Fig. 3: Targeting of MCL-1 in bCAFs promotes tubulogenesis of endothelial cells in vitro and angiogenesis in ovo.

A bCAFs were treated for 18 h with MCL-1 inhibitor (S63845 500 nM) or not. The treatment was removed and cells were rinsed and cultured for additional 72 h in EGM2 (Endothelial Cell Growth Medium-2) medium supplemented with 1% of FBS. After 72 h, the conditioned media (CM) were applied on HUVECs for tubulogenesis assay. B, C (Top) Representative images of tubulogenesis assay of HUVECs under (B) CM from bCAFs treated or not with MCL-1 inhibitor (S63845 500 nM) or (C) CM from bCAFs expressing MCL-1 or not. (Bottom) Quantification of meshes, master junctions, and master segments of endothelial cells under (B) CM from bCAFs (untreated and S63845) n = 4 or (C) CM from bCAFs sgCTRL or sgMCL-1 (n = 5). Student t-test, *P < 0.5, **P < 0.01. D Chronological timeline of CAM model experimentation. After 10 days of embryonic development (ED10), T47D luminal breast cancer cell line with bCAFs expressing or not MCL-1 (bCAFsgCTRL or bCAFsgMCL-1) were xenografted. The tumours were treated with VEGF inhibitor (bevacizumab, BVZ 100 µg/CAM) every 2 days during one week until day 17 of embryonic development (ED17). E (Left) Representative pictures of engrafted tumours on CAM at ED17 are shown (Top: untreated or Bottom: treated with bevacizumab, BVZ 100 µg/CAM). (Right) Quantification of blood vessels density around the tumours (within a 5 mm radius of the tumour). Two-way ANOVA, ***P < 0.001, **P < 0.01, ns non-significant.