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Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures.

Valyi-Nagy K, Kormos B, Ali M, Shukla D, Valyi-Nagy T - Mol. Vis. (2012)

Bottom Line: Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome.We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271.In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA.

ABSTRACT

Purpose: Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype.

Methods: The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry.

Results: We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271.

Conclusions: These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells.

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Related in: MedlinePlus

CD271 stem cell marker expression in C918 uveal melanoma cultures. The upper row of pictures demonstrates the morphology of two-dimensional (2D; A and C) and three-dimensional (3D; E and G) cultures of C918 uveal melanoma with the formation of prominent VM in 3D cultures. Arrows in E and G point to VM. Asterisks in E and G point to cells growing in monolayer on the Matrigel surface. Arrowheads in E and G point to cells growing on the bottom of the culture dish. Panel H demonstrates detection of CD271 expression by VM-forming tumor cells by immunofluorescence in 3D cultures. No CD271 expression was detected in 2D cultures (D). CD271 expression was also not detected in control 2D and 3D cultures where no primary antibody was used (B and F). Magnification: 200×.
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f2: CD271 stem cell marker expression in C918 uveal melanoma cultures. The upper row of pictures demonstrates the morphology of two-dimensional (2D; A and C) and three-dimensional (3D; E and G) cultures of C918 uveal melanoma with the formation of prominent VM in 3D cultures. Arrows in E and G point to VM. Asterisks in E and G point to cells growing in monolayer on the Matrigel surface. Arrowheads in E and G point to cells growing on the bottom of the culture dish. Panel H demonstrates detection of CD271 expression by VM-forming tumor cells by immunofluorescence in 3D cultures. No CD271 expression was detected in 2D cultures (D). CD271 expression was also not detected in control 2D and 3D cultures where no primary antibody was used (B and F). Magnification: 200×.

Mentions: To detect CD271 expression in C918 and OCM1 cultures, uveal melanoma cells were seeded onto plastic culture dishes to establish 2D cultures and onto Matrigel matrix surface to form 3D structures (Figure 1). Both C918 and OCM1 cell cultures became confluent in the plastic dish and formed monolayer. C918 melanoma cells cultured in the presence of Matrigel matrix formed a variety of morphologically distinguishable tumor cell subpopulations including VM (Figure 1A,B and Figure 2E,G). Specifically and as described in detail previously (Valyi-Nagy et al. [18]), these subpopulations included cells that grew in monolayer on the Matrigel surface, cells that formed VM on the Matrigel surface and tumor cells that grew as a monolayer on the bottom of the culture dish under the Matrigel layer (Figure 1A,B and Figure 2E,G). OCM1 cells in 3D cultures formed cell aggregates on the Matrigel surface and invaded Matrigel without forming VM. Both 2D and 3D cultures were labeled with CD271 antibody, then FITC-conjugated secondary antibody. For control cultures, no primary antibody was added. We found that C918 uveal melanoma cells cultured under 2D conditions did not express CD271 (Figure 2D). In contrast, a subpopulation of C918 cells cultured in 3D, specifically VM-forming cells expressed CD271 at clearly detectable level (Figure 2H). Interestingly, other subpopulations of uveal melanoma cells in 3D cultures, including monolayer forming cells on the Matrigel surface and on the bottom of the culture dish were CD271 negative (Figure 2H). Neither 2D nor 3D cultures of OCM1 uveal melanoma cells expressed CD271 (data not shown). Control 2D and 3D uveal melanoma cultures exposed only to secondary antibody remained CD271 negative (Figure 2B,F, respectively).


Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures.

Valyi-Nagy K, Kormos B, Ali M, Shukla D, Valyi-Nagy T - Mol. Vis. (2012)

CD271 stem cell marker expression in C918 uveal melanoma cultures. The upper row of pictures demonstrates the morphology of two-dimensional (2D; A and C) and three-dimensional (3D; E and G) cultures of C918 uveal melanoma with the formation of prominent VM in 3D cultures. Arrows in E and G point to VM. Asterisks in E and G point to cells growing in monolayer on the Matrigel surface. Arrowheads in E and G point to cells growing on the bottom of the culture dish. Panel H demonstrates detection of CD271 expression by VM-forming tumor cells by immunofluorescence in 3D cultures. No CD271 expression was detected in 2D cultures (D). CD271 expression was also not detected in control 2D and 3D cultures where no primary antibody was used (B and F). Magnification: 200×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298423&req=5

f2: CD271 stem cell marker expression in C918 uveal melanoma cultures. The upper row of pictures demonstrates the morphology of two-dimensional (2D; A and C) and three-dimensional (3D; E and G) cultures of C918 uveal melanoma with the formation of prominent VM in 3D cultures. Arrows in E and G point to VM. Asterisks in E and G point to cells growing in monolayer on the Matrigel surface. Arrowheads in E and G point to cells growing on the bottom of the culture dish. Panel H demonstrates detection of CD271 expression by VM-forming tumor cells by immunofluorescence in 3D cultures. No CD271 expression was detected in 2D cultures (D). CD271 expression was also not detected in control 2D and 3D cultures where no primary antibody was used (B and F). Magnification: 200×.
Mentions: To detect CD271 expression in C918 and OCM1 cultures, uveal melanoma cells were seeded onto plastic culture dishes to establish 2D cultures and onto Matrigel matrix surface to form 3D structures (Figure 1). Both C918 and OCM1 cell cultures became confluent in the plastic dish and formed monolayer. C918 melanoma cells cultured in the presence of Matrigel matrix formed a variety of morphologically distinguishable tumor cell subpopulations including VM (Figure 1A,B and Figure 2E,G). Specifically and as described in detail previously (Valyi-Nagy et al. [18]), these subpopulations included cells that grew in monolayer on the Matrigel surface, cells that formed VM on the Matrigel surface and tumor cells that grew as a monolayer on the bottom of the culture dish under the Matrigel layer (Figure 1A,B and Figure 2E,G). OCM1 cells in 3D cultures formed cell aggregates on the Matrigel surface and invaded Matrigel without forming VM. Both 2D and 3D cultures were labeled with CD271 antibody, then FITC-conjugated secondary antibody. For control cultures, no primary antibody was added. We found that C918 uveal melanoma cells cultured under 2D conditions did not express CD271 (Figure 2D). In contrast, a subpopulation of C918 cells cultured in 3D, specifically VM-forming cells expressed CD271 at clearly detectable level (Figure 2H). Interestingly, other subpopulations of uveal melanoma cells in 3D cultures, including monolayer forming cells on the Matrigel surface and on the bottom of the culture dish were CD271 negative (Figure 2H). Neither 2D nor 3D cultures of OCM1 uveal melanoma cells expressed CD271 (data not shown). Control 2D and 3D uveal melanoma cultures exposed only to secondary antibody remained CD271 negative (Figure 2B,F, respectively).

Bottom Line: Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome.We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271.In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA.

ABSTRACT

Purpose: Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype.

Methods: The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry.

Results: We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271.

Conclusions: These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells.

Show MeSH
Related in: MedlinePlus