A Novel In Vitro Culture Model System to Study Merkel Cell Polyomavirus–Associated MCC Using Three-Dimensional Organotypic Raft Equivalents of Human Skin

Abstract

Merkel cell polyomavirus (MCPyV) is a human polyomavirus causally linked to the development of Merkel cell carcinoma (MCC), an aggressive malignancy that largely arises within the dermis of the skin. In this study, we recapitulate the histopathology of human MCC tumors in vitro using an organotypic (raft) culture system that is traditionally used to recapitulate the dermal and epidermal equivalents of skin in three dimensions (3D). In the optimal culture condition, MCPyV+ MCC cells were embedded in collagen between the epidermal equivalent comprising human keratinocytes and a dermal equivalent containing fibroblasts, resulting in MCC-like lesions arising within the dermal equivalent. The presence and organization of MCC cells within these dermal lesions were characterized through biomarker analyses. Interestingly, co-culture of MCPyV+ MCC together with keratinocytes specifically within the epidermal equivalent of the raft did not reproduce human MCC morphology, nor were any keratinocytes necessary for MCC-like lesions to develop in the dermal equivalent. This 3D tissue culture system provides a novel in vitro platform for studying the role of MCPyV T antigens in MCC oncogenesis, identifying additional factors involved in this process, and for screening potential MCPyV+ MCC therapeutic strategies.

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

“…Given their location on top of the keratinized epithelium, limited access to nutrients could explain the cell death. Our findings are consistent with a previous report concluding that the MKL1 cell line does not survive within the epithelial equivalent of the raft [ 27 ].…”

“…Lambert et al recently reported on the development of a novel in vitro 3D culture system for the co-culture of the MKL-1 (MCPyV + ) cell line [ 27 ]. MCPyV + MCC cells embedded in a collagen layer between the epidermal equivalent (comprising human keratinocytes) and a dermal equivalent (containing human fibroblasts) were identified as the optimal culture conditions, leading to MCC-like lesions arising within the dermal equivalent.…”

“…When MCPyV + MCC cells were co-cultured together with keratinocytes within the epidermal equivalent of the raft, human MCC morphology could not be reproduced, as MKL1 cells did not survive within such a raft. Furthermore, Loke et al [ 27 ] found that keratinocytes were not necessary for the formation of MCC-like lesions in the dermal equivalent. Although we and Loke et al both used a 3D culture system, their findings and ours differ, which could be explained by differences in the methodology and in the setup of the rafts.…”

“…The epidermis, which constitutes the upper part of the skin, is continuously renewed by proliferating keratinocytes, and can hence be divided into different layers: (1) the basal layer, or stratum basale, contains proliferating keratinocytes that differentiate as they move up in the epidermis; (2) in the spinous layer, or stratum spinosum, the keratinocytes reinforce their cytoskeleton and interact with one another via desmosomes; (3) the granular layer, or stratum granulosum, contains flat anuclear keratinocytes with numerous proteins, such as filaggrins, which aggregate in granules; (4) the stratum corneum is the outermost layer, and is formed by dead keratinocytes or corneocytes sealed together by secreted lipids and rich in keratin filaments [ 25 , 26 ]. Recently, a novel in vitro system using 3D organotypic raft cultures to study MCPyV-associated MCC was reported [ 27 ]. In this model, when the MCPyV + MCC MKL1 cell line was embedded in a collagen layer placed between the dermal equivalent (containing human fibroblasts) and the epidermal equivalent (comprising human keratinocytes), MCC-like lesions arose within the dermal equivalent independently of the presence of keratinocytes.…”

“…In this model, when the MCPyV + MCC MKL1 cell line was embedded in a collagen layer placed between the dermal equivalent (containing human fibroblasts) and the epidermal equivalent (comprising human keratinocytes), MCC-like lesions arose within the dermal equivalent independently of the presence of keratinocytes. However, co-culture of MCPyV + MCC and keratinocytes within the epidermal equivalent did not reproduce the morphology of human MCC, as MKL1 cells did not survive within such a raft [ 27 ].…”