Vasculogenic mimicry (VM) patterns are present in a wide variety of malignant tumors, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome (Maniotis et al., 1999; Folberg et al., 2000; Hendrix et al., 2003; Folberg and Maniotis, 2004; Döme et al., 2007). Mechanisms by which VM may contribute to adverse outcome are not well understood. Previous observations in our laboratory indicated that VM-forming tumor cells have increased resistance to herpes simplex virus-mediated oncolysis in three-dimensional (3D) uveal melanoma cultures (Valyi-Nagy et al., 2010). To determine whether VM-forming tumor cell subpopulations also have increased resistance against cytotoxic drugs, traditional two-dimensional (2D) and extracellular matrix (ECM, Matrigel)-containing 3D cultures of C918 uveal melanoma cells were established. In 2D cultures, C918 cells grew in monolayers. In 3D cultures, C918 cells formed a number of morphologically distinct tumor cell subpopulations that included cells that grew in monolayers on the Matrigel surface, cells that formed VM patterns, and cells that formed monolayers on the bottom of the culture dish. Following exposure to cisplatin or cadmium chloride, VM-forming tumor cells demonstrated prolonged survival relative to other tumor cell subpopulations in 3D cultures and cells grown in 2D. As presented in detail below, these findings suggest that increased drug resistance is a mechanism by which VM-forming tumor cells contribute to adverse outcome.
1.1. Vasculogenic mimicry
VM patterns represent the formation of perfusion pathways by tumor cells (Maniotis et al., 1999). VM is composed of patterned networks of laminin-rich basement membranes lined by tumor cells. VM patterns are present in a wide variety of malignant tumors and the presence of VM in tumors is associated with adverse outcome (Folberg et al., 1992 and 1993; Folberg and Maniotis, 2004; Gosh et al., 2005; Folberg et al., 2000 and 2007; Hendrix et al., 2003; Döme et al., 2007). While VM formation is clearly a marker of highly invasive tumor phenotype, mechanisms by which these structures may contribute to adverse outcome are not well understood. It has been proposed that VM formation may facilitate tumor perfusion and the physical connection between VM and blood vessels may also facilitate hematogeneous dissemination of tumor cells (Folberg and Maniotis, 2004). Interestingly, uveal melanoma cells within VM patterns assume a spindle A morphology and the expression of the Ki67 proliferation marker is significantly reduced in these cells (Folberg et al., 2006). These findings raise the possibility that VM-forming tumor cells have increased resistance against radiation and chemotherapeutic agents that target highly proliferative tumor cell populations.
Recent studies also indicate that malignant melanoma initiating cells (MMIC) are specifically associated with VM and it has been proposed that one mechanism by which MMIC promote tumor growth is by the induction of VM formation by MMIC (Frank et al., 2011). Previous work in our laboratory indicated that VM-forming tumor cells in 3D tumor cell cultures have increased resistance against at least one form of therapy: oncolytic virotherapy (Valyi-Nagy et al., 2010). To test the hypothesis that VM-forming tumor cells also have increased resistance against cytotoxic agents, in the current study we utilized 3D cultures of uveal melanoma cells as an experimental platform.
1.2. Three-dimensional tumor cultures
The introduction of 3D tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that can not be explored in traditional two dimensional (2D) monolayer cultures. The behavior of cells
Uveal melanoma cells form several morphologically distinct cell populations under 3D culture conditions (Maniotis et al., 1999, 2005; Valyi-Nagy et al., 2010)
2. Experimental approach
C918 uveal melanoma cells of high invasive potential were maintained in Eagle’s Minimal Essential Medium (EMEM, BioWhittaker Inc., Walkersville, MD) supplemented with heat inactivated 15% fetal bovine serum (Fisher, Ontario, Canada) without the addition of exogenous extracellular matrix molecules or growth factors. This cell line has been described in detail previously (Folberg et al., 2008).
2.2. Establishment of 2D and 3D melanoma cultures
For the establishment of 2D and 3D cultures used in experiments, C918 uveal melanoma cells were grown on 24-well plates in EMEM medium either in the presence (3D cultures) or in the absence (2D cultures) of extracellular matrix rich in laminin (Matrigel, BD Biosciences, Bedford, MA) as described previously (Valyi-Nagy et al., 2007 and 2010). For 3D cultures, Matrigel was poured onto the bottom of the tissue culture wells to a depth of approximately 0.2 mm and was allowed to polymerize for 1 hour at 37oC. Following polymerization, C918 melanoma cells were placed on the Matrigel surface and the cultures were incubated in repeatedly refreshed culture medium for up to 4 weeks. Cultures were observed daily under an inverted microscope (Leica, Bannockburn, IL).
