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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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Related in: MedlinePlus

Hierarchical clustering of transcription profiles of the genes related to extracellular factors in HPM, BLM, and NCSC (A). Results of RT-qPCR verification of the transcriptional activity of IL-8, CXCL-1, GFG-2, and VEGF-A in BLM, NSCC, and A2058, G361 melanoma lines as well as in primary cultures of ascitic melanoma cells (Asc) after 1st and 3rd passage in relation to HPM (B). Statistically significant differences from HPM at p < 0.05 significance level are marked by asterisk.
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Fig4: Hierarchical clustering of transcription profiles of the genes related to extracellular factors in HPM, BLM, and NCSC (A). Results of RT-qPCR verification of the transcriptional activity of IL-8, CXCL-1, GFG-2, and VEGF-A in BLM, NSCC, and A2058, G361 melanoma lines as well as in primary cultures of ascitic melanoma cells (Asc) after 1st and 3rd passage in relation to HPM (B). Statistically significant differences from HPM at p < 0.05 significance level are marked by asterisk.

Mentions: Analysis of microarrays with transcriptomes of HPM, BLM and NCSC at the whole genome scale demonstrated distinct differences among tested cell types (Figure 4A). As transcription profiles of pure cultures were well conserved in different media (Figure 4A), the biological effect in co-culture could only be attributed to particular cell types in transwells. Because this experimental setting excludes direct physical interaction of cells, we further focused our interest on extracellularly released cell products that could be responsible for paracrine regulation of the environment. In the co-culture experiments, we observed that only BLM and NCSC significantly affected the phenotype in co-cultured keratinocytes, thus we searched for the cell products that were excessively produced by these cells and not by HPM. However, the reciprocal effect of keratinocytes on pigment cell keratinocyte axis cannot be excluded. The differentially regulated factors are listed in Additional file7: Table S3.Figure 4


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)

Hierarchical clustering of transcription profiles of the genes related to extracellular factors in HPM, BLM, and NCSC (A). Results of RT-qPCR verification of the transcriptional activity of IL-8, CXCL-1, GFG-2, and VEGF-A in BLM, NSCC, and A2058, G361 melanoma lines as well as in primary cultures of ascitic melanoma cells (Asc) after 1st and 3rd passage in relation to HPM (B). Statistically significant differences from HPM at p < 0.05 significance level are marked by asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Hierarchical clustering of transcription profiles of the genes related to extracellular factors in HPM, BLM, and NCSC (A). Results of RT-qPCR verification of the transcriptional activity of IL-8, CXCL-1, GFG-2, and VEGF-A in BLM, NSCC, and A2058, G361 melanoma lines as well as in primary cultures of ascitic melanoma cells (Asc) after 1st and 3rd passage in relation to HPM (B). Statistically significant differences from HPM at p < 0.05 significance level are marked by asterisk.
Mentions: Analysis of microarrays with transcriptomes of HPM, BLM and NCSC at the whole genome scale demonstrated distinct differences among tested cell types (Figure 4A). As transcription profiles of pure cultures were well conserved in different media (Figure 4A), the biological effect in co-culture could only be attributed to particular cell types in transwells. Because this experimental setting excludes direct physical interaction of cells, we further focused our interest on extracellularly released cell products that could be responsible for paracrine regulation of the environment. In the co-culture experiments, we observed that only BLM and NCSC significantly affected the phenotype in co-cultured keratinocytes, thus we searched for the cell products that were excessively produced by these cells and not by HPM. However, the reciprocal effect of keratinocytes on pigment cell keratinocyte axis cannot be excluded. The differentially regulated factors are listed in Additional file7: Table S3.Figure 4

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