Figure 1.
INV and REF breast cancer cell invasion and migration.
Cells (2.5×105) were added to the upper side of each 8 µm insert of Boyden chambers coated (A) or not (B) with Matrigel for invasion or migration assays respectively; cells were then counted as described in Materials and Methods. MMP-9 activity secreted by REF (lane 1) and INV (lane 2) cells (C) was assessed by subjecting aliquots of lyophilized conditioned media normalized to the number of cells to 10% SDS-polyacrylamide gels containing 1 mg/mL gelatin. For QRT-PCR analysis (D), total RNA (1 µg) was reverse-transcribed using MMLV RT and subjected to qRT-PCR as described in Material and Methods. Representative data was normalised to PPIA is given. Each column represents a mean (± SD) of three independent experiments. *P<0.05.
Table 1.
List of genes that are down-regulated in INV cells versus REF cells.
Table 2.
List of genes that are up-regulated in INV cells versus REF cells.
Figure 2.
REF and INV cell adhesion and transmigration.
Morphology of INV and REF cells (A) revealed by optical microscopy at ×400 magnification. Arrows indicate cell junctions and asterisks indicate cell shape. Adhesion to fibronectin (B) and hCMEC/D3 endothelial cell adhesion assay (C). Transmigration assays (D) involved growing hCMEC/D3 endothelial cells to confluence on the upper side of 8 µm insert Boyden chambers. CMFDA-stained cells that transmigrated through the endothelial cells were counted as described in “Materials and Methods”. For adhesion to fibronectin, the data were normalized to BSA adhesion. Each column shows means (± SD) of three independent experiments. *P<0.05.
Figure 3.
REF and INV metastasis formation.
INV and REF cells (105) were injected into the left ventricle of nude mice (n = 7, A). Ex vivo data confirm tissue metastasis from INV (right panel) and REF cells injection (left panel) (B). Quantification of metastatic sites (C). Mouse survival following injection of REF and INV cells (D). Each column shows means (±SD) of representative experiments. *P versus control<0.05.
Figure 4.
INV and REF cells were injected subcutaneously near mammary gland into nude mice as described in “Materials and Methods”. After 1 week, tumors appeared in each group (A). Each point represents the mean tumor volume (mm3) (± SD, n = 7). Dead cells in REF and INV tumor sections were stained (B). Arrows indicate apoptotic and necrotic cells (magnification ×200). Percentage of cell death was determined as described in “Materials and Methods” (C).
Figure 5.
Cells (2×105) were cultured for 96 h in serum-free medium or were incubated with 2.5 µM Doxorubicin for 72 h in a serum-containing medium (A) or were cultured for 96 h in serum-free medium (B). For qRT-PCR analysis (C), total RNA (1 µg) was reverse-transcribed and the results were normalized as described above. (D) REF cells (Lanes 1 to 3) and INV cells (lanes 4 to 6) were treated (lane 1 and 4) with Serum free DMEM for 1 h or for 3 days (lanes 2 and 5) or with 10 ng/ml of IGF-1 (lanes 3 and 6), to detected AKT and pAKT by western blotting. Each column shows means (± SD) of three independent experiments. *P<0.05.
Figure 6.
REF and INV tumor angiogenesis.
Endothelial cells in REF and INV tumor sections were stained as described in “Materials and Methods”. Microvessels are revealed by brown staining (magnification ×100) and endothelial cells were quantified (B). QRT-PCR was used to quantify VEGF, VEGFR2, VEGFR1 and NRP-1 gene expression in INV and REF cells (C). VEGF (D) and NRP-1 (E) proteins were detected by ELISA tests and by western blotting, respectively. Each column represents a mean (± SD) of three independent experiments. *P<0.05. Gene functions from the GO Description provided by NetAffx™ Analysis Center Affymetrix (https://www.affymetrix.com/analysis/netaffx/index.affx)* Validated by QRT-PCR.