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Additional file 3: Supplementary Figure 1. Validation of the ectopic expression of CD44 and/or TrkA in COS-7 cells. The expression of all CD44 isoforms (A), CD44 variant 3 (B), CD44 variant 6 (C) and TrkA (hyaluronan [HA]; D) was evaluated by RT–qPCR (normalized using PUM-1). No expression of CD44, CD44 variants [3 or 6] or TrkA was detected in COS-7 cells (A-D) compared with that in cells transfected with the expression plasmid carrying each protein. Supplementary figure 2. TrkA does not interact with CD44s or CD44v6. Wild-type cos7 cells (A) or CD44S- (B) or CD44v6 (C) cells transfected with TrkA were treated with 100 ng/ml NGF (0, 5 or 30 min) and fixed. Quantification of the PLA results was performed using ImageJ software (30 randomly chosen fields per condition of three different experiments). Statistical analyses were performed using one-way ANOVA followed by Bonferroni’s posttest. The error bars represent the standard error of the mean (S.E.M.); ns, not significant. Supplementary Figure 3. CD44v3 and TrkA are recruited to the plasma membrane through NGF stimulation. CD44v3 and CD44v6 levels at the plasma membrane were assessed by flow cytometry (A-D). The plasma membrane and total TrkA and CD44v3 levels were also quantified by confocal microscopy (E-H). Statistical analyses were performed using one-way ANOVA followed by Bonferroni’s posttest. The error bars represent the standard error of the mean (S.E.M.); * p

ORAL; International audience; With nearly 2.3 million cases diagnosed worldwide each year and an estimated 685 000 deaths by 2020, breast cancer is the leading cause of cancer-related death in women. Brain metastases cause severe cognitive complications that severely impair quality of life. In TN breast cancer, the prognosis for brain metastases is particularly poor with a median survival of no more than 6 months. It is therefore crucial to know the molecular actors that promote the metastatic dissemination of triple negative breast cancer to the brain but also to prevent the proliferation of brain micrometastases that can cause fatal recurrences. In order to recapitulate several final stages of brain metastasis, we used both human Blood- Brain-Barrier in vitro model, human organotypic 3D extracellular in vitro matrix, mice brain slices organotypic ex vivo culture and in vivo mice xenograft. These models are coupled with single cell, 3D and real time imaging techniques, including FRET biosensing. This unique experimental approach allows us to study the involvement of signaling pathways in these different biological processes. Using this innovative method, we have identified that inhibition neurotrophins receptor leads to a decrease in the activity of the underlying signaling pathways and consequently to a decrease in the ability of breast cancer cells to pass through the BBB, to grow and to colonize the brain parenchyma.