Optimized zymogram protocol from 3D spheroid cultures to study MMP-2 and -9 activities in tumor cells.

Abstract

Three-dimensional spheroids are more representative of tumors than cell-cultured monolayers. As in tumors, gradients of oxygen, nutrients and wastes are found in spheroid cultures but not in classical cultured monolayers. On the other hand, cell-based assays on the latter are hardly applicable to spheroid cultures. Such is the case for zymogram assays, which are classically used to measure MMP-2 and MMP-9 activities, and for immunoblots to measure the phosphorylation of proteins involved in ligand-induced intracellular signaling in normal and tumor cells. In this study we used two renal cancer cell lines as models, the first derived from a pediatric rhabdoid tumor and the second from an adult clear cell renal cell carcinoma. Using these two cell lines, we successfully developed a simple inexpensive assay to measure MMP-2 and MMP-9 activities in spheroids established in the presence of methylcellulose. After washing, 1 to 5 spheroids were pooled and stimulated with collagen I for 24 h before analysis. MMP-2 and MMP-9 activities were measured in supernatants using a standard but enhanced zymogram assay. Both pro-MMP-9 and MMP-2 activities were detected in spheroids established from both cell lines. In contrast with our previous data using classical cultures monolayers, collagen I stimulation decreased pro-MMP-9 activity without affecting MMP-2 activity. On the other hand, we could not accurately measure AKT intracellular signaling pathways from spheroids stimulated with collagen I. Finally, we adapted our 3D protocol to analyze the MAPK/ERK pathway in kidney tumor cells following induction by EGF. In conclusion, this zymogram assay for analyzing MMP-2 and MMP-9 activities in spheroids paves the way for novel experimentations in tumor biology.