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

Abstract

Background: Standard 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.

Methods: 3D 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.

Results: Both 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.

Discussion: The 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.

Keywords: Cancer; Cell line; Cell migration; Invasion; Melanoma; Metastasis; Skin cancer; Skin equivalent model; Skin model; Three dimensional model.

Figure 1. Three-dimensional representation of melanoma progression.

(A) Schematic representation of the RGP phase, associated with melanoma cells in the epidermal region only and the metastatic phase, associated with melanoma cells that move away from the primary site. The cells in the metastatic phase are able to cross the basement membrane, enter the dermis and move into the blood vessels. This illustration is adapted, with permission, from Zaidi, Day & Merlino (2008). (B) and (C) H&E staining of native human skin and HSE respectively, showing a well-defined epidermis and dermis. The scale bar corresponds to 100 µm.

Figure 2. HSE and MSE preparation.

(A) Time frame for cell culture and DED preparation to construct HSE and MSE models. (B) Time intervals at which the HSE and MSE models are cultured and inspected. (C) Schematic of the circular barrier assay showing how cells are placed inside the barrier on a DED within a 24-well tissue culture plate. (D) DED with cells submerged in full Green’s medium. (E)–(F) Schematic and image of the HSE and/or MSE models lifted to the air-liquid interface on a sterile stainless steel grid with full Green’s medium placed in a 6-well plate. Scale in (F) bar corresponds to 6 mm.

Figure 3. MTT assay.

Experimental images of the MTT assay shows viable cells (purple) on the HSE (A)–(B). The MSE with WM35 melanoma cells is shown in (C)–(D). The MSE with SK-MEL-28 melanoma cells is shown in (E)–(F). Results in the left column are at day 0, and results in the right column are at day 9. The magnified central region of the HSE and MSE with melanoma cell colonies is shown in the insets in (A), (C) and (E). Scale bars in the main image show approximately 2 mm, whereas the scale bar in the insets show approximately 1.5 mm.

Figure 4. Proliferation, migration and invasion of skin cells and WM35 melanoma cells.

(A)–(D) Proliferating cells (brown) highlighted by Ki-67 at day 0, 9, 15 and 20. (E)–(H) Migrating cells (brown) highlighted by vimentin. Dermal cells with elongated morphology are fibroblasts, and colonies of cells are migrating WM35 melanoma cells. (I)–(L) WM35 melanoma cells (brown) highlighted by S100 at day 0, 9, 15 and 20. Black arrows and inset images highlight positive staining. The scale bar in the main images shows 100 µm, and the width of the insets are approximately 75 µm.

Figure 5. Proliferation, migration and invasion of skin cells and SK-MEL-28 melanoma cells.

(A)–(D) Proliferating cells (brown) highlighted by Ki-67 at day 0, 9, 15 and 20. (E)–(H) Migrating cells (brown) highlighted by vimentin. Dermal cells with elongated morphology are fibroblasts, and colonies of cells are SK-MEL-28 melanoma cells. (I)–(L) SK-MEL-28 melanoma cells (brown) highlighted by S100 at day 0, 9, 15 and 20. Black arrows and inset images highlight positive staining. The scale bar in the main images shows 100 µm, and the width of the insets are approximately 75 µm.

Figure 6. Proliferation, migration and invasion of skin cells.

(A)–(D) Proliferating cells (brown) highlighted by Ki-67 at day 0, 9, 15 and 20. (E)–(H) Migrating fibroblast cells (brown) highlighted by vimentin. (I)–(L) No specific melanoma staining is highlighted by S100 at day 0, 9, 15 and 20. Black arrows and inset images highlight positive staining. Scale bar corresponds to 100 µm.

Figure 7. Quantification of melanoma cell invasion depth.

Depth of melanoma invasion for the WM35 (red) and SK-MEL-28 (blue) cell lines. Data points show the average depth of invasion. The error bars measure the variability, as given by the sample standard deviation. In each case the sample mean and sample standard deviation is calculated using measurements from at least nine (n = 9) identically prepared experiments.