Fig. 1. Mechanical stimulation promotes RUNX2 expression in the engineered tumor model.
(a) The bones are exposed to cyclic tissue deformation resulting from the mechanical loads generated mainly by body weight and muscle tension. Cancer cell growing in the bone are exposed to a mechano-environment where extracellular forces, resulting from pressurization of interstitial fluids, flow-induced shear and matrix strain, are dynamically superimposed and alter cell survival and drug response through the activation of transcription factors (RUNX2). To generate the bone tumor models, ES cells were seeded onto a 3D porous matrix, placed in a tissue culture dish and grown in free-swelling conditions for 48 hours. Plates were lodged in the bioreactor for 2 hours of stimulation followed by 24 hours of culture in free-swelling conditions. For longer experiments stimulation was repeated every 24 hours. (b) Representative images of ES cells cultured on a 2D tissue culture dish (top) or on a 3D porous scaffold (middle) compared to ES tumor sections obtained from patients (bottom). Scale bars: phase contrast 50 μm; H&E 100 μm. (c) Gene expression analysis of engineered ES models exposed to bone-like (1% strain) or cartilage-like (10% strain) mechanical loadings. mRNA levels of RUNX2 and several target genes (OPN, MMP9, PTHrP, MMP13 and BSP) was normalized to GAPDH and to the 2D culture. (average ± sd; n= 9; * p<0.05; ** p<0.01; ns not statistically significant). P values are determined by Student’s t-test (two-tailed). (d) Immunostaining revealed RUNX2 nuclear localization (black arrows) in cells exposed to stimulation. Scale bar: 20μm. (e,f) Western blot analysis of RUNX2 protein levels in 2D culture or in the ES models (1% strain or control). Quantified RUNX2 protein levels (f) are represented as relative changes in band density normalized to GAPDH and to RUNX2 levels in 2D cultures. (Average ± sd; n= 9; * p<0.05; ** p<0.01; ns not statistically significant). P values are determined by Student’s t-test (two-tailed). (g–i) Immunostaining analysis of RUNX2 transcriptional targets Survivin (g), BSP (h) and ES diagnostic marker CD99 (i). Scale bars: 50μm.