Fig. 1

PaCa-EVs reprogram macrophages to M2-like tumor-associated macrophage (TAM) phenotype. a–c Representative confocal microscopy images of murine BMDMs treated with PKH67-labeled EVs (green), showing the uptake of EVs: BMDMs were stained with cell mask deep red (red), and nuclei were stained with Hoechst stain (blue). a A single stack of the 3D constructed image. b Birds-eye view. c Perpendicular view of macrophage. All images were captured after 24 h of EVs treatment at 63x magnification with pseudo color selection for each dye channel. PBS without any EVs mixed with PKH67 dye was used as a negative control. d Flow cytometry analysis shows time-dependent uptake of PKH67-labeled EVs by BMDMs at different time intervals (3, 6, 12, and 24 h post EV treatment). e Flow cytometry analysis shows time-dependent uptake of PKH67-labeled EVs by THP-1 macrophages at different time intervals (3, 6, 12, and 24 h post EV treatment). f Schematic of the strategy used to evaluate in vivo EV uptake by murine peritoneal cavity macrophages (PCMs). g Representative flowcytometry histogram and data showing uptake of PKH-67 labeled EVs by murine peritoneal cavity macrophages (PCMs) 72 h following intraperitoneal (I.P.) injection of PKH-67 labeled EVs. h Western blots of EVs, showing increased expression of CD9 and CD81 in hTERT-HPNE, PANC-1 and PPCL-68 EV cargo. i Schematic overview of EV treatment to BMDMs for M1 and M2 polarization analysis. j Representative flow cytometry plots. k, l Bar graphs showing CD206 and CD86 expression on EV (10 μg/mL) treated BMDMs after 72 h of treatment. m Concentration of anti-inflammatory (free active TGF-β1, IL-10, CCL22 (MDC), CCL17 (TARC), GM-CSF, and IL-6). n Pro-inflammatory cytokines (TNF-α, IL-12p40, IL-1β, IL-18, and IL-23) in the cellular supernatant of EVs treated BMDMs. o Schematic representation of targeted metabolomics experiment. p PCA analysis of metabolomic profiles of EV treated BMDMs. q Venn-diagram displaying the number of differentially altered metabolites in BMDMs after PANC-1 and PPCL-68 EV treatment as compared to the hTERT-HPNE EV treatment (FDR-adj. p ≤ 0.05 or q ≤ 0.05). r Heatmap of significantly altered metabolites of hTERT-HPNE, PANC-1, and PPCL-68 EVs treated BMDMs (q ≤ 0.05; n = 4). s Bar graphs highlighting the differences in intracellular abundance of selected metabolites of arginine and proline metabolic pathway in BMDMs after 72 h of EV treatment. t Arginase 1 (ARG1) gene expression in EV treated BMDMs. All data are presented as Mean ± SE, where n ≥ 4 per condition. ns = p > 0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, and **** = p ≤ 0.0001 by one-way ANOVA, two-way ANOVA (EVs uptake experiment) and unpaired t-test (metabolomics data)