Figure 1.
Effects of GLPG0187 and ITGAV knockdown on adherence to tissue culture plastic.
Representative images of cells treated for 24 hours with a concentration series of GLPG0187 (dosage between 0–500 ng/ml, indicated underneath the images). Treatment resulted in a dose-dependent loss of adherence to tissue culture plastic in both UM-UC-3luc2 cells (A) and RT-4 cells (C). After 48 hours of GLPG0187 treatment, cells cultured for 4 days in GLPG0187-free medium regained their adherence to the tissue culture plastic in UM-UC-3luc2 cells (B) and RT-4 cells (D). Loss of adherence was also observed in UM-UC-3luc2 and RT4 cells stably transduced with a short hairpin targeted against ITGAV (respectively F–G for UMUC3luc2 sh ITGAV clones 1 and 2 and J–K for RT4 shITGAV clones 1 and 2). As a control, cells stably transduced with a non-targeting short hairpin (NT) were used (UMUC3 (E) and RT4 (I)). Flow cytometric analysis of relative ITGAV expression levels in UM-UC-3luc2 (H) and RT4 (L) cells (% of positive cells * mean fluorescence intensity). Data are presented as mean ± SEM, n = 3, the percentage of ITGAV positive cells is indicated above the bars.
Figure 2.
Effects of αv integrin on migration and EMT.
Effects of knockdown of ITGAV and 48 hrs of GLPG0187 treatment on migratory capacity of UM-UC-3luc2 and RT-4 cells as determined by Transwell Boyden chamber migration assays. Mean numbers of migrated cells per area were measured. Mean number of migrated cells of the control NT cells are depicted above the respective bars (A). Effects of ITGAV knockdown and 48 hrs of GLPG0187 treatment on the CDH1/CDH2 ratio (B). Data were normalized to the NT or control conditions (n = 3) and are presented as mean ± SEM. qPCR (C) and protein analysis of SNAI1 (D). qPCR analysis (E) and protein analysis of SNAI2 (F). qPCR analysis (G) and protein analysis of ZEB1 (H). qPCR analysis (I) and protein analysis of ZEB2 (J). Relative expression levels are shown compared to respectively NT or vehicle treated cells. All qPCR values were normalized for GAPDH and presented as mean ± SEM.
Figure 3.
Effects of αv integrin on clonogenicity and stem cell/metastasis markers.
The relative percentage and size distribution of colony-forming cells in a 96-wells plate clonogenic assay of single-cell diluted cultures after 2 weeks in the αv kd or NT cells and cells treated with a dose range of GLPG0187 for 48 hrs and plated afterwards. The area of the colonies was measured with Image J software and divided according to size. Small colonies are between 0.5 and 1.5 mm2, medium sized colonies are between 1.5 and 4 mm2 and large colonies are bigger than 4 mm2. Data were normalized to the NT or control conditions and are presented as mean ± SEM. Percentage of colony-forming cells in the control NT cells are depicted above the respective bars (A). UM-UC-3 cells were seeded 100 cells/cm2 in an ultra-low attachment plate in serum-starved conditions. The percentage of cells with sphere forming capacity (P0) was measured after 10 days of culture (B). P0 spheres were dissociated into single cells and seeded in ultra-low attachment 96 wells. The percentage of cells with sphere forming capacity (P1) was measured after 10 days of culture (B). The area of the spheres was measured with Image J software (D). Percentage of cells with high ALDH activity (ALDHhi) as measured with Aldefluor assay. Data are normalized to the NT or vehicle treated cells. Percentages of ALDHhi cells in the NT and control cells are depicted above the respective bars (E). qPCR analysis of NANOG (F) qPCR analysis of BMI1 (G). Relative expression levels are shown compared to respectively NT or vehicle-treated cells. All values were normalized for GAPDH and presented as mean ± SEM.
Figure 4.
Effect of αv integrin on expression levels of CD24 and urothelial differentiation markers and on senescence.
Relative expression levels of CD24 (A) and CD227 (B). Relative expression levels (% of positive cells * Mean fluorescence intensity) were measured by flow cytometry and normalized to the NT or vehicle treated cells. Data are represented as mean ± SEM. Percentages of positive cells are depicted above the respective bars. qPCR analysis of KRT20. Relative expression levels are shown compared to respectively NT or non-treated cells. All values were normalized for GAPDH and presented as mean ± SEM (C). UM-UC-3 luc2 and RT-4 cells were seeded into a six-well plate and exposed to a concentration series of GLPG0187 (0–500 ng/ml). 48 h after incubation, cells were harvested and senescence associated acid β-galactosidase activity was measured. Data are represented as fold change in fluorescence intensity of the signal (D).
Figure 5.
ITGAV knockdown in UM-UC-3luc2 cells affects intra-bone growth in a preclinical model.
A) Percentage of mice with tumors after intra-bone inoculation of either αv-kd-UM-UC-3luc2 or NT-UM-UC-3luc2 cells B) Total tumor burden of the mice injected with αv-kd-UM-UC-3luc2 (closed circles) or NT-UM-UC-3luc2 cells (open circles). C) Representative images of mice intra-osseously inoculated with either αv-kd-UM-UC-3luc2 or NT-UM-UC-3luc2 cells 7 days after inoculation (n = 10/group *P<0.05).
Figure 6.
Effect of systemic administration of GLPG0187 on tumor growth and metastasis in a preventive and curative protocol.
A) Schematic representation of the preventive protocol. Mice were treated daily with either IP administrated vehicle or GLPG0187 (100 mg/kg/day) from day -1 onwards. At day 0, 100,000 UM-UC-3luc2 cells were inoculated into the left heart ventricle and once a week BLI images were taken. B) Number of metastasis per mouse. C) Total tumor burden for the mice treated with 100 mg/kg/day GLPG0187 (closed circles) or vehicle (open circles). In the insert, the first 14 days are shown. D) Representative images of mice treated with vehicle or 100 mg/kg/day GLPG0187 taken at day 28 after inoculation. E) Schematic representation of the curative protocol. At day -21, 100,000 UM-UC-3luc2 cells were injected into the left heart ventricle and once a week BLI images were taken. At day 0, mice were divided into groups with equal total tumor burden. Mice were daily treated with an IP dosage of either vehicle or GLPG0187 (100 mg/kg/day) from day 0 onwards. F) Number of metastasis per mouse. G) Total tumor burden for the mice treated with 100 mg/kg/day GLPG0187 (closed circles) or vehicle (open circles). H) Representative images of mice treated with vehicle or 100 mg/kg/day GLPG0187 taken at day 15 after start of treatment.