485 Vaspin (serpin A12) increases melanoma cell invasion through extracellular matrix models

Abstract

The development of targeted therapies and immune checkpoint inhibitors has revolutionised melanoma treatment over the past decade. However, there remains an urgent need to identify new pro-metastatic pathways in melanoma. Vaspin was identified in a phage display screen against two in vitro melanoma models. The effect of recombinant human vaspin (rhVaspin) was tested on a range of melanoma lines including. Phenotypic changes were assessed using 2D cell migration assays and monitoring cell invasion through Matrigel-coated Boyden chambers or fluorescently labelled gelatin-coated glass coverslips. 3D melanoma spheroids were transferred to gels composed of Matrigel or type I collagen before time-lapse imaging. Protein expression was examined by Western blotting and immunofluorescence microscopy. From the peptide phage display hits we identified vaspin as a candidate protein for further investigation. rhVaspin treatment of A375, M202, SKMEL5, SKMEL28 cell models caused a temporal increase in phospho-Akt levels and increased the invasion of A375 and M202 cells (1.4- and 2.0-fold, respectively) through Matrigel coated Boyden chambers, but not M229 cells. Overexpression of vaspin in melanoma cells did not significantly affect cell proliferation, cell cycle progression or adhesion. In contrast, time-lapse microscopy revealed that the invasion of A375 and SKMEL28 cells from 3D melanoma spheroids into Matrigel and collagen gels was significantly increased. The accelerated invasion of A375 cells through Matrigel coated Boyden chambers was reversible by treatment with the pan-MMP inhibitor CT1746 (10 μM). Moreover, the expression levels of MMP-2 and -3 were increased in A375 cells following treatment with rhVaspin. Our studies show that vaspin expression in melanoma models is associated with increased invasive capacity through an MMP-dependent mechanism. Ongoing work aims to examine the pro-invasive phenotype in primary melanoma cultures using in vivo models.