Organotypic Epithelial Raft Cultures as a Three-Dimensional In Vitro Model of Merkel Cell Carcinoma

Abstract

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.

Current In Vitro and In Vivo Models to Study MCPyV-Associated MCC

“…In this model, MCC cells were either added directly on top of the dermal equivalents, mixed with keratinocytes at different ratios before seeding, or co-cultured with fibroblasts in the dermal equivalent ( Figure 3 B). Interestingly, intraepidermal MCC-like lesions developed with MCPyV+ MCC cell lines within these rafts and not in the dermal equivalents when co-cultured with fibroblasts, which differs from our results [ 157 ]. These discordant results may reflect different rafting techniques, the use of different keratinocyte/fibroblast cells, and/or the heterogeneity of patient-derived MCC cell lines used in the two studies [ 108 , 158 , 159 , 160 ].…”

“…We and others have developed models using organotypic rafts to study MCPyV+ MCC [ 156 , 157 ] ( Figure 3 ). We adapted our laboratory’s established protocol for studying the HPV life cycle by incorporating the MKL-1 cell line to develop a raft-based model for the co-culture of MCPyV+ MCC cell lines [ 155 , 158 ].…”

“…Temblador et al also recently reported an organotypic raft-based model for MCC that included both MCPyV- (MCC13, MCC12/2 and MCC26) and MCPyV+ MCC (WAGA, MS-1, and MKL-1) cell lines [ 157 ]. In this model, MCC cells were either added directly on top of the dermal equivalents, mixed with keratinocytes at different ratios before seeding, or co-cultured with fibroblasts in the dermal equivalent ( Figure 3 B).…”

“…These discordant results may reflect different rafting techniques, the use of different keratinocyte/fibroblast cells, and/or the heterogeneity of patient-derived MCC cell lines used in the two studies [ 108 , 158 , 159 , 160 ]. Using this in vitro system, Temblador et al observed significantly higher expression of several genes associated with tumorigenesis in cells grown within the rafts, such as extracellular matrix, cell adhesion molecules, and growth factors, compared to cells cultured in a monolayer [ 157 ]. This finding highlights the potential biological importance of using organotypic rafts, whereby cell-cell interactions can be elucidated in a biologically relevant context.…”

Evaluation of Biological Activity of Natural Compounds: Current Trends and Methods

“…From the array of cell cultures available for in vitro testing that offer diverse degrees of intricacy and similarity to the in vivo setting, organotypic cultures are tissue slices that maintain cell interactions and the extracellular matrix composition of the original tissue and tissue function [ 52 , 53 , 54 , 55 ]. However, this system lacks intercommunication with the circulatory and immune systems and is inadequate for medium- to high-throughput analysis [ 11 ].…”