Figure 4. Effect of GSE on the growth promoting potential and chemotactic properties of adipocytes towards CRC cells.

(A) 3D model of adipocyte-CRC no-contact co-culture. Pre-adipocytes (3T3-L1 fibroblasts) were plated on the bottom of 8 well-BD-chamber slide and then allowed to differentiate in matrigel for 8 days. Mature adipocytes (3T3-L1-AC) were then treated with GSE (10 μg/ mL) for 2 days (late-acute treatment on day 8 for 48h). GSE was next washed off and the top of the matrigel was overlaid with a single layer of CRC HT29 cells in serum free media for 6 days. Cells were exposed to BrdU for 24 h, before study end; strong perilipin and BrdU –ve staining identified differentiated adipocytes. (B) Representative photomicrographs (X 200 magnification) indicate the decrease in the number of BrdU +ve cells-green, after 6 days of co-culture. DAPI-blue was used to stain nuclei. (C) GSE decreases the chemotactic properties of adipocytes towards HCT116 CRC cell invasion. Adipocyte-conditioned media (CM: collected for 48 h) after late-acute pre-treatment with GSE was used as the chemo-attractant in BD matrigel invasion chamber. After 48 h, matrigel invaded CRC cells were methanol fixed, H&E stained and counted. Adipocytes used were 3T3-L1-AC and human type II diabetic visceral adipocytes (HDP-AC). Representative photomicrographs of H&E stained (X 100 magnification) matrigel invaded HCT116 CRC cells are shown. Columns, mean values; error bars, SEM.