Fig 1.
BBB properties and cancer cells adhesion assay on animal in vitro BBB models.
BBB Integrity measurement. The bovine (A) and the murine (C) models present a continuous staining of tight junction proteins ZO-1 (left panel) and Claudin-5 (right panel) associated with a PeLY of respectively 0.39 ± 0.07 x 10−3 cm/min and 0.36 ± 0.19 x 10−3 cm/min. Nuclei are stained with Hoechst (blue), bar = 50 μm. Quantification of cancer cells adhesion. The number of adherent MDA-MB-231 was set up to 100% and equal to 286 for the bovine model (B) and 442 the murine model (D); PeLY: Lucifer Yellow Endothelial Permeability; ZO-1: Zonula-Occludens-1, NS: Non Significant. The results are mean of triplicate and representative of two independent experiments. ***p<0.001.
Fig 2.
BBB properties and cancer cells adhesion assay on HUVECs in vitro model.
(A) Integrity measurement. The HUVECs model presents a discontinuous staining of tight junction proteins ZO-1 (left panel) and Claudin-5 (right panel) associated with a high PeLY of 2.35 ± 0.19 x 10−3 cm/min. Interruptions of the staining are indicated by white arrows. Nuclei are stained with Hoechst, bar = 50 μm. (B) Quantification of cancer cells adhesion. The number of adherent MDA-MB-231 was set up to 100% and equal to 1748. HUVECs: Human Umbilical vein endothelial cells; PeLY: Lucifer Yellow Endothelial Permeability; ZO-1: Zonula-Occludens-1. The results are mean of triplicate and representative of three independent experiments. N.S.: Non significant.
Fig 3.
BBB properties and cancer cells adhesion assay on human BBB in vitro model.
(A) Integrity measurement. The BLECs model presents a continuous staining of tight junction proteins ZO-1 (left panel) and Claudin-5 (right panel) associated with a low PeLY of 0.58 ± 0.07 x 10−3 cm/min. Nuclei are stained with Hoechst, bar = 50 μm. (B) Quantification of cancer cells adhesion. The number of adherent MDA-MB-231 was set up to 100% and equal to 706. BLECs: Brain Like Endothelial Cells; PeLY: Lucifer Yellow Endothelial Permeability; ZO-1: Zonula-Occludens-1. The results are mean of triplicate and representative of six independent experiments. The results are mean of triplicate and representative of three independent experiments. ***p<0.001.
Table 1.
Comparative results obtained for BBB characteristics and cancer cells adhesion.
Fig 4.
Adhesion-transmigration analysis of breast cancer cell lines representative of different molecular cancer subtypes on the BLECs model.
Quantification of cancer cells adhesion. (A) The number of adherent MDA-MB-231 was set up to 100% and equal to 672. The results are mean of triplicate and representative of three independent experiments. Quantification of cancer cells transmigration. (B) The number of transmigrated MDA-MB-231 was set up to 100% and equal to 251. The results are mean of triplicate and representative of two independent experiments. N.S: Non Significant; **p<0.01; ***p<0.001. Basal-like (Triple negative) molecular subtype (open bars), luminal molecular subtype (filled bars).
Fig 5.
Adhesion analysis of breast cancer cell lines after glycosphingolipid biosynthesis inhibition.
(A) Visualization of glycosphingolipid synthesis inhibition. In control condition, MDA-MB-231 expressed the glycosphingolipid GM1 (green) at the cell surface (left panel). After 10 μM PPMP treatment during 5 days, GM1 expression at the cell surface was significantly reduced (right panel). Nuclei are stained with Hoechst, bar = 50 μm. (B) Quantification of MDA-MB-231 cells adhesion after PPMP treatment. The number of adherent non-treated MDA-MB-231 was set up to 100%. (C) Quantification of MCF-7 cells adhesion after PPMP treatment. The number of adherent non-treated MCF-7 was set up to 100%. The results are mean of triplicate and representative of three independent experiments. ***p<0.001.