Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2-to 3-day pulse stimulates angiogenesis by inducing an endothelial "tip cell" phenotype. TNF induces the known tip cell genes plateletderived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reversetranscription-polymerase chain reaction IntroductionNeovascularization, or the formation of new blood vessels, is a critical component of many physiologic as well as pathologic conditions, including development, reproduction, wound healing, diabetic retinopathy, and tumor growth. During wound healing, new vessel growth by angiogenesis is a necessary early step in rebuilding tissue, however the coordination of angiogenesis with the resolution of the acute inflammatory stage is not well understood. The earliest events after tissue damage include the generation of a fibrin clot and the bursting of platelets to release numerous growth factors. Fibrin provides a provisional matrix that promotes the accumulation of blood-derived monocytes that then differentiate into tissue macrophages. Activated macrophages synthesize several cytokines, including tumor necrosis factor (TNF), which activate local endothelial cells (ECs) and promote leukocyte recruitment. After 3 to 4 days, when the initial infection has been cleared, there is a switch toward tissue repair and concomitant with this is the acceleration of angiogenesis. 1,2 TNF is a major inflammatory mediator that induces multiple changes in EC gene expression including induction of adhesion molecules, integrins, and matrix metalloproteinases (MMPs). Its effects on angiogenesis have been the subject of some controversy. For example, TNF blocks EC proliferation and migration in vitro [3][4][5] and has been reported to down-regulate activity 6 and expression 7,8 of vascular endothelial cell growth factor receptor-2 (VEGFR2). On the other hand, TNF has also been shown to up-regulate VEGFR2 expression 9 and promote EC migration. 10 In vivo the situation is no clearer: TNF promotes angiogenesis in the cornea, 3,11 whereas loss of TNFR1 (p55 receptor) leads to enhanced angiogenesis in both retina 12 and wounded skin. 13 Further studies with TNF receptor-deficient mice have demonstrated enhanced hind limb angiogenesis after temporary ischemia in T...

The requirement for notch signaling during vascular development is well-documented but poorly understood. Embryonic and adult endothelial cells (EC) express notch and notch ligands; however, the necessity for cell-autonomous notch signaling during angiogenesis has not been determined. During angiogenesis, EC display plasticity, whereby a subset of previously quiescent cells loses polarity and becomes migratory. To investigate the role of notch in EC, we have used a three-dimensional in vitro system that models all of the early steps of angiogenesis. We find that newly forming sprouts are composed of specialized tip cells that guide the sprout and trunk cells that proliferate and rearrange to form intercellular lumens. Furthermore, we find that notch acts cell-autonomously to suppress EC proliferation, thereby regulating tube diameter. In addition, when notch signaling is blocked, tip cells divide, and both daughter cells take on a tip cell phenotype, resulting in increased branching through vessel bifurcation. In contrast, notch signaling is not required for re-establishment of EC polarity or for lumen formation. Thus, notch is used reiteratively and cell-autonomously by EC to regulate vessel diameter, to limit branching at the tip of sprouts, and to establish a mature, quiescent phenotype.

The function of CUB domain-containing protein 1 (CDCP1), a recently described transmembrane protein expressed on the surface of hematopoietic stem cells and normal and malignant cells of different tissue origin, is not well defined. The contribution of CDCP1 to tumor metastasis was analyzed by using HeLa carcinoma cells overexpressing CDCP1 (HeLa-CDCP1) and a high-disseminating variant of prostate carcinoma PC-3 naturally expressing high levels of CDCP1 (PC3-hi/diss). CDCP1 expression rendered HeLa cells more aggressive in experimental metastasis in immunodeficient mice. Metastatic colonization by HeLa-CDCP1 was effectively inhibited with subtractive immunization-generated, CDCP1-specific monoclonal antibody (mAb) 41-2, suggesting that CDCP1 facilitates relatively late stages of the metastatic cascade. In the chick embryo model, time-and dose-dependent inhibition of HeLa-CDCP1 colonization by mAb 41-2 was analyzed quantitatively to determine when and where CDCP1 functions during metastasis. Quantitative PCR and immunohistochemical analyses indicated that CDCP1 facilitated tumor cell survival soon after vascular arrest. Live cell imaging showed that the function-blocking mechanism of mAb 41-2 involved enhancement of tumor cell apoptosis, confirmed by attenuation of mAb 41-2-mediated effects with the caspase inhibitor z-VAD-fmk. Under proapoptotic conditions in vitro, CDCP1 expression conferred HeLa-CDCP1 cells with resistance to doxorubicin-induced apoptosis, whereas ligation of CDCP1 with mAb 41-2 caused additional enhancement of the apoptotic response. The functional role of naturally expressed CDCP1 was shown by mAb 41-2-mediated inhibition of both experimental and spontaneous metastasis of PC3-hi/diss. These findings confirm that CDCP1 functions as an antiapoptotic molecule and indicate that during metastasis CDCP1 facilitates tumor cell survival likely during or soon after extravasation.

