developed, but, their therapeutic efficacy is not dramatic and they have also been reported to cause toxic side effects. To develop ideal antiangiogenic therapies, understanding tumor endothelial cell abnormalities is important. We have isolated tumor endothelial cells from mouse tumor xenografts and have shown that tumor-associated endothelial cells are abnormal. Tumor-associated endothelial cells upregulate many genes, such as epidermal growth factor receptor (EGFR). Tumor-associated endothelial cells are also more sensitive to EGF. They also have relatively large, heterogeneous nuclei. Unexpectedly, tumor endothelial cells are cytogenetically abnormal. Fluorescence in situ hybridization (FISH) analysis showed that freshly isolated uncultured tumor endothelial cells were aneuploid and had abnormal multiple centrosomes. The degree of aneuploidy was exacerbated by passage in culture. In marked contrast, freshly isolated normal skin and adipose endothelial cells were diploid. They had normal centrosomes and remained cytogenetically stable in culture even up to 20 passages. We conclude that tumor endothelial cells can acquire cytogenetic abnormalities while in the tumor microenvironment. Questions as to whether or not tumor endothelial cells become resistant to antiangiogenic drugs are thus raised. Our preliminary data show that tumor endothelial cells are more resistant to certain chemotherapeutic drugs. Studies to evaluate the mechanism for cytogenetic abnormalities in tumor endothelial cells are underway. It is becoming quite clear that the tumor vasculature is much more complex and unpredictable than initially perceived. Here, we provide an overview of the current studies on tumor endothelial cell abnormalities.     

Tumor-associated endothelial cells with cytogenetic abnormalities

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels have been shown to differ from normal counterparts, for example, by changes in morphology. An important concept in tumor angiogenesis is that tumor endothelial cells are assumed to be genetically normal, although these endothelial cells are structurally and functionally abnormal. However, we hypothesized that given the phenotypic differences between tumor and normal blood vessels, there may be genotypic alterations as well. Mouse endothelial cells were isolated from two different human tumor xenografts, melanoma and liposarcoma, and from two normal endothelial cell counterparts, skin and adipose. Tumor-associated endothelial cells expressed typical endothelial cell markers, such as CD31. They had relatively large, heterogeneous nuclei. Unexpectedly, tumor endothelial cells were cytogenetically abnormal. Fluorescence in situ hybridization (FISH) analysis showed that freshly isolated uncultured tumor endothelial cells were aneuploid and had abnormal multiple centrosomes. The degree of aneuploidy was exacerbated by passage in culture. Multicolor FISH indicated that the structural chromosomal aberrations in tumor endothelial cells were heterogeneous, indicating that the cytogenetic alterations were not clonal. There was no evidence of human tumor-derived chromosomal material in the mouse tumor endothelial cells. In marked contrast, freshly isolated normal skin and adipose endothelial cells were diploid, had normal centrosomes, and remained cytogenetically stable in culture even up to 20 passages. FISH analysis of tumor sections also showed endothelial cell aneuploidy. We conclude that tumor endothelial cells can acquire cytogenetic abnormalities while in the tumor microenvironment.     

Endothelial differentiation of hematopoietic stem and progenitor cells from patients with multiple myeloma.

