Figure 1. Metformin Inhibits Colonization of the Omentum Ex Vivo and Invasion of the 3D TME In Vitro.

(A) Schematic: ex vivo adhesion of GFP-tagged HeyA8 OvCa cells to fresh human omental biopsies from patients without cancer taking metformin for type 2 diabetes or control patients not taking metformin.

(B) Omental explants from control or metformin patients were seeded with GFP-tagged HeyA8 OvCa cells. Tumor cells were allowed to adhere to omental explants for 1.5 h before imaging and quantification (n = 3 patients per group in triplicate). Images are representative and were taken at 10× magnification; scale bar represents 200 μm.

(C and D) Ex vivo adhesion and colonization: human omental explants were pretreated with metformin (250 μM) for 72 h before seeding of GFP-tagged HeyA8 OvCa cells, and the number of tumor cells that adhered to (1.5 h, C) or colonized (72 h, D) per omentum were quantified (n = 2 patients in triplicate for both experiments).

(E) Migration of HeyA8 OvCa cells through transwell chamber (15 h) toward omental explants that had been pretreated with metformin (250 μM, 72 h). During the assay, metformin was removed, and all media were replaced with serum-free media containing 0.1% BSA (n = 2 patients in triplicate).

(F) Invasion through 3D organotypic model (36 h) after seeding of TYKnu OvCa cells. Each cell type was individually treated with metformin (250 μM, 72 h) before the 3D model was constructed. HPMC, human primary mesothelial cells; NOF, normal omental fibroblast; n = 3 patients in triplicate.

(G) Invasion of TYKnu cells through 3D model (36 h) treated with the indicated compounds: metformin (1 mM), AMPK inhibitor compound C (CompC, 1 μM), and AMPK activator AICAR (1 mM) (n = 3 patients in triplicate).

Data represent mean values ± SDs. *p < 0.05, **p < 0.01, and ***p < 0.005; n.s., not significant.