Fig. 3: EVs from neuroinvasive cancer cells promote increased migration of mSCs and enriched with p75NTR.
a NTA analysis of P100 fractions from KPC and TPAC cells. Data represent the mean ± S.E.M. of ten replicates. b Western blot analysis of EV and non-EV markers of proteins from whole cell lysates (Cells), EV-depleted supernatant (S100) and EV pellets from pancreatic cell lines (P100). c Live-tracking analysis of PKH67-labelled KPC- and TPAC-EV (green fluorescent spots) uptake by hSCs. Representative image of green fluorescent spots accumulated in the cells (red border) at time points 0 and 19. Line graphs illustrated the mean total intensity of Alexa Fluor 488 over 19 h. Measurements and analysis were performed by the Operetta CLSTM high-content analysis system. d 3D migration assay of mSCs mixed with EVs from KPC or TPAC cells embedded in a single Matrigel drop and monitored for 4 days. Images (pseudo-colour) were taken by digital phase contrast of the Operetta system. Quantification was performed by measuring the area covered by cells (µm2). The data are mean ± S.E.M. from three independent experiments. e qRT-PCR analysis of the targets in KPC and TPAC cells as well as their respective EVs. The heatmap (left) depicting neurotrophin-related targets in KPC and TPAC cells was generated based on 2-ΔΔCT relative to KPC cells. The heatmap (right) showing the raw CT values of the targets in KPC- and TPAC-derived EVs. Log of the raw values was used for the generation of a heatmap using R software version 4.1.2. Data were obtained based on four independent replicates. f Western blot analysis of p75NTR and NGF expression levels in whole cell lysate (Cells), EV-depleted supernatant (Sup) and EVs (EV) from KPC and TPAC cells. Data were obtained based on four independent replicates and shown as mean ± S.E.M. Statistical difference of all data here was analysed by a two-tailed unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