PURPOSE: Vasculogenesis is a physiologic process typical of fetal development in which new blood vessels develop from undifferentiated precursors (or angioblasts). In tumors, near angiogenesis, vasculogenesis contributes to the formation of the microvascular plexus that is important for diffusion. Here, we show that hematopoietic stem and progenitor cells (HSPC) of multiple myeloma (MM) patients are able to differentiate into cells with endothelial phenotype on exposure to angiogenic cytokines. EXPERIMENTAL DESIGN: Circulating HSPCs were purified with an anti-CD133 antibody from patients with newly diagnosed MM before autologous transplantation and exposed to vascular endothelial growth factor (VEGF), fibroblast growth factor-2 and insulin-like growth factor in a 3-week culture. RESULTS: HSPCs gradually lost CD133 expression and acquired VEGF receptor-2, factor VIII-related antigen, and vascular endothelial-cadherin expression. The expression pattern overlapped with paired MM endothelial cells (MMEC). During culture, cells adhered to fibronectin, spread, and acquired an endothelial cell shape. Differentiated HSPCs also became capillarogenic in the Matrigel assay with maximal activity at the third week of culture. Bone marrow biopsies revealed HSPCs inside the neovessel wall in patients with MM but not in those with monoclonal gammopathy of undetermined significance. CONCLUSIONS: In patients with MM, but not in those with monoclonal gammopathy of undetermined significance, HSPCs contribute to the neovessel wall building together with MMECs. Therefore, besides angiogenesis, HSPC-linked vasculogenesis contributes to neovascularization in MM patients. Tentatively, we hypothesize that in HSPC cultures a multipotent cell population expressing low VEGF receptor-2 levels corresponds to the endothelial progenitor cell precursor and seems to be the MMEC precursor.     

人内皮细胞对正常人和再生障碍性贫血患者粒系造血的研究

以人脐静脉内皮细胞作铺层，进行体外ＣＦＵ－ＧＭ半固体培养，观察内皮细胞对正常人和再生障碍性贫血患者粒系造血的影响。在有内皮细胞的各培养体系，ＣＦＵ－ＧＭ的产率均显著高于无内皮细胞的对照体系。在无内皮细胞存在的体系中，需添加ＧＭ－ＣＳＦ才能表现出对粒系造血的刺激作用。对１５例初诊慢性再障患者骨髓粒系祖细胞的培养结果表明，无内皮细胞存在时，无论向培养体系中加何种细胞因子均无集落形成，而在有内皮细胞的双层培养中，部分患者骨髓培养中有ＣＦＵ－ＧＭ集落形成。研究结果表明内皮细胞对正常人和再障患者粒系造血均有明显影响。     

Heptocyte Growth Factor-Pleiotropic Cytokine Produced by Human Leukemia cells

Hepatocyte growth factor (HGF) was identified. purified and molecularly cloned as a potent mitogen for mature rat hepatocytes in primary culture. It is one of the largest cytokines and is composed of disulfide-linked subunits of approximately 60 (heavy chain) and 35 kilodaltons (light chain). Recent observations revealed that HGF is mitogenic to various epithelial cells other than hepatocytes and to endothelial cells, and that it also acts as a motogen, morphogen and tumor-suppressor as well as a mitogen. These various biological activities of HGF are presumably transduced through the same reccptor, c-Met, which is a member of the tyrosine kinase receptor family. Although it shows multiple biological activities on cells in culture. HGF is most likely the physiological hepatotrophic factor which triggers liver regeneration. It may also function as a renotrophic and pulmotrophic factor after tissue injury. HGF production in the liver. kidney and lung increases after injury to these organs. An elevated HGF level may act as an inducer of compensatory DNA synthesis. The regulation of HGF production is. therefore, important for the control of organ regeneration. HGF is produced mainly by mesenchymal cells such as fibroblasts and vascular smooth musclc cells. Various types of human leukemia cells also secrete HGF both in virru and in riw. Some biological activities of HGF on hematopoietic cells, including co-mitogenic activity on myeloid leukemia cell lines. were recently demonstrated. HGF gene expression and the protein production in leukemia and fibroblast cells are modulated by various cytokines and hormones. Those modulators may indirectly affect organ regeneration and other biological processes by controlling HGF production.     

Predicting treatment responses and disease progression in myeloma using serum vascular endothelial growth factor and hepatocyte growth factor levels

Angiogenesis is a crucial process in the progression of multiple myeloma (MM). Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are multifunctional cytokines that potently stimulate angiogenesis during tumor neovascularization. VEGF is secreted by MM cells. It induces proliferation of MM cells and stimulates IL-6 expression by microvascular endothelial cells and bone marrow stroma cells, suggesting both autocrine and paracrine functions for VEGF in MM. HGF and the HGF receptor, c-Met, are expressed simultaneously in MM cell lines and in freshly isolated MM cells, suggesting a possible role for HGF in MM cell proliferation. This review focuses on the clinical significance of serum levels of VEGF and HGF in MM.     

