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. 2014 May 19;5(11):3711–3727. doi: 10.18632/oncotarget.1987

Figure 5. The anti-tumoral efficacy of MMP12-engineered ECFC in vitro and in vivo.

Figure 5

Panel A: Characterization of MMP12-engineered ECFC. RT-PCR shows MMP12 expression of engineered ECFC and Western blotting analysis detects MMP12 released in conditioned medium of ECFC containing empty vector (CM-ECFC MOCK, lane 1) or containing lentiviral vector encoding MMP12 molecule (CM-ECFC MMP12, lane 2). Panel B: Western blotting analysis of standard uPAR (st) incubated with aliquots of CM-ECFC MOCK (lane 1) and of CM-ECFC MMP12 (lane 2) in the presence of anti-MMP12 antibody (lane 3) or an irrelevant IgG (lane 4). Panel C: Capillary morphogenesis at 6h of ECFC-MOCK (picture1) and ECFC-MMP12 (Figure 2), in the presence of anti-MMP12 antibody (picture 3) or irrelevant IgG (picture 4). Numbers: percent field occupancy, taking control as 100%. Data are from 3 experiments performed in triplicate. Panel D: Anti-tumor property of ECFC-MMP12. Matrigel invasion of A375 cells in control conditions (picture 1) and upon addition, in the lower well of the migration chamber, of CM ECFC-MMP12 (picture 2), in the presence of anti-MMP12 antibody (picture 3) or irrelevant IgG (picture 4). The histogram represents the quantification of Matrigel invasion experiments, evaluated as in Fig.1A. Asterisks (*: P<0.05) indicate significant difference between the indicated experimental condition. Full length and truncated form of uPAR in lysates of A375 melanoma cells, shown on the right, was evaluated by Western blotting in the same conditions described above. Data result from three independent experiments. Panel E: Anti-angiogenic property of ECFC-MMP12. Capillary morphogenesis of endothelial cells treated with CM-ECFC MOCK (picture 1) or CM-ECFC MMP12 (picture 2) in the presence of anti-MMP12 antibody (picture 3) or irrelevant IgG (picture 4). Numbers: percent field occupancy, taking control as 100%. The pictures are representative of 3 different experiments performed in triplicate that gave similar results. Full length and truncated form of uPAR in lysates of endothelial cells, shown on the right, was evaluated by Western blotting in the same conditions described above. Panel F: Histogram represents number of A375 cells allowed to invade across the Matrigel pre-incubated overnight with CM ECFC MOCK and CM ECFC MMP12.