Extended Data Fig. 8: Fisetin suppresses EV uptake by neurons and PDAC progression.
a, Flow cytometry analysis showing differential neuronal uptake of Dil-labelled EVs derived from KPC cells after pretreatment with inhibitors compared with vehicle. Neurons were pretreated with the indicated inhibitors (10 µM) or vehicle, followed by incubation with Dil-labelled EVs (1 µg/105 cells) derived from KPC cells (n = 3 independent experiments). b, An example that shows the gating strategy for the analysis of Dil+ EV uptake. c and d, Fisetin pretreatment significantly suppressed neuronal uptake of GFP-labelled EVs (c, green) or Dil-labelled EVs (d, Orange) from KPC cells by quantification of the EV uptake area (n = 6 independent experiments). Neurons were pretreated with Fisetin (10 µM) or vehicle, followed by incubation with GFP-labelled EVs (c) or Dil-labelled EVs (d) (1 µg/105 cells) from KPC cells. Representative images of fluorescence and white light were acquired at the indicated times (n = 6 independent experiments). e–g, Fisetin treatment (10 µM) had no significant effect on the migration (e, n = 5 independent experiments), invasion (f, n = 5 independent experiments) and proliferation (g, n = 4 independent experiments) ability of KPC cells. h–j, Fisetin pretreatment (10 µM) did not alter EV uptake of fibroblasts (h, n = 5 independent experiments), macrophages (i, n = 5 independent experiments) and CD3+ lymphocytes (j, n = 5 independent experiments) isolated from orthotopic PDAC tumor tissue. Fibroblasts, macrophages and CD3+ lymphocytes were pretreated with Fisetin (10 µM) or vehicle, followed by incubation with Dil-labelled EVs (1 µg/105 cells) derived from KPC cells. EV uptake was assessed by flow cytometry (n = 5 independent experiments). k, Fisetin treatment (20 mg/kg, i.p.) significantly decreased Ki67 expression on stressed mouse allograft tumors (n = 5). l and m, Fisetin treatment (20 mg/kg, i.p.) significantly decreased stress-induced sympathetic nerve (l, TH+) and sensory nerve (m, Trpv1+) innervation in mouse allograft tumors (n = 5 mice). Scale bars, 200 μm (c, d, k–m). All bar data are the means ± s.e.m., with P by two-tailed Student’s t-test in a, c–m.