Cerebellar capillary hemangioblastoma: its histogenesis studied by organ culture and electron microscopy.

A capillary hemangioblastoma from the vermis of a 21-year-old man with von Hippel-Lindau's disease was successfully maintained in organ culture systems on Millipore filter platforms and gelatin sponge foam matrices up to 48 days. Three cell types--endothelial cells, pericytes, and stromal cells--identified by their fine structural features and their architectural relationships to vascular lumens and to the extracellular space were recognized in the original tumor. By light microscopy the explants showed, in the late stages, increased lipid droplets in the stromal cells and perivascular hyaline thickening. By electron microscopy, endothelial cells, pericytes, and stromal cells remained distinguishable as cell types. However, the stromal cells demonstrated, in addition to an increase of their normal complement of microfilaments and lipid droplets, features that were more characteristic of the two other cell types, i.e. micropinocytotic vesicles and the formation of zonula occludens junctions and hemidesmosomes. Basement membranes also became more apparent around the stromal cells. With increasing time in vitro, there was a striking increase in mature collagen fibers in the extracellular space. The roles of the different cell types in capillary hemangioblastoma and the histogenesis of the stromal cells are discussed in light of these observations. It is concluded that the capillary hemangioblastoma consists of multiple cell lines--endothelial cells, pericytes, stromal cells and, occasionally, hematopoietic cells--all of which are neoplastic and replicate in parallel with one another. Stromal cells may be regarded as an aberrant monopotential cell type which shares with the endothelial cell and pericyte a common mesenchymal, presumably angiogenic, ancestry, and may, on occasion, display morphological features, such as increased basement membrane formation and the formation of zonula occludens junctions, which recall its angiogenic lineage. However, interconvertibility between endothelial cells and stromal cells does not appear to occur in vivo or in vitro.     

Role of nitric oxide in lysis of tumor cells by cytokine-activated endothelial cells

The purpose of these studies was to determine whether nitric oxide produced by cytokine-activated murine lung vascular endothelial cells plays a role in their lytic destruction of M-5076 reticulum cell sarcoma. Vascular endothelial cells harvested from perfused lungs of mice were adapted to grow in culture. Cloned lines ascertained to be of endothelial origin were incubated in vitro with interferon gamma and tumor necrosis factor. Lysis of radiolabeled tumor cells and accumulation of nitrite in the culture medium were determined at several time points. The concentration of nitrite in the culture medium directly correlated with endothelial cell-mediated tumor cell lysis. Endothelial cells cultured in L-arginine-free medium did not produce significant tumor cell lysis nor accumulation of nitrite in the medium. Both tumor cell lysis and nitrite accumulation were observed when the deficient medium was reconstituted with L-arginine, suggesting that endothelial cell-mediated tumor lysis was dependent on L-arginine, a precursor of nitric oxide. Moreover, specific inhibition of nitric oxide synthesis by NG-methyl-L-arginine resulted in complete inhibition of endothelial cell-mediated lysis of the M-5076 reticulum cell sarcoma. Similarly, treatment of cytokine-activated endothelial cells with dexamethasone inhibited both target cell lysis and production of nitrite. Collectively, these results suggest that nitric oxide plays a major role in the lysis of tumor cells mediated by cytokine-activated endothelial cells.     

