Subprojeto 12 – Invadopodia formation in malignant cell lines treated by laminin-derived peptides. Study by 4D microscopy

Participante: Ruy Gastaldoni Jaeger (ICB-USP)

Invadopodia are actin-rich protrusions playing a key role during migration, invasion and protease activity of tumor cells. We will study by 4D confocal microscopy invadopodia formation dynamics in malignant cells treated by laminin-derived peptides. We have already demonstrated that laminin-derived peptides, such as YIGSR, SIKVAV, AG73 and C16 are involved in migration, invasion and protease activity of tumor cells (Oral Oncol. 40: 483, 2004; Am J Pathol. 171: 124, 2007; Matrix Biol. 27: 402, 2008; J. Cell Mol. Medicine. 2008 (submitted); Oral Oncol. 43: 987, 2007). Malignant cell lines will be treated with laminin peptides and their scrambled controls, followed by growth in fluorescent substrates (either Matrigel or gelatin). Growth on these substrates will determine digestion spots, characterizing invadopodia functional activity. Counting and measuring of these spots will inform whether a peptide stimulated invadopodia formation. Dynamics of invadopodia will be addressed in cell lines treated by laminin-derived peptides. Control and treated tumor cells will be transfected with vectors encoding fluorescently-tagged invadopodia proteins, such as mCherry-actin and cortactin-GFP. After that, cells will grow on fluorescent substrates and analyzed by multichannel 4D confocal microscopy, with z-sections taken at different time points. We will also study putative receptors and signaling pathways involved in invadopodia formation induced by laminin-derived peptides. Receptors such as integrin and syndecan-1 will be silenced by RNAi, followed by peptide treatment, growth in fluorescent substrates and analysis by 4D confocal microscopy. To assess signaling pathways cells will be treated with specific inhibitors, followed by peptide treatment, growth in fluorescent substrates and analysis by 4D confocal microscopy.