Figure 2. Galectin-3 secretion by cancer cells increases sprouting angiogenesis via JAG1 ligand.

(A–C) HUVECs spheroids embedded in a fibrinogen gel were cultured for 24 hrs with MCF7-scramble or shRNA-gal-3 cells seeded on the top of the gel. After this period, spheroids were (A) photographed and (B) the average number and (C) sprouts length were quantified. (D) In vitro cell fate assay. Prior to spheroids formation, HUVECs were labeled with cell tracker green or cell tracker red and then mixed in a 1:1 ratio. Alternatively, red-labeled HUVECs were incubated for 15 min with rhgal-3 (37 nM) prior to spheroid formation. Spheroids were then embedded in a fibrinogen gel and cultured for 24 hrs. Arrowheads indicate the tip cells position and graph shows the percentage of green or red-labeled tip cells found per spheroid. (E–I) HUVECs previously transfected with JAG1 or DLL4 siRNA were grown into spheroids overnight in the presence/absence of rhgal-3 or rhgal-3C (37 nM). After this period spheroids were embedded in fibrinogen gel and cultured for additional 24 hrs (E) representative images are shown. (F and H) Mean number of sprouts and (G and I) sprouts length of HUVECs spheroids were measured. Controls are the same for F-I and all conditions were run simultaneously for each replicates Data are (A, D and E) representative images or (B–D and F–I) the mean (S.D.) of three independent experiments, n = 20. **p < 0.01, ****p < 0.0001 by 2-way ANOVA or two-tailed paired Student's t-test