To analyze the process of tumor cell intravasation, we used the human tumor-chick embryo spontaneous metastasis model to select in vivo high (PC-hi/diss) and low (PC-lo/diss) disseminating variants from the human PC-3 prostate carcinoma cell line. These variants dramatically differed in their intravasation and dissemination capacities in both chick embryo and mouse spontaneous metastasis models. Concomitant with enhanced intravasation, PC-hi/diss exhibited increased angiogenic potential in avian and murine models. PC-hi/diss angiogenesis and intravasation were dependent on increased secretion of vascular endothelial growth factor (VEGF), since treating developing tumors with a function-blocking anti-VEGF antibody simultaneously inhibited both processes without affecting primary tumor growth. PC-hi/diss cells were also more migratory and invasive, suggestive of heightened ability to escape from primary tumors due to matrix-degrading activity. Consistent with this suggestion, PC-hi/diss cells produced more of the serine protease urokinase-type plasminogen activator (uPA) as compared with PC-lo/diss. The functional role of uPA in PC-hi/diss dissemination was confirmed by inhibition of invasion, angiogenesis, and intravasation with specific function-blocking antibodies that prevented uPA activation and blocked uPA activity. These processes were similarly sensitive to aprotinin, a potent inhibitor of serine proteases, including uPA-generated plasmin. Thus, our comparison of the PC-3 intravasation variants points to key roles for the uPA-plasmin system in PC-hi/diss intravasation, possibly via (1) promoting tumor cell matrix invasion and (2) facilitating development of VEGF-dependent angiogenic blood vessels. (Am J Pathol

In order to better understand the molecular and cellular determinants of tumor cell intravasation, our laboratory has generated a pair of congenic human HT-1080 fibrosarcoma variants (i.e. HT-hi/diss and HT-lo/diss) differing 50 -100-fold in their ability to intravasate and disseminate. To investigate the molecular differences underlying the distinct dissemination capacities of these HT-1080 variants, we performed a comparative analysis of the cell surface proteomes of HT-hi/diss and HT-lo/diss. Cell membrane proteins were enriched by biotinylation and avidin precipitation and analyzed by tandem mass spectrometry employing multidimensional protein identification technology. By this approach, 47 cell surface-associated molecules were identified as differentially expressed between the HT-1080 intravasation variants. From these candidates, four targets (i.e. TIMP-2, NCAM-1, JAM-C, and tissue factor (TF)) were selected for further biochemical validation and in vivo functional verification. Western blot analysis of the cell surface enriched fractions confirmed the proteomic array data, demonstrating that, in vitro, TIMP-2 protein was increased in the HT-lo/diss variant, whereas NCAM-1, JAM-C, and TF levels were increased in the HT-hi/diss variant. Corresponding in vivo differences in levels of TIMP-2, JAM-C, and TF were demonstrated in primary tumors grown in the chick embryo. Finally, functional inhibition of one selected protein (i.e. TF) by small interfering RNA silencing or ligation with a function-blocking antibody significantly reduced HT-hi/diss intravasation, thus clearly implicating TF in the early steps of tumor cell dissemination. Overall, our cell surface proteomic analysis provides a powerful tool for identification of specific cell membrane molecules that contribute functionally to intravasation and metastasis in vivo.One of the early and possibly rate-limiting steps in cancer progression from a localized tumor to systemic metastatic disease is intravasation (i.e. the entry of metastatic cells into the vasculature) (1-5). Molecules involved in intravasation represent attractive therapeutic targets, since preventing or inhibiting this process would confine tumor cells to their primary site and provide a more focused target for clinical intervention (6). To identify cellular attributes that functionally contribute to tumor cell intravasation and metastasis, including escape from the primary site, invasion of local stoma, and entry into the vasculature, we have employed a pair of congenic human fibrosarcoma HT-1080 cell variants, differing 50 -100-fold in their ability to intravasate and disseminate (HT-hi/diss and HT-lo/diss) while having similar capacities to form primary tumors (7). These cell variants display a distinct differential during spontaneous metastasis but behave comparably in experimental metastasis models where cells are inoculated intravenously and only the later steps of the metastatic cascade are recapitulated. Therefore, comparative analysis of the HT-hi/diss and HT-lo/diss variants can...

Increased metastatic and angiogenic potentials of aggressive human colon carcinoma cells were verified in independent chick embryo models by comparing in vivo highly metastatic SW620 colon carcinoma cell line with its isogenic, non-metastatic SW480 cell variant. In the experimental metastasis model, both cell types rapidly arrested in the chorioallantoic membrane (CAM) vasculature as demonstrated by quantitative PCR and immunohistochemistry. Live cell imaging also indicated that both SW620 and SW480 cells efficiently extravasated from the CAM capillary system. However, only few SW480 cells were present in the CAM tissue after 24–48 h. In contrast, the numbers of SW620 cells increased exponentially, indicating proliferative and survival advantages of metastatic colon carcinoma cells in vivo. Multicellular SW620 foci were identified in close proximity to CAM blood vessels. A positive correlation between increased metastatic ability and VEGF-expression of colon carcinoma SW620 cells was demonstrated by the substantial inhibitory effects of anti-VEGF treatment on the levels of metastatic colonization and density of blood vessels adjacent to tumor cell foci. Furthermore, the chick embryo angiogenesis model confirmed high levels of VEGF-dependent angiogenesis induced by SW620 cells, but not SW480 cells. Thus, chick embryo experimental metastasis and CAM angiogenesis models appear to coordinately reflect critical features of advanced colon carcinomas, i.e., the acquisition of enhanced survival and increased angiogenic potentials, both constituting critical determinants of colon cancer progression. The use of rapid and quantitative chick embryo models might provide alternative approaches to conventional mammalian model systems for screening anti-cancer agents.

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