Calpain is involved in cisplatin-induced endothelial injury in an in vitro three-dimensional blood vessel model

To study endothelial injury in vitro, we established a three-dimensional (3-D) blood vessel model in which human umbilical vein endothelial cells were grown in the presence of basic fibroblast growth factor and vascular endothelial growth factor. We then performed comparative studies on cisplatin (cis-platinum-diammine-dichloride, CDDP)-induced endothelial injury in 3-D and monolayer cultures. In 3-D culture, CDDP induced cell death and tube breakdown without DNA damage, whereas CDDP induced apoptosis accompanied by DNA damage in monolayer culture. CDDP also induced caspase-3 activation in a concentration-dependent manner in both cultures. A broad-spectrum caspase inhibitor, zVAD-fmk, failed to prevent CDDP-induced cell death and tube breakdown in 3-D culture, whereas zVAD-fmk suppressed CDDP-induced apoptosis in monolayer culture. A calpain inhibitor, MDL28170, attenuated CDDP-induced cell death and tube breakdown in 3-D culture, but not apoptosis in monolayer culture. These results showed that calpain is involved in CDDP-induced endothelial injury in 3-D culture and there are significant differences in signaling pathways between 3-D and monolayer cultures.     

Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy.

Vascular endothelial growth factor A (VEGF-A), the founding member of the vascular permeability factor (VPF)/VEGF family of proteins, is an important angiogenic cytokine with critical roles in tumor angiogenesis. This article reviews the literature with regard to VEGF-A's multiple functions, the mechanisms by which it induces angiogenesis, and its current and projected roles in clinical oncology. VEGF-A is a multifunctional cytokine that is widely expressed by tumor cells and that acts through receptors (VEGFR-1, VEGFR-2, and neuropilin) that are expressed on vascular endothelium and on some other cells. It increases microvascular permeability, induces endothelial cell migration and division, reprograms gene expression, promotes endothelial cell survival, prevents senescence, and induces angiogenesis. Recently, VEGF-A has also been shown to induce lymphangiogenesis. Measurements of circulating levels of VEGF-A may have value in estimating prognosis, and VEGF-A and its receptors are potential targets for therapy. Recognized as the single most important angiogenic cytokine, VEGF-A has a central role in tumor biology and will likely have an important role in future approaches designed to evaluate patient prognosis. It may also become an important target for cancer therapy.     

In 2D and 3D Cell Culture Models,Effects of Endothelial Cells on E-cadherin / β-catenin Expression Levels and Spheroid Sizes in Ishikawa Cells

Objective: Increasing evidence shows that three dimensional cell culture models better reflect the in vivo tumor microenvironment than two dimensional cell culture models. Co-culture models are ideal cell culture models for understanding the communication between cells and the in vivo microenvironment. Changes in expression levels of E-cadherin are closely related to cancer metastasis and progression. β-catenin mediates cell adhesion of E-cadherin. Endothelial cells are stromal cell components in the tumor microenvironment. It is known that there is little or no expression of E-cadherin in endothelial cells. Methods: In our study, both two-dimensional and three dimensional mono-culture and co-culture models were created using Huvec and Ishikawa cells (endometrial cancer cell lines) to better reflect cell interactions. Spheroids were followed for three days in the three-dimensional cell culture model. E-cadherin and β-catenin expression levels of two-dimensional and three dimensional mono-culture and co-culture models were measured by immunofluorescence staining. Spheroid images were recorded using a Z-stack. Intensity measurements in both two-dimensional and three-dimensional mono-culture and co-culture models were made using the Image J software. Study groups were evaluated by one-way analysis of variance (One-Way ANOVA). Values of p <0.05 were considered statistically significant. Results: The size of the co-culture spheroids was recorded significantly larger than the mono-culture spheroids (p <0.0001). In two (p = 0.0175) and three dimensional models (p <0.0001), expression levels of E-cadherin in the mono-culture of Ishikawa cells were recorded significantly higher than in Huvec and co-culture cells. Likewise, while the expression levels of β-catenin were higher in the mono-culture of Ishikawa cells in two-dimensional models (p <0.05), no significant difference was observed in three dimensional models (p> 0.05). Conclusion: In summary, it has been noted that the expression levels of E-cadherin are significantly reduced in co-cultures of Ishikawa cells with Huvec cells in both two and three dimensions . These results support the idea that endothelial cells may cause changes in endometrial cancer progression by suppressing E-cadherin expression in Ishikawa cells.     

