Additional file 1 of ProNGF promotes brain metastasis through TrkA/EphA2 induced Src activation in triple negative breast cancer cells
Creators: name: Cicero, Julien
name: Trouvilliez, Sarah
name: Palma, Martine
name: Ternier, Gaetan
name: Decoster, Laurine
name: Happernegg, Eloise
name: Barois, Nicolas
name: Van Outryve, Alexandre
name: Dehouck, Lucie
name: Bourette, Roland P.
name: Adriaenssens, Eric
name: Lagadec, Chann
name: Tarhan, Cagatay Mehmet
name: Collard, Dominique
name: Souguir, Zied
name: Vandenhaute, Elodie
name: Maubon, Grégory
name: Sipieter, François
name: Borghi, Nicolas
name: Shimizu, Fumitaka
name: Kanda, Takashi
name: Giacobini, Paolo
name: Gosselet, Fabien
name: Maubon, Nathalie
name: Le Bourhis, Xuefen
name: Van Seuningen, Isabelle
name: Mysiorek, Caroline
name: Toillon, Robert-Alain
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Mental Health

description: Additional file 1: Figure S1. proNGF increases TrkA/EphA2 complexes in TNBC regardless of p75NTR. Representative (A) pictures and associated quantification (B) of TrkA/EphA2 PLA signal following proNGF stimulation (60 min) in MDA-MB-231 TrkA treated with p75NTR targeting siRNA. Unpaired 2-tailed t test for B. ***P ≤ 0.001, ****P ≤ 0.0001, ns (non-significant). For A, scale bar = 10 µm. Figure S2. proNGF induces Src activation independently of TrkA phosphorylation in TNBC. Immunoblotting (A) and associated quantification (B) from MDA-MB-231 TrkA after proNGF stimulation (60 min) with Entrectinib (doses range from 1 to 500 nM) for TrkA, Src (and their phosphorylation form) and actin. (C) Immunoblotting for EphA2 from MDA-MB-231 TrkA treated with EphA2-targeting siRNA. Figure S3. proNGF induces Src activation regardless of p75NTR in TNBC. (A-F) Representative confocal images from MDA-MB-231 TrkA (A) and MDA-MB-231 HA-TrkA KD (D) of Src active form (Red) and EphA2 (Blue) staining with pSrc/EphA2 colocazation matrix with quantification associated for Src activity (B and E) and for EphA2/pSrc colocalization (C and F). Data in A-F are representative of 3 independents experiments on 30 cells/condition each. Data in B and E are presented with violin plots demonstrating the median (red bold line), quartiles (dots lines), variability and density. Data in C and E are represented by column bar graph with SD. Unpaired 2-tailed t test for B, C, E and F. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001. For C, scale bare = 15 µm. Figure S4. Real time monitoring of Src activation induced by proNGF in TNBC cells. (A) Scheme of FRET Src reporter from Ouyang et al. that is characterized by an ECFP/YPET pair, an SH2 domain, a Src substrate domain and a prenylation site (22). (B) Emission ratio images and profiles along the indicated white lines of the ECFP/YPet-based Src biosensor in response to proNGF stimulation with or without prior transfection of siEphA2 on MDA-MB-231 TrkA KD. (C-E) Cellular-scale activity of Src is quantified by photobleaching of the FRET reporter acceptor (AB-FRET) in MDA-MB-231 TrkA KD (C &amp; D) and MDA-MB-231 TrkA (E) after proNGF stimulation (60 min) with or without prior inhibitors treatments (siEphA2 and/or Entrectinib). Data in B-C are representative of 3 independents with 3 cells/conditions for A to C. Data in D and E are representative of 1 experiment with 3 cells/conditions. Data in B and D are represented by column bar graph with SD. One-way ANOVA followed by Dunnett’s test for B and D. **P ≤ 0.01, ***P ≤ 0.001, ns (non-significant). For A scale bar = 10 µm. Figure S5. ProNGF induced TNBC cells trans-BBB migration doesn't affect BBB integrity. (A) The permeability (Pe) coefficient of the endothelial layer is evaluated by the low-molecular-weight integrity marker Lucifer yellow (LY) on 3 µm pores insert after a 16 hours trans-BBB migration of MDA-MB-231 TrkA with or without proNGF abluminal stimulation and/or prior siEphA2 transfection. (B) Representative confocal images showing Claudin-5 or VE-Cadherin (white) and Hoechst (blue) signal on endothelium according to proNGF abluminal stimulation. Data in A to C are representative of 3 independents experiments with all condition realized in duplicate. Data in A are represented by column bar graph with SD. ns (non-significant). Figure S6. TrkA/EphA2 does not affect liver tissue invasion of TNBC. (A-B) Representative pictures (A) and associated quantification (B) from the monitoring of CellTracker™ stained MDA-MB-231 HA-TrkA KD mouse liver slices invasion. Data in B are presented as a column bar graph with SD. Unpaired 2-tailed t test for B. ns (non-significant). The dot line in A represent the limit of the Matrigel® plug. For A, scale bar = 100 µm. Figure S7. Combinatorial treatment with TrkA- and EphA2- targeting inhibitor does not affect tumoral development in TNBC cells maintained in live-like matrix. (A) Scanning electron microscopy picture of cut view from brain-like 3D ECM BIOMIMESYS®. (B) After 21 days of culture, the shape of fixed MDA-MB-21 WT/TrkA/TrkA KD are revealed with dapi (blue) and phalloidin (green) to allow cells and colonies segmentation after images acquisition with confocal microscope. (C-H) According to the TrkA overexpression status in MDA-MB-231, cell number (C &amp; E) and colonies volumes (F - H) are quantified after entrectinib and/or EphA2 targeting siRNA of 21 days culture in 3D liver-like ECM. Unpaired 2-tailed t test for C, D, F and G. Two-way ANOVA followed by Tukey’s test for E and GH. **P ≤ 0.01, ns (non-significant). For B, scale bar = 300 µm. Figure S8. Combinatorial treatment with TrkA- and EphA2- targeting inhibitor delays tumor growth and does not affect mice health condition. After tumor relapse from MDA-MB-231 HA-TrkA were xenografted in SCID mice, mice are treated with Entrectinib and/or EphA2-targeting siRNA 5 times every 2-3 days. (A) Tumor volume was measured every 2-3 days. Mice weight were follow-up for each treatment (B). Data in A are represented by connecting line graph with mean ± SEM. Data in B are presented as points graph with median. Two-way ANOVA followed by Dunnet’s test for A was performed, the control condition was compared with entrectinib (a), siEPhA2 (b) or combinatorial treatment (c). One-way ANOVA followed by Tukey’s test for B. *P ≤ 0.05, ***P ≤ 0.001, ns (non-significant).