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Melanoma cells influence the differentiation pattern of human epidermal keratinocytes.

Kodet O, Lacina L, Krejčí E, Dvořánková B, Grim M, Štork J, Kodetová D, Vlček Č, Šáchová J, Kolář M, Strnad H, Smetana K - Mol. Cancer (2015)

Bottom Line: Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively.Differentially expressed candidate genes were verified by RT-qPCR.We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro.

View Article: PubMed Central - PubMed

Affiliation: 1st Faculty of Medicine, Institute of Anatomy, Charles University in Prague, U Nemocnice 3, CZ-12800 Prague, Czech Republic. strnad@img.cas.cz.

ABSTRACT

Background: Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).

Methods: Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK.

Results: Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.

Conclusion: We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

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Detection of K14 (red signal) in colonies of normal human keratinocytes (Control, A), and after the application of FGF-2 (B), VEGF-A (C), IL-8 and CXCl-1 (D), and simultaneous application of all tested substances (E). Green signal represents keratin, bar denotes 50 μm. Panel (F) shows the difference of the thickness of K14-positive layer cells surrounding the keratinocyte colonies. All stimulated cell colonies have significantly different margin extent when compared to control colonies. The cells stimulated simultaneously by all factors have significantly wider margin than the cells treated by individual factors (p < 0.01). The peripheral cells positive for K14 are presented in higher magnification.
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Fig5: Detection of K14 (red signal) in colonies of normal human keratinocytes (Control, A), and after the application of FGF-2 (B), VEGF-A (C), IL-8 and CXCl-1 (D), and simultaneous application of all tested substances (E). Green signal represents keratin, bar denotes 50 μm. Panel (F) shows the difference of the thickness of K14-positive layer cells surrounding the keratinocyte colonies. All stimulated cell colonies have significantly different margin extent when compared to control colonies. The cells stimulated simultaneously by all factors have significantly wider margin than the cells treated by individual factors (p < 0.01). The peripheral cells positive for K14 are presented in higher magnification.

Mentions: Keratinocytes strongly positive for K14 were located predominantly at the periphery of colonies (Figure 5A). When either FGF-2, VEGF-A and/or a mixture of IL-8 with CXCL-1 was supplemented to the culture medium, the K14-positive cells border of the colonies was significantly wider than in non-treated cultures (Figure 5B-D, F). Simultaneous addition of all tested factors to keratinocyte culture significantly increased the thickness of K14-positive edge of colony (Figure 5E, F). K14-positive cells at the border of treated colonies exhibited more elongated shape more resembling fibroblasts than usually seen in epithelial cells. These cells were not anymore in tight contact (Figure 5F).Figure 5


Melanoma cells influence the differentiation pattern of human epidermal keratinocytes.

Kodet O, Lacina L, Krejčí E, Dvořánková B, Grim M, Štork J, Kodetová D, Vlček Č, Šáchová J, Kolář M, Strnad H, Smetana K - Mol. Cancer (2015)

Detection of K14 (red signal) in colonies of normal human keratinocytes (Control, A), and after the application of FGF-2 (B), VEGF-A (C), IL-8 and CXCl-1 (D), and simultaneous application of all tested substances (E). Green signal represents keratin, bar denotes 50 μm. Panel (F) shows the difference of the thickness of K14-positive layer cells surrounding the keratinocyte colonies. All stimulated cell colonies have significantly different margin extent when compared to control colonies. The cells stimulated simultaneously by all factors have significantly wider margin than the cells treated by individual factors (p < 0.01). The peripheral cells positive for K14 are presented in higher magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4325966&req=5

Fig5: Detection of K14 (red signal) in colonies of normal human keratinocytes (Control, A), and after the application of FGF-2 (B), VEGF-A (C), IL-8 and CXCl-1 (D), and simultaneous application of all tested substances (E). Green signal represents keratin, bar denotes 50 μm. Panel (F) shows the difference of the thickness of K14-positive layer cells surrounding the keratinocyte colonies. All stimulated cell colonies have significantly different margin extent when compared to control colonies. The cells stimulated simultaneously by all factors have significantly wider margin than the cells treated by individual factors (p < 0.01). The peripheral cells positive for K14 are presented in higher magnification.
Mentions: Keratinocytes strongly positive for K14 were located predominantly at the periphery of colonies (Figure 5A). When either FGF-2, VEGF-A and/or a mixture of IL-8 with CXCL-1 was supplemented to the culture medium, the K14-positive cells border of the colonies was significantly wider than in non-treated cultures (Figure 5B-D, F). Simultaneous addition of all tested factors to keratinocyte culture significantly increased the thickness of K14-positive edge of colony (Figure 5E, F). K14-positive cells at the border of treated colonies exhibited more elongated shape more resembling fibroblasts than usually seen in epithelial cells. These cells were not anymore in tight contact (Figure 5F).Figure 5

Bottom Line: Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively.Differentially expressed candidate genes were verified by RT-qPCR.We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro.

View Article: PubMed Central - PubMed

Affiliation: 1st Faculty of Medicine, Institute of Anatomy, Charles University in Prague, U Nemocnice 3, CZ-12800 Prague, Czech Republic. strnad@img.cas.cz.

ABSTRACT

Background: Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).

Methods: Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK.

Results: Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.

Conclusion: We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

Show MeSH
Related in: MedlinePlus