Sarcomas are rare, aggressive, and highly heterogeneous neoplastic malignancies that typically arise in tissues of mesenchymal origin. Cell motility leading to metastasis is responsible for about 90% of cancer deaths, including sarcoma. Understanding the molecular and metabolic mechanisms of cell motility leading to sarcomas metastasis is essential to improve treatment strategies. Single-cell morphokinetic plasticity was analyzed using infrastructure based on 1) scatter and wound healing fluorescent cells assays; 2) time-lapse microscopy (IncuCyte), cell segmentation (Imaris) and single-cell morphokinetic analysis (MATLAB and Python) infrastructure developed in our lab.
We have analyzed the effect of MET activator and inhibitor on different human sarcoma cell lines. We showed that MET activation in U2OS, SKLMS-1, Saos-2 and RD18 cell lines increases average speed, MSD and displacement2.
We performed a single-cell motility analysis to understand further the effect of Glucose, Glutamine, 2DG, and the cell line on cell morphokinetics. The analysis demonstrates that U2OS cells with FCS are characterized by dominantly increasing cumulative kinetics values (MSD, displacement2) and higher mean values in non-cumulative parameters (Instantaneous speed, Velocity y) and their shape is less spherical and more elongated. While without FCS that are characterized by dominantly increasing cumulative kinetics values (MSD, displacement2) and higher mean values in non-cumulative parameters (Instantaneous speed, Velocity x). To summarize, In U2OS cells with or without FCS, Glucose and Glutamine free environment, and 5mM Glucose/ 5mM 2DG/0mM Glutamine significantly decreases in cell motility.
To study the role of specific metabolic pathway on MET induced cell motility we used specific inhibitors and single cell morphokinetic analysis. Rotenone (Ox Phos - Complex 1 inhibitor) and BPTES (Glutaminolysis - Glutaminase GLS1 inhibitor) is highly effective against the U2OS cell line on motility. Rotenone treated cells demonstrated a decrease in velocity, displacement2, MSD, area, and confinement ratio and loss in the highly dynamic cell subpopulation. Although the U2OS cell line undergoes a significant reduction of cell motility and it is unable to close the wound. BPTES treated cells demonstrated a decrease in velocity, displacement2, MSD, area, directionality, and confinement ratio and lose in the highly dynamic cell subpopulation but it prevents the wound from closing as compared to the control. Except for the treatment HGF/SF combine with BPTES, it causes an increase in velocity and displacement2, however, the decrease in area, directionality, and the ratio between displacement and track length results in an overall reduction of cell motility and the inability to close the wound.
These results shed a light on the interplay between signaling and metabolism in altering the motility phenotype of the cell. The signaling/metabolic interplay induces motility plasticity of the sarcoma cells promoting its metastatic potential. The motility plasticity molecular and metabolic pathways can serve as new targets for therapy and precision medicine.