Characterization of an in vitro model of human melanoma invasion based on reconstructed human skin

Abstract

The purpose of this study was to compare the invasive properties of normal human cutaneous melanocytes and of a cutaneous melanoma cell line (HBL) in a three-dimensional model of reconstructed human skin. Specifically, we asked to what extent the pigmentary and invasive behaviour of both cells is influenced by their interaction with adjacent skin cells (keratinocytes and fibroblasts) and the basement membrane (BM). In the presence of a BM, normal human melanocytes within this model remained within the basal layer of keratinocytes and did not pigment spontaneously. When the BM was removed, melanocytes were found suprabasally and pigmented extensively. No significant invasion of melanocytes into the dermis was detected in the presence or absence of the BM. HBL melanoma cells showed no significant ability to invade into the dermis in the absence of other cells, irrespective of the presence or absence of the BM. However, when added to keratinocytes and fibroblasts, HBL cells showed a capacity to invade into the dermis, both in the presence and absence of the BM. Associated with HBL invasion into the dermis, we noted significant keratinocyte entry into the dermis. On their own, keratinocytes entered the dermis in the absence of the BM but showed no significant penetration into the dermis when the BM was present. In summary, this model demonstrates clear differences between melanocytes and a melanoma cell line with respect to their invasive properties. It also allows demonstration of interactions between cells, and between cells and the BM. The study also provides evidence for a synergistic interaction between this melanoma cell line and keratinocytes in penetrating the BM.

TNF-α increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes

“…There is also evidence from our laboratory that melanoma invasion in a model of human reconstructed skin requires the presence of host skin cells (namely fibroblasts and keratinocytes) that express characteristics of 'wounded' skin (Eves et al, 2000;Eves et al, in press). We recently reported that TNF-a upregulated integrin expression in a human melanoma cell line and also increased the ability of cells to bind to substrates and to invade through fibronectin (Zhu et al, 2002).…”

P-Cadherin Promotes Cell-Cell Adhesion and Counteracts Invasion in Human Melanoma

“…In the former, tumor cells were confronted with myocardium of embryonic chick heart fragments, whereas the latter model, consisting of human keratinocytes, basement membrane, dermal fibroblasts, and dermal collagen, more closely recapitulates the microenvironment of a melanocytic tumor in vivo. Whereas others have mainly applied this model in a qualitative or semiquantitative way (36,44), we quantified the invasion of our tested cells. We counted the cells that had invaded into the dermal collagen and measured the minimal invasion depth of each cell relative to the basement membrane as an in vitro correlate of the Breslow index (tumor invasion depth), a prognostic factor routinely assessed in the clinical setting (2).…”

“…Skin reconstructs were generated using published techniques (35,36) with modifications. First, dead de-epidermized dermis (DED) was produced from human skin fragments derived from plastic surgery (abdominoplasty or breast reconstructions).…”

Transient TNF regulates the self-renewing capacity of stem-like label-retaining cells in sphere and skin equivalent models of melanoma

“…This finding suggests that TNF imprints transferable molecular changes that permanently affect the functionality of the melanoma GFP high SC compartment. To gain insight into the possible cellular mechanism(s) by which TNF maintained its effect, we recapitulated melanoma in an in vivo-like [32,33] skin equivalent (SE) model, which is an alternative to animal models, using sorted quiescent (GFP high ) and fast-cycling (GFP low ) cells in the presence or absence of systemic TNF for 3 weeks. Interestingly, control GFP high HBL-H2B-GFP cells ( Figure 4D) and, to a lesser extent, SK-Mel28-H2B-GFP (not shown) cells were capable of developing into the highly pigmented assembly of cells resembling melanoma tumor in vivo.…”