Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model

Abstract

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.

Progress in melanoma modelling in vitro

“…After the formation of each compartments, the reconstructed skin is left at the air‐liquid interface allowing keratinocyte differentiation and stratification . Histologically, HSE architecture and composition closely resemble human skin . HSE has been used as an alternative to animal testing for studies of skin barrier function, skin irritation, wound healing, ultraviolet light‐induced damage, for the treatment of burns and other wounds .…”

“…DED is placed on a sterile stainless steel rings with a diameter of 6 mm in a 24‐well tissue plate. Primary keratinocytes, fibroblasts and melanoma cells are seeded together on DED and grown submerged for 24 hours, followed by 5‐20 days of culture at the air‐liquid interface to allow keratinocyte differentiation and stratification …”

“…which could help to understand the intricate process that leads to switch from radial to vertical growth and the invasion into the underlying tissue. In particular, melanoma invasion can be easily quantified by measuring the distance in depth reached by cells in the dermis . Finally, it is possible to test the antimigratory effect of different drugs.…”

Revisiting the Fisher-KPP equation to interpret the spreading-extinction dichotomy

“…We find that Stefan boundary condition [47,48]. In more realistic applications of invasion, such as malignant cellular invasion into surrounding tissues, there will be a conversion of consumed tissue into malignant cells at the interface [34,60,61,62]. One way of interpreting this conversion from tissues to cells is that there is a loss of one species, in this case the surrounding tissue, that is converted into another species, in this case invasive cells.…”

Cancer growth, metastasis and control likewise Go gaming: an Ising model approach

“…However, studies in vitro of metastatic melanoma showed the appearance of probable melanoma cell colonies that later cannot be found in the tissue after some days possibility invading tissue underneath (Haridas et al, 2017). In fact, our simulations are in 2D and resemble a lot the spreading of the cancer cell in a monolayer tissue, the introduction of the new dimension to make the model 3D would enhance our simulation by allowing us to study the vertical invasion of cancer cells.…”