Affiliations of authors: Childrens Memorial Research Center, Northwestern University Feinberg School of Medicine, Robert H. Lurie Comprehensive Cancer Center, Chicago, IL (REBS, EAS, ARH, LMG, DAK, MJCH); Angiogenesis Laboratory, Department of Pathology/Internal Medicine, Research Institute for Growth and Development (GROW), Maastricht University and Hospital, Maastricht, The Netherlands (DWJVDS, AWG); Department of Pharmacology, University of Minnesota, Minneapolis (YY)
Correspondence to: Mary J. C. Hendrix, PhD, Childrens Memorial Research Center, Northwestern University Feinberg School of Medicine, 2300 Childrens Plaza, Box 222, Chicago, IL 60614-3394 (e-mail: mjchendrix{at}childrensmemorial.org)
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ABSTRACT |
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Additional studies have demonstrated that melanoma cells have a vascular phenotype that is characterized by their ability to form tubular networks when grown in three-dimensional (3-D) culture, concomitant with their expression of genes typically expressed by endothelial cells, phenomena referred to as vasculogenic mimicry (15,16). These observations have prompted us to investigate the potential relevance of a plastic tumor cell phenotype because they challenge our current thinking about how to identify and target tumor cells that might masquerade as endothelial cells or other cell types. Thus, we examined the effects of specific angiogenesis inhibitors on melanoma cells to see if they inhibit vasculogenic mimicry in a manner similar to the way they inhibit endothelial celldriven angiogenesis.
To test this premise, we treated human endothelial and melanoma cells (seeded at an initial density of 100 000 cells/well on 12-well plates containing 3-D Matrigel or collagen I) with one of three angiogenesis inhibitors of differing specificitiesanginex (17), TNP-470 (18), or endostatin (19)or the aqueous-based vehicle onlyfor various times and examined the effects of the treatments on the ability of the cells to form vascular cords and tubular networks. We used human metastatic melanoma MUM-2B (uveal) and C8161 (cutaneous) cells, which form tubular structures with lumen(s) and networks when cultured on 3-D collagen matrices (15,16), and human microvascular endothelial cells [HMEC-1; (20)] and human umbilical vein endothelial cells (HUVECs), which form cords and vascular networks when cultured on Matrigel or 3-D collagen matrices (15,16). MUM-2B cells were cloned from a heterogeneous MUM-2 uveal melanoma cell line derived from a liver metastasis (21); C8161 cells were isolated from an abdominal wall metastasis (22). Data were photographically recorded daily; the endothelial cell cultures were morphologically assessed using bright-field microscopy; the melanoma cultures were stained with periodic acid Schiff to visualize tubular networks. Histologic cross-sections of these networks were stained with hematoxylineosin to assess luminal integrity. The average total length and mean total number of junctions for treated and control endothelial cords were further analyzed using two-sided MannWhitney U tests (17,18).
As shown in Fig. 1 (panels A, D, G, J, and M), HMEC-1 cells treated with each of the three angiogenesis inhibitors had statistically significantly shorter cords and fewer junctions (a measure of the extent of vascular network formation) (23) than HMEC-1 cells treated with the corresponding vehicle. For example, compared with vehicle control, 25 µM anginex and 10 µM anginex decreased mean cord length by 71% (95% confidence interval [CI] = 44% to 98% decrease; P<.001) and 63% (95% CI = 38% to 88% decrease; P = .001), respectively; 25 µM anginex and 10 µM anginex decreased the mean number of junctions by 73% (95% CI = 52% to 94% decrease; P<.001) and 73% (95% CI = 50% to 93% decrease; P<.001), respectively. Compared with vehicle control, 100 ng of TNP-470/mL, 30 ng of TNP-470/mL, and 10 ng of TNP-470/mL decreased the average cord length by 57% (95% CI = 42% to 72% decrease; P = .007), 31% (95% CI = 18% to 45% decrease; P = .008), and 21% (95% CI = 12% to 30% decrease; P = .038), respectively. Compared with vehicle control, 100 ng of TNP-470/mL, 30 ng of TNP-470/mL, and 10 ng of TNP-470/mL decreased the mean number of junctions by 46% (95% CI = 29% to 63% decrease; P<.001), 53% (95% CI = 33% to 74% decrease; P = .011), and 33% (95% CI = 22% to 45% decrease; P = .035), respectively. Compared with vehicle control, 10 µg of endostatin/mL decreased the average cord length by 94% (95% CI = 86% to 100% decrease; P = .005) and the mean number of junctions by 75% (95% CI = 49% to 100% decrease; P<.001). We obtained identical results with HUVECs under similar experimental conditions (data not shown).
