Comparative analysis of metastasis variants derived from human prostate carcinoma cells: roles in intravasation of VEGF-mediated angiogenesis and uPA-mediated invasion.

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.

Evaluation of metastatic and angiogenic potentials of human colon carcinoma cells in chick embryo model systems.

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.

Functional role of cell surface CUB domain-containing protein 1 in tumor cell dissemination.

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.

Cell surface proteomics identifies molecules functionally linked to tumor cell intravasation.

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.