Fig. 6: Chemotherapy modulates the pro-angiogenic properties of bCAFs through NOXA-mediated MCL-1 degradation and subsequent activation of NF-κB signaling.

A (Left) Representative experiments of proteins expression level (MCL-1, BCL-xL, and NOXA) in bCAFs after 18 h of chemotherapy treatment (5-Fluorouracil 22 µM, Cisplatin 11 µM, and Doxorubicin 1 µM) or not evaluated using western blots, Actin expression was used as loading control. (Right) Quantification of the amounts of MCL-1 and BCL-xL protein band relative to Actin in bCAFs after chemotherapy treatment (n = 3), results are expressed as a ratio relative to untreated bCAFs. One sample t-test, **P < 0.01, ns non-significant. B RT-qPCR of VEGF-A, FGF2, and ANGPT1 mRNA in bCAFs after 18 h of chemotherapy treatment or not normalized on RPLPO mRNA expression. Mean and SEM of three independent experiments are represented as relative quantity of mRNA. Student t-test, *P < 0.05, ns not significant. C VEGF-A was analysed by ELISA in bCAFs CM after 18 h of chemotherapy. Student t-test, **P < 0.01. D bCAFs were treated or not with chemotherapy for 18 h. The treatment was removed and cells were rinsed and cultured for additional 72 h in EGM2 medium supplemented with 1% of FBS. After 72 h, conditioned media (CM) were recovered, and a portion was incubated with VEGF inhibitor (BVZ, 2 mg/mL) for 2 h −37 °C before adding on HUVECs for tubulogenesis assay. E Representative images of tubulogenesis assay of HUVECs cultured in conditioned media from bCAFs treated with chemotherapy or not +/− BVZ (2 mg/mL). F Quantification of meshes, master junctions, and master segments of HUVECs cultured in conditioned media from bCAFs treated or not with chemotherapy +/− BVZ (2 mg/mL) (n = 3). Two-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns non-significant. G (Left) Representative pictures of engrafted tumours on CAM at ED17, tumours are composed of T47D cells and bCAFs pre-treated or not with chemotherapy for 18 h before xenograft (Top). The tumours were treated with VEGF inhibitor (bevacizumab, BVZ 100 µg/CAM) every 2 days during one week until ED17 (Bottom). (Right) Quantification of blood vessels density around the tumours (within a 5 mm radius of the tumour). Two-way ANOVA, **P < 0.01, *P < 0.05, ns non-significant. H MCL-1, BCL-xL, and NOXA proteins expression levels in bCAFs, surexpressing MCL-1 (CAFpLvxMCL-1) or BCL-xL (CAFpLvxBCL-xL) or not (CAFpLvxCTRL) after 18 h of chemotherapy were evaluated using Western blots analysis. Actin expression was used as loading control. I VEGF-A secretion was analysed by ELISA in bCAFs CM in the same conditions as (H) (n = 3) Two-way ANOVA, ****P < 0.0001; ns non-significant. J MCL-1 and NOXA proteins expression levels in bCAFs expressing NOXA (CAFsgCTRL) or not (CAFsgNOXA) after 18 h of chemotherapy were evaluated using Western blots analysis. Actin expression was used as loading control. K Quantification (percentage of cells positive for nuclear p65) (right) and confocal image (left) of p65 staining (red) in bCAFs expressing NOXA (CAFsgCTRL) or not (CAFsgNOXA) and treated with chemotherapy (1 µM) for 18 h or not. The nuclei were counterstained with DAPI (blue) (n = 3). Scale bar = 50 μm. Two-way ANOVA, ****P < 0.0001, *P < 0.05, ns not significant. L VEGF-A secretion was analysed by ELISA in bCAFs CM in the same conditions as (J). M qRT-PCR of VEGFA, CXCL8, IL-1β, and CXCL1 mRNA in bCAFs treated with chemotherapy (1 µM) for 18 h or not in combination with an IKKβ inhibitor (AS602868, 10 µM) or not normalized on RPLPO mRNA expression (n = 3). Two-way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns non-significant.