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The process of angiogenesis requires cell growth and migration as well as tube assembly. Therefore, we examined the effect of the three angiogenesis inhibitors on endothelial and melanoma cell growth over the course of 6 days, starting with 5000 cells seeded per well. As shown in Fig. 2, A, anginex and TNP-470 resulted in statistically significant inhibition of endothelial cell proliferation (25 µM anginex versus control: 83% decrease in proliferation [95% CI = 76% to 89%; P = .002]; 100 ng of TNP-470/mL versus control: 80% decrease in proliferation [95% CI = 72% to 89%; P = .003]); by contrast, endostatin had no statistically significant effect on the proliferation of endothelial cells or melanoma cells. However, 25 µM anginex statistically significantly inhibited the proliferation of C8161 cells compared with control (64% decrease, 95% CI = 52% to 77% decrease; P = .045), and 100 ng of TNP-470/mL statistically significantly inhibited the proliferation of MUM-2B cells compared with control (60% decrease compared with control, 95% CI = 47% to 73% decrease; P = .047). The observation that TNP-470 increased the proliferation of C8161 cells (166% increase compared to control, 95% CI = 156% to 176% increase; P = .016) is contrary to what occurred with endothelial cells in this study and merits further examination.
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Finally, to address a possible mechanistic basis for the different effects of the inhibitors on endothelial and melanoma cells, we used semiquantitative reverse transcriptionpolymerase chain reaction, Western blot, and fluorescence-activated cell sorter analyses to examine whether endothelial cells and melanoma cells expressed different levels of the recently reported receptors for endostatin, 5
1 integrin (25) and heparin sulfate proteoglycan 2 (HSPG2; also known as perlecan, 26,27). We found that endothelial HUVEC cells displayed robust expression of HSPG2 and the integrin
5-subunit at the mRNA and protein levels (Fig. 2, CF). By contrast, the melanoma cells expressed only modest levels of integrin
5-subunit mRNA and protein and barely detectable levels of HSPG2 mRNA and protein. Thus, these data reveal that the receptors for one of the angiogenesis inhibitors tested, endostatin, are differentially expressed in the endothelial and melanoma cells. This observation of vastly different levels of the integrin
5-subunit and HSPG2 may provide important clues as to the disparate responses of these cell types to antivas-cular therapy. HSPG2 is considered an excellent target for antiangiogenic therapy because it has been shown to play a key role in mediating basic fibroblast growth factor stimulation of endothelial celldriven angiogenesis (28). The
5 integrin is critical for endothelial blood vessel formation, and thus, agents targeting this integrin are likely to be effective against angiogenesis (29). Our data reveal biologically significant differences in the response of endothelial cells and aggressive melanoma cells that are engaged in vasculogenic mimicry to select angiogenesis inhibitors. Because vasculogenic mimicry has been reported in several other tumor models, including breast, prostatic, ovarian, and lung carcinoma [reviewed in Hendrix et al. (30)], these findings may offer new insights for designing rational antivascular therapeutic approaches in a broader context.
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Manuscript received January 30, 2004; revised July 13, 2004; accepted July 21, 2004.
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