Periprostatic adipose tissue-derived extracellular vesicles modulate prostate cancer cell behaviour in vitro according to tumour grade

Figures

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  Fig. 1. Characteristics of EVs derived from Low- and High-risk human PPAT tissues. A Representative TEM images of EVs isolated from human High-PPAT tissue. B Representative Western blot showing the EV markers TSG101, CD9 and α-tubulin in EVs derived from low-risk (Low-PPAT EVs) and high-risk (High-PPAT EVs) PPAT samples, and from total adipocyte cell extract. C Western blot analysis showing the absence of apolipoprotein A1 (ApoA1) in EVs derived from Low- and High-PPAT tissues. Total human serum protein was used as a positive control. D Particle concentration (normalized per gram of adipose tissue) and EVs size distribution from low- and high- risk PPAT samples (n = 4, independent samