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A Novel Fully Humanized 3D Skin Equivalent to Model Early Melanoma Invasion.

ABSTRACT: Metastatic melanoma remains incurable, emphasizing the acute need for improved research models to investigate the underlying biologic mechanisms mediating tumor invasion and metastasis, and to develop more effective targeted therapies to improve clinical outcome. Available animal models of melanoma do not accurately reflect human disease and current in vitro human skin equivalent models incorporating melanoma cells are not fully representative of the human skin microenvironment. We have developed a robust and reproducible, fully humanized three-dimensional (3D) skin equivalent comprising a stratified, terminally differentiated epidermis and a dermal compartment consisting of fibroblast-generated extracellular matrix. Melanoma cells incorporated into the epidermis were able to invade through the basement membrane and into the dermis, mirroring early tumor invasion in vivo. Comparison of our novel 3D melanoma skin equivalent with melanoma in situ and metastatic melanoma indicates that this model accurately recreates features of disease pathology, making it a physiologically representative model of early radial and vertical growth-phase melanoma invasion.

SUBMITTER: Hill DS

PROVIDER: S-EPMC4637991 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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A Novel Fully Humanized 3D Skin Equivalent to Model Early Melanoma Invasion.

Hill David S DS Robinson Neil D P ND Caley Matthew P MP Chen Mei M O'Toole Edel A EA Armstrong Jane L JL Przyborski Stefan S Lovat Penny E PE

Molecular cancer therapeutics 20150901 11

Metastatic melanoma remains incurable, emphasizing the acute need for improved research models to investigate the underlying biologic mechanisms mediating tumor invasion and metastasis, and to develop more effective targeted therapies to improve clinical outcome. Available animal models of melanoma do not accurately reflect human disease and current in vitro human skin equivalent models incorporating melanoma cells are not fully representative of the human skin microenvironment. We have develope ...[more]

PMID: 26330548

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Project description:BackgroundStandard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected.Methods3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data.ResultsBoth HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells.DiscussionThe MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details.

Project description:Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.

Dataset Information

A Novel Fully Humanized 3D Skin Equivalent to Model Early Melanoma Invasion.

Publications

A Novel Fully Humanized 3D Skin Equivalent to Model Early Melanoma Invasion.

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