Figure 2. Saos-2 cells drive aerobic glycolysis in adjacent MSC.

(A) Upregulation of PKM2 in MSC activated by OS cells. Cell lysates were prepared from MSC activated by conditioned medium from serum-starved Saos-2 cells or untreated, non-activated MSC. Lysates were analyzed by Western blot with an anti-PKM2 specific antibody. Note the upregulation of PKM2 in tumor-activated MSC, as compared to untreated MSC. Normalization was done by actin immunoblot. (B) Gene expression analysis of GLUT1 (left plot) and MCT-4 (right plot) by RT-PCR. Note that MSC cultured with CM from Saos-2 cells, show significantly higher levels of the GLUT1 and MCT-4 genes, versus non-activated MSC. Glut1, p=0.0092; MCT-4, p=0.0071. (C) Upregulation of MCT-4 expression was validated by immunofluorescence analysis. Homotypic cultures of MSC (top row) and Saos-2 GFP cells (middle row) and Saos-2-MSC heterotypic cultures (lower row) were immunostained with MCT-4 antibodies. DAPI was used to stain nuclei (blue). Note that the MCT-4 expression (red) is clearly increased in MSC in co-culture condition, as compared to MSC cultured alone. Importantly, images were acquired using identical exposure settings. Original magnification, 20x. Scale bar 40 μm. (D) Tumor-activated MSC show increased lactate secretion. MSC were incubated with CM obtained from OS cells (Saos-2 or HOS) for 48 hours. Then, cells were carefully washed and incubated in fresh serum-free medium for an additional 24 hours. Lactate assay was performed on this culture medium. Note that lactate production is significantly increased in MSC, after activation with conditioned media from Saos-2 cells (left plot) and HOS cells (right plot). Values were normalized by cell numbers *p<0.05.