Melanoma cells in distinct growth phases retain specific invasive qualities during brain metastasis in vivo
2011-10-04

Dear Editor, The Clark model describes the progression from normal melanocytes to malignant melanoma based on histopathological changes. The model suggests a sequence of five distinct steps: (i) benign nevus to, (ii) dysplastic nevus to, (iii) radial growth phase (RGP) melanoma to, (iv) vertical growth phase (VGP) melanoma to, and (v) metastatic melanoma (reviewed by Miller and Mihm, 2006). However, research efforts of the past 10 yrs clearly pointed out that the Clark model by itself does not reflect the vast complexity of melanoma initiation and progression. Melanoma cells from RGP and metastatic cells are characterized by unique RNA signatures (Sousa and Espreafico, 2008) and distinct expression of major histocompatibility complex molecules (Degenhardt et al., 2010). The increase in cytomorphologic atypia from RGP to VGP cells is reflected by an increase in NRAS and BRAF mutations (Greene et al., 2009). The switch from RGP to VGP and the metastatic phenotype is associated with increased CD1, loss of E-Cadherin, c-KIT and AP-2, expression of N-Cadherin, MMP-2, osteonectin, survivin, βFGF, and αVβ3 integrin (reviewed by Bar-Eli, 2001; Meier et al., 2000, 2003; Sturm et al., 2002). Further, telomeric crisis is rare in pigmented lesions less progressed than the VGP (Soo et al., 2011). To assess, whether cell lines from different growth phases (not originating from the same patient, thus having distinct genetic backgrounds) retain the growth pattern of the original parental tumors, we examined migration and invasion in vitro and in vivo. For detailed description, see Materials and Methods in Data S1. Migration and invasion increased from SBCL2 (RGP) to WM115 (VGP) to metastatic BLM cells (Figure S1A). Next, we asked whether the different migration and invasion properties of the melanoma cells were also present in human epidermal skin reconstruct. SBCL2 cells were confined to the basal layer of the stratified epithelium, depicting little proliferation (Figure S1B, upper panel). A similar image was observed on WM115 cells: no invasion, but an increased proliferation and tumor formation within the keratinizing stratified epithelium (Figure S1B, middle panel). In contrast, BLM cells had invaded the skin reconstruct (Figure S1B, lower panel). To scrutinize a possible suitability of melanoma cell lines from distinct growth phases for the study of melanoma progression in vivo, we generated a novel model for brain metastasis (C. Busch and U. Drews, unpublished data). Melanoma cells were transplanted into the IVth ventricle of the developing chick embryonic hindbrain, and embryos were further incubated for 4 days. The brain vesicle as transplantation site allowed the melanoma cells to invade the neural epithelium to form a metastasis. Histological examination yielded that SBCL2 cells had formed demarcated metastases in the dorsal neural epithelium and the surrounding brain tissue. No single SBCL2 cell infiltrated the brain. In contrast to SBCL2 cells in the skin reconstruct, almost all cells were proliferating (Figure 1, upper panel). WM115 cells also formed demarcated, proliferating metastases in the dorsal neural epithelium and surrounding tissues; however, single MIB1-positive WM115 cells invaded the brain (Figure 1, middle panel). For metastatic BLM cells, the proliferating BLM metastases were diffusely embedded into the brain tissue. A large number of BLM cells invaded the brain (Figure 1, lower panel). Our results confirm that the melanoma cell lines from distinct growth phases retain their specific properties in a complex in vivo brain metastasis model, highlighting that RGP, VGP, and metastatic melanoma cells are suitable to study aspects of melanoma progression. Given that only one cell line per representative growth phase was used in this study, it cannot be excluded that other cell lines might behave differently in the same experimental setup. Metastatic BLM cells extensively invade the chick embryonic brain. Melanoma cells were transplanted into the IVth ventricle of the developing rhombencephalon (hindbrain) of stage 13 chick embryos. After 4 days of further incubation, tumor formation and invasion into the brain tissue was assessed. Radial growth phase SBCL2 cells formed well demarcated, highly proliferating tumors (approximately 90% of cells MIB-1-positive) in the dorsal neural epithelium and surrounding brain tissues. No invasion was observed (upper panel). Vertical growth phase WM115 cells formed less proliferative (approximately 50% MIB-1 positive), demarcated tumors in the same location; single cells start to penetrate the adjacent brain tissues (middle panel). Metastatic BLM cells formed irregular, diffusely proliferating (approximately 80% MIB-1 positive) tumors destroying the dorsal brain. Single cells and clusters of BLM cells invaded the neural tissues (lower panel). Figure S1. Migration and invasion in Boyden chamber and human epidermal skin reconstructs increase from RGP to VGP to metastatic melanoma cells. Data S1. Supplementary Materials and Methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.