Luteolin抑制血管生成的机制研究

目的:探讨luteolin对血管的抑制机制。方法:采用不同浓度luteolin处理人微血管内皮细胞,观察luteolin对内皮细胞生长,乳腺癌细胞MDA-MB 231培养液介导的内皮细胞趋化抑制作用。并探讨luteolin对内皮细胞中IL-8信号激活的抑制作用,及luteolin对血管生成抑制作用机制。结果:Luteolin对人微血管内皮细胞细胞增殖抑制作用明显(P<0.01)。Luteolin可抑制乳腺癌细胞MDA-MB 231培养液介导的内皮细胞趋化作用(P<0.01),并明显抑制IL-8对内皮细胞ERK及AKT的激活。结论:Luteolin可抑制人微血管内皮细胞增殖及乳腺癌细胞MDA-MB 231培养液介导的趋化作用,并可抑制IL-8对人微血管内皮细胞的信号激活作用,luteolin抗血管生成作用在预防恶性肿瘤复发及转移中可能有重要的作用。     

Induction of vasculogenesis in breast cancer models

Recently, there have been reports of postnatal vasculogenesis in cases of ischaemia models. The aim of the present study is to provide evidence of postnatal vasculogenesis in breast-cancer-bearing mice. Based on cell surface antigen expression, we isolated endothelial precursor cells from bone marrow, peripheral blood and tumour-infiltrating cells from mice that had received six human breast cancer xenografts. In all three areas (bone marrow, peripheral blood and tumour-infiltrating cells), endothelial precursor cell population was elevated in all transplanted mice. Differentiation and migration activities of endothelial precursor cells were measured by comparing levels of the endothelial precursor cell maturation markers Flk-1, Flt-1, Tie2, VE-cadherin and CD31 among these three areas. The endothelial precursor cell population was 14% or greater in the gated lymphocyte-size fraction of the inflammatory breast cancer xenograft named WIBC-9, which exhibits a hypervascular structure and de novo formation of vascular channels, namely vasculogenic mimicry (Shirakawa et al, 2001). In vitro, bone marrow-derived endothelial precursor cells from four human breast cancer xenografts proliferated and formed multiple clusters of spindle-shaped attaching cells on a vitronectin-coated dish. The attaching cells, which incorporated DiI-labelled acetylated low-density lipoprotein (DiI-acLDL) and were negative for Mac-1. The putative bone marrow derived endothelial precursor cell subset, which was double positive of CD34 and Flk-1, and comparative bone marrow derived CD34 positive with Flk-1 negative subset were cultured. The former subset incorporated DiI-acLDL and were integrated with HUVECs. Furthermore, they demonstrated significantly higher levels of murine vascular endothelial growth factor and interleukin-8 in culture supernatant on time course by enzyme-linked immunosorbent assay. These findings constitute direct evidence that breast cancer induces postnatal vasculogenesis in vivo.     

Bone marrow angiogenesis in multiple myeloma.

Angiogenesis is a constant hallmark of multiple myeloma (MM) progression and has prognostic potential. It is induced by plasma cells via angiogenic factors with the transition from monoclonal gammopathy of undetermined significance (MGUS) to MM, and probably with loss of angiostatic activity on the part of MGUS. The pathophysiology of MM-induced angiogenesis is complex and involves both direct production of angiogenic cytokines by plasma cells and their induction within the microenvironment. The latter are secreted by stromal cells, endothelial cells (EC) and osteoclasts, and promote plasma cell growth, survival and migration, as well as paracrine cytokine secretion and angiogenesis in the bone marrow milieu. Angiogenesis is also supported by inflammatory cells following their recruitment and activation by plasma cells. Finally, circulating EC and endothelial precursor cells (EPC) contribute to the neovascularization, and the presence of EPC suggests that vasculogenesis (new vessel formation from EPC) may also contribute to the full MM vascular tree.     