2.3. Exposure of 2D and 3D melanoma cultures to cytotoxic agents
Cells were first exposed to various concentrations of cytotoxic agents four to five days after the initiation of cultures, when 2D cultures formed monolayers and cells in 3D cultures formed morphologically distinct tumor cell subpopulations including VM patterns. Tested cytotoxic agents included cisplatin [cis-diammineplatinum(II)dichloride, Sigma] and cadmium chloride [CdCl2 hemi (pentahydrate), Sigma]. Culture media containing either cisplatin or cadmium chloride were changed daily. Cultures were observed daily under an inverted fluorescence microscope (Leica, Bannockburn, IL) for evidence of drug toxicity. The day when at least 99% of a given morphologically distinct tumor cell subpopulation was destroyed was noted. Cell death was confirmed by the uptake of the charged cationic dye Trypan blue (Mediatech Inc., Manassas, VA) by more than 99% of cells following incubation of cultures with Trypan blue (0.2%) for 10 minutes at 37 °C.
3.1. Uveal melanoma cells form morphologically distinct cell populations under 3D conditions
As expected based on previous observations (Valyi-Nagy et al, 2010), C918 cells grew in monolayers in 2D cultures (Fig.1) and formed several morphologically distinct cell populations under 3D culture conditions (Fig.2)
3.2. VM-forming melanoma cells have increased resistance against cisplatin
Starting 4 to 5 days after the initiation of cultures, when 2D cultures formed monolayers and cells in 3D cultures formed morphologically distinct tumor cell subpopulations including VM patterns, cultures were exposed to various concentrations of cisplatin (Del Bello et al., 2003; Feldman et al., 2004; Bowden et al., 2010) and observed daily for toxicity. Cisplatin concentrations tested in the experiments ranged from 30 to 300 µM and fresh culture media containing cisplatin were added to the cultures daily. Control cultures not exposed to cisplatin remained viable and demonstrated growth. However, cultures exposed to cisplatin demonstrated dose dependant toxicity (Table 1). Interestingly, we found that VM-forming tumor cells demonstrated prolonged survival following cisplatin treatment relative to other tumor cell subpopulations in 3D cultures and relative to cells grown in 2D (Table 1, Figs 3 to 8). For instance, it took 6 days until 300 µM cisplatin destroyed more than 99% of cells forming VM in 3D cultures while other cell subpopulations in 3D cultures and cells grown in 2D cultures were destroyed by 4 and 3 days, respectively (Table 1). Even more pronounced differences were detected at 100 µM cisplatin concentrations as destruction of VM-forming tumor cells never reached 99% in 3D cultures during a 7-day observation period while other cell subpopulations in 3D cultures and cells grown in 2D cultures were destroyed by 4 and 3 days, respectively (Table 1). These findings indicate that VM-forming tumor cells have increased resistance against the cytotoxic effects of cisplatin in 3D uveal melanoma cultures.
3.3. VM-forming melanoma cells have increased resistance against cadmium
Starting 4 to 5 days after the initiation of cultures, when 2D cultures formed monolayers and cells in 3D cultures formed morphologically distinct tumor cell subpopulations including VM patterns, cultures were exposed to various concentrations of cadmium chloride (Yokouchi et al., 2007) and observed daily for toxicity. Cadmium chloride concentrations tested in the experiments ranged from 20 to 1000 µM. Fresh culture media containing cadmium chloride were added to the cultures daily for up to 4 weeks. Control cultures not exposed to cadmium chloride remained viable and demonstrated growth for several weeks. However, cultures exposed to cadmium chloride in the culture medium demonstrated dose dependant toxicity (Table 2). Similarly to the observations made with cisplatin, VM-forming tumor cells demonstrated prolonged survival following cadmium treatment relative to other tumor cell subpopulations in 3D cultures and cells grown in 2D (Table 2, Figs 9 through 14). Specifically, at 200 µM cadmium chloride concentration, destruction of VM-forming tumor cells never reached 99% in 3D cultures during a 16-day observation period while other cell subpopulations in 3D cultures and cells grown in 2D cultures were completely destroyed by 4 and 3 days, respectively (Table 2). Interestingly, as presented in the next section in detail, if cadmium chloride treatment was stopped sixteen days after initiation of drug treatment, residual still viable cells in VM patterns served as foci of new tumor growth (Fig. 16 through 18). These findings indicate that VM-forming tumor cells have increased resistance against the cytotoxic effects of cadmium chloride in 3D uveal melanoma cultures.
3.4. VM-forming melanoma cells may serve as foci of renewed growth once drug treatment is stopped
Findings presented above indicate that VM-forming tumor cells have increased resistance against the cytotoxic effects of cadmium chloride in 3D uveal melanoma cultures. Interestingly, if cadmium chloride treatment was stopped sixteen days after initiation of drug treatment, residual still viable cells associated with VM patterns appeared to serve as foci of new tumor growth in the 3D cultures (Fig. 15 through 18).
While VM formation is clearly a marker of highly invasive tumor phenotype
This work was supported by fellowship grants to Andras Voros and Eva Gagyi by the Rosztoczy Foundation.
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