Abstract
Simple SummaryMerkel cell carcinoma (MCC) is an extremely aggressive type of skin cancer. Nevertheless, the development of new therapeutic approaches to treat this malignancy is hampered by the absence of in vitro culture models. Furthermore, prospective clinical trials are scarce due to the rarity of the disease. Thus, the aim of the present study was to generate organotypic epithelial raft cultures (OERCs) of MCC by growing primary human keratinocytes and MCC cell lines on artificial dermal equivalents. Histological and immunohistochemical analyses confirmed the proliferation of MCC cell lines. In addition, gene expression analysis showed genes that were differently expressed in the tumor cells and the keratinocytes. In brief, we developed a three-dimensional cell culture model of MCC that can potentially be used for evaluating the efficacy and selectivity of new drug candidates against this disease.Merkel cell carcinoma (MCC) is a rare type of skin cancer for which an in vitro model is still lacking. MCC tumorigenesis is associated either with the integration of Merkel cell polyomavirus into the host genome, or with the accumulation of somatic mutations upon chronic exposure to UV light. Transgenic animals expressing the viral oncoproteins, which are constitutively expressed in virus-related MCC, do not fully recapitulate MCC. Although cell-line-derived xenografts have been established for the two subtypes of MCC, they still present certain limitations. Here, we generated organotypic epithelial raft cultures (OERCs) of MCC by using primary human keratinocytes and both virus-positive and virus-negative MCC cell lines. The primary human keratinocytes and the tumor cells were grown on top of a dermal equivalent. Histological and immunohistochemical examination of the rafts confirmed the growth of MCC cells. Furthermore, gene expression analysis revealed differences in the expression profiles of the distinct tumor cells and the keratinocytes at the transcriptional level. In summary, considering the limited availability of patient samples, OERCs of MCC may constitute a suitable model for evaluating the efficacy and selectivity of new drug candidates against MCC; moreover, they are a potential tool to study the oncogenic mechanisms of this malignancy.
Highlights
Merkel cell carcinoma (MCC) is an aggressive type of skin tumor with a mortality rate between 33 and 46% [1]
Hematoxylin and eosin (H&E) staining of organotypic epithelial raft cultures (OERCs) derived from primary human keratinocytes (PHKs) seeded on top of dermal equivalents showed that these cells were able to proliferate and form a differentiated epithelium resembling the normal epithelium seen in vivo (Figure 2A), as previously reported [28]
The results essentially point to one cell line (MCC14/2) growing in all three types of raft, and the WAGA, MS-1, and MKL-1 Merkel cell polyomavirus (MCPyV)+ MCC cells and the MCC13 and MCC26 MCPyV− MCC cells only growing when mixed with the PHK cells in the epithelial layer (Table 1)
Summary
Merkel cell carcinoma (MCC) is an aggressive type of skin tumor with a mortality rate between 33 and 46% [1]. MCC’s incidence is higher among elderly individuals of Caucasian origin with prolonged exposure to UV light. Despite MCC’s incidence being rather low (i.e., 0.7 cases/100,000 individuals in the US), several studies have reported increasing incidence, which is projected to persist in the coming years [2]. Little was known about MCC tumorigenesis until the identification of a new human polyomavirus in 2008—Merkel cell polyomavirus (MCPyV), clonally integrated in the majority of the tumors [3]. Similar to other polyomaviruses capable of inducing tumors in animal models (e.g., SV40) [4], a truncated form of the LT of MCPyV, which conserves the LXCXE motif, maintains cell growth by inhibiting retinoblastoma (Rb) activity [5,6]. MCPyV-negative (MCPyV−) MCC is predominant in Australia and New Zealand [1,7], supposedly due to the role of sun irradiation in the accumulation of the somatic mutations that give rise to this subgroup [8–10]
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