Cultured tumor cells produce chemotactic factors specific for endothelial cells: a possible mechanism for tumor-induced angiogenesis

The production of stimulants of endothelial cell motility by cultured tumor cells was studied. Spontaneously transformed murine fibroblasts in culture produced activity that stimulated migration of endothelial cells, while this kind of activity was not detected in media from cultures of the normal counterparts of the transformed cells. Furthermore, a line of murine tumor cells (HB4), known to induce vasoformative sarcomas in vivo, was found to produce in culture a strong chemoattractant specific for endothelial cells. Since the tumor-derived material also caused vessel ingrowth when implanted in the rabbit eye, these results suggest that the angiogenesis observed during tumor growth may involve chemoattractants for endothelial cells produced by tumor cells.     

Preferential adhesion of prostate cancer cells to bone is mediated by binding to bone marrow endothelial cells as compared to extracellular matrix components in vitro.

We have demonstrated previously that the preferential adhesion of prostate cancer cells to human bone marrow endothelial (HBME) cells may contribute to their preferential metastasis to bone. Although a subject of debate, it has been postulated that the endothelial cells of the bone marrow are fenestrated. It is unknown therefore whether prostate cancer cells adhere preferentially to the extracellular matrix (ECM) or the endothelial cells. It has also been demonstrated in other organ systems that the types of cell adhesion molecules on the surface of endothelial cells lining the organ microvasculature are determined, in part, by the ECM of the organ. We investigated how prostate cancer cell adhesion to HBME cells is affected by growing HBME cells on selected organ-derived ECM proteins in vitro. Growth of HBME cells and immortalized human aortic endothelial cells on bone, kidney, and placenta ECM proteins significantly increased their ability to bind PC-3 cells. This increased adhesion was not dose dependent and was not demonstrated with human dermal microvascular endothelial cells. Scanning electron microscopic analysis demonstrated that prostate cancer cells adhered directly to the endothelial cells and not to the underlying substrata. These results suggest that unidentified cell adhesion molecules are expressed or up-regulated on the apical surfaces of human aortic endothelial cells and HBME cells grown on bone, kidney, and placenta ECMs. These results also strongly demonstrate that the adhesion of prostate cancer cells to bone may be initiated by direct binding to endothelial cells rather than direct binding to exposed ECM components.     

Interaction with endothelial cells is a prerequisite for branching ductal-alveolar morphogenesis and hyperplasia of preneoplastic human breast epithelial cells: regulation by estrogen

Although there is experimental evidence supporting the involvement of angiogenesis in the pathogenesis of breast cancer, the exact nature and effects of interaction between human breast epithelial cells (HBECs) and endothelial cells (ECs) have not been described thus far. This approach requires an assay system that permits growth and differentiation of both epithelial and endothelial cells. Here, we report the development of a three-dimensional in vitro culture system that supports growth and functional differentiation of preneoplastic HBECs and ECs and recapitulates estrogen-induced in vivo effects on angiogenesis and the proliferative potential of MCF10AT xenografts. MCF10A and MCF10AT1-EIII8 (referred to as EIII8) cell lines used in this study are normal or produce preneoplastic lesions, respectively. When MCF10A or EIII8 cells are seeded on reconstituted basement membrane (Matrigel), both lines organize into a three-dimensional tubular network of cells; however, tubes produced by EIII8 cells appear multicellular in contrast to unicellular structures formed by MCF10A cells. However, when MCF10A or EIII8 cells are cocultured with human umbilical vein endothelial cells (HUVECs) on Matrigel, rather than interacting with extracellular matrix, the ECs exhibit preferential adherence to epithelial cells. Although both MCF10A and EIII8 cells provide preferential substrate for EC attachment, only EIII8 cells facilitate sustained proliferation of ECs for prolonged periods that are visualized as "endothelial cell enriched spots," which express factor VIII-related antigen. At regions of endothelial-enriched spots, preneoplastic HBECs undergo branching ductal-alveolar morphogenesis that produce mucin, express cytokeratins, and proliferating cell nuclear antigen. The presence of actively proliferating and functional endothelial cells is essential for ductal-alveolar morphogenesis of preneoplastic HBECs because without ECs, the epithelial cells formed only tubular structures. This ability to establish functional ECs and ductal-alveolar morphogenesis is facilitated only by preneoplastic HBECs because normal MCF10A cells fail to elicit similar effects. Thus, a cause-effect relationship that is mutually beneficial exists between EC and preneoplastic HBECs that is critical for generation of functional vascular networks and local proliferative ductal alveolar outgrowths with invasive potential. Both these processes are augmented by estrogen, whereas antiestrogens inhibit these processes. Induction and maintenance of angiogenic phenotype is associated with up-regulation in expression of interleukin 8 and matrix metalloproteinase-2 and estrogen-induced increases in vascular endothelial growth factor and vascular endothelial growth factor receptor 2. This three-dimensional culture model offers a unique opportunity to study endothelial- and epithelial cell-specific factors that are important for ductal-alveolar morphogenesis, angiogenesis, and progression to malignant phenotype.     

