MULTIMODAL MICROSCOPY IMAGING AS A TOOL TO UNDERSTAND MOLECULAR MECHANISMS OF AMOEBOID CELL MOTILITY

Migrating cells show a versatile repertoire of migration modes with remarkable plasticity, allowing them to switch between different migration strategies in response to changing environmental conditions and activation of distinct molecular pathways. Amoeboid cell motility of tumor cells was shown to be important for tumor progression and metastasis; however, its molecular and morphokinetic mechanism are poorly understood.

Met tyrosine kinase growth factor receptor overexpression and deregulation is often observed in cancer and its inhibitors were approved as therapy. We study the role of Met and its downstream signaling molecules Gab1 and Grb2 in amoeboid cell motility. We previously showed that aggressive breast cancer cells expressing high levels of Met protein and display Met-dependent amoeboid-blebbing. Similarly, human embryo kidney (HEK293T) cells over expressing fluorescent Met, exhibit constitutive Met activation and amoeboid cell motility. Co-expression of Met and Gab1 induced high dynamic and large area blebs in a dose-dependent manner. Co-expression of Grb2 and Met altered bleb morphology to a long podium structure with high local concentration of Met and Grb2 that were co-localized at the tip of the podia. The co-localized Met-Grb2 were shown to be phosphorylated. Actin is organized in fibers that may contribute to the stability of these podia structures. Using WB analysis and phosphoprotein array we found that Met, Met-Gab1 and Met Grb2 over expression, as was found in human tumors, activate different Met signaling network pathways that alters cell morphology. To study if these unique networks elicit different motility modes we established a morphokinetic profile based on time lapse microscopy of single cell evaluating 35 parameters. The parameters include Velocity (V), Collectivity (C), Displacement² (Dis), Area (A) and Sphericity (S). Each parameter was defined as high (H) medium (M) or low (L), compared to control cells. Met expression alone induced rounded cell structures with medium kinetics (V^M/C^M/Dis^2M/ /A^H/S^M). Met-Gab1 expression dramatically increased cell kinetics (V^H/C^H/Dis^2M/A^M/S^M), whereas Met-Grb2 expression decreased kinetics (V^L/C^M/Dis^2M/A^M/S^H). Our results demonstrate that alterations of Met downstream signaling induce alteration of the signaling network that intern induce various amoeboid motility modes that enable cancer cells to invade for migration and metastasis. This model can shed a light of the molecular and cellular mechanisms of motility plasticity, a major hallmark of metastasis and could serve as a tool to develop new inhibitors that will work together with the clinically improved Met inhibitors to reduce resistance development and inhibit motility plasticity leading to metastasis.