Androgen-induced gene 1 correlates with extracellular vesicle-associated cholesterol metabolism and hepatic metastasis of pancreatic cancer

Figure 7 Androgen-induced gene 1 loss reduces small extracellular vesicle-mediated cholesterol release and alters macrophage polarization. A: Heatmap showing the relative expression of vesicle biogenesis genes between groups based on bulk RNA sequencing analysis; B: Representative transmission electron microscopy images of small extracellular vesicles (sEVs) isolated from mouse pancreatic cancer cells. Scale bars: 200 nm (left) and 100 nm (right). sEVs exhibit typical cup-shaped morphology; C: Western blot validation of sEV markers. Isolated vesicles expressed the characteristic sEV markers CD9 and TSG101; D: Nanoparticle tracking analysis of sEVs isolated from equal volumes of conditioned medium collected at comparable cell confluence. Particle diameter ranged from 50 nm to 200 nm. Total particle counts were lower in the androgen-induced gene 1 knockdown group than in the control group. Particle counts were normalized to cellular protein content; E: Measurement of cholesterol content in sEVs normalized to sEV protein levels; F: Quantitative real-time polymerase chain reaction analysis of bone marrow-derived macrophages cultured for 16 hours with conditioned medium from non-targeting control single-guide RNA or androgen-induced gene 1 knockdown cells. The control group refers to bone marrow-derived macrophages cultured in 1% fetal bovine serum medium without tumor-derived conditioned medium. All quantitative real-time polymerase chain reaction and cholesterol measurements were conducted using three independent biological replicates. Statistical significance was determined using a two-tailed unpaired t-test or one-way ANOVA followed by Tukey’s multiple comparisons test. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. sgControl: Non-targeting control single-guide RNA; AIG1-KD: Androgen-induced gene 1 knockdown; sEV: Small extracellular vesicle; IRF1: Interferon regulatory factor 1; CXCL10: C-X-C motif chemokine ligand 10; TNF: Tumor necrosis factor; IL: Interleukin; ARG1: Arginase 1; CM: Conditioned medium.