Caveolin-1 is required for vascular endothelial growth factor-triggered multiple myeloma cell migration and is targeted by bortezomib

We recently demonstrated that caveolae, vesicular flask-shaped invaginations of the plasma membrane, represent novel therapeutic targets in multiple myeloma. In the present study, we demonstrate that vascular endothelial growth factor (VEGF) triggers Src-dependent phosphorylation of caveolin-1, which is required for p130(Cas) phosphorylation and multiple myeloma cell migration. Conversely, depletion of caveolin-1 by antisense methodology abrogates p130(Cas) phosphorylation and VEGF-triggered multiple myeloma cell migration. The proteasome inhibitor bortezomib both inhibited VEGF-triggered caveolin-1 phosphorylation and markedly decreased caveolin-1 expression. Consequently, bortezomib inhibited VEGF-induced multiple myeloma cell migration. Bortezomib also decreased VEGF secretion in the bone marrow microenvironment and inhibited VEGF-triggered tyrosine phosphorylation of caveolin-1, migration, and survival in human umbilical vascular endothelial cells. Taken together, these studies demonstrate the requirement of caveolae for VEGF-triggered multiple myeloma cell migration and identify caveolin-1 in multiple myeloma cells and human umbilical vascular endothelial cells as a molecular target of bortezomib.     

Endothelial-like malignant glioma cells in dynamic three dimensional culture identifies a role for VEGF and FGFR in a tumor-derived angiogenic response

Aims: Recent studies have observed that cells from high-grade glial tumors are capable of assuming an endothelial phenotype and genotype, a process termed ‘vasculogenic mimicry’ (VM). Here we model and manipulate VM in dynamic 3-dimensional (3D) glioma cultures. Methods: The Rotary Cell Culture System (RCCS) was used to derive large macroscopic glioma aggregates, which were sectioned for immunohistochemistry and RNA extracted prior to angiogenic array-PCR. Results: A 3D cell culture induced microenvironment (containing only glial cells) is sufficient to promote expression of the endothelial markers CD105, CD31 and vWF in a proportion of glioma aggregates in vitro. Many pro-angiogenic genes were upregulated in glioma aggregates and in primary explants and glioma cells were capable of forming tubular-like 3D structures under endothelial-promoting conditions. Competitive inhibition of either vascular endothelial growth factor or fibroblast growth factor receptor was sufficient to impair VM and downregulate the tumor-derived angiogenic response, whilst impairing tumor cell derived tubule formation. Glioma xenografts using the same cells reveal tumor-derived vessel-like structures near necrotic areas, consistent with widespread tumor-derived endothelial expression in primary glioma tissue. Conclusions: Our findings support studies indicating that tumor-derived endothelial cells arise in gliomas and describe a dynamic 3D culture as a bona fide model to interrogate the molecular basis of this phenomenon in vitro. Resistance to current anti-angiogenic therapies and the contribution of tumor derived endothelial cells to such resistance are amenable to study using the RCCS.