Fig. 4: Amyloid β is the driver of CAF-induced t-NETosis.

A Quantification of the relative NET coverage of neutrophils that were added to lung CAFs treated with or without FB CMed, CAF CMed or PMA for 3 h. B Quantification of the relative NET coverage of neutrophils treated with lung CAF CMed or CAF CMed-derived microvesicles (MV) or CMed depleted of MV. C Quantification of the relative NET coverage of neutrophils treated with lung CAF CMed or the metabolite or protein fractions of the CAF CMed. D Differentially secreted proteins in pancreatic FB and CAF CMed analyzed by mass spectrometry. The relative intensity of each protein is indicated by the colored bar under the heatmap. E Spectral counts for NET-related factors secreted by pancreatic FBs and CAFs. F Levels of Amyloid β in basal media and pancreatic CAF CMed generated in the presence or absence of an inhibitor of amyloid-beta secretion (BACEi). G Confocal microscopy of amyloid precursor protein (APP) and Podoplanin (PDPN) expressed by CAFs in pancreatic tumors. Insets depict diffuse vs. aggregated patterns of APP/Amyloid β distribution. H Quantification of the relative NET coverage of neutrophils treated with pancreatic and lung CAF CMed generated with or without 24-h pre-treatment of the CAFs with a β-secretase 1 and 2 (BACE1-2) inhibitor. I Quantification of the relative NET coverage of bone marrow neutrophils taken from wild-type mice intravenously infused with pancreatic CAF CMed taken from cells treated with or without BACE1-2 inhibitor for 24 h or recombinant Amyloid β. J Schematic of BACEi treatment regime of skin tumor-bearing mice and relative endpoint volume of skin tumors (calculated relative to the volume of each tumor at the start of treatment—indicated by dotted line on the graph) on mice after treatment with vehicle or BACEi (Z-VLL-CHO). K Growth of orthotopically implanted B16.F10 tumor cells with vehicle, CAF CMed or recombinant Amyloid β treatment. Quantification of the relative NET coverage after 3-h treatment with pancreatic CAF CMed with or without L CD11b or M TLR2 blocking antibodies. Data are mean ±  SEM; **p < 0.01 and ***p < 0.001 using (A and H) one-way ANOVA with a Dunnett post hoc test, B–C, I and L–M one-way ANOVA with a Tukey post hoc test and J a Mann–Whitney test. Assays were performed on A n = 6 (Untreated and CAF CMed treated) n = 3 (PMA and FB CMed treated), B n = 7, C n = 5, (E) n = 3, F n = 3–6, G Representative images of n = 3 tumors, H n = 6 (in triplicate), I n = 7 (in duplicate or triplicate), J n = 9 (Vehicle) and n = 3 (BACEi) male and female, 8–24-wk-old mice, K n = 6 8-wk-old female C57BL/6 mice and L–M n = 3 (in triplicate) independent experiments. Scale bars are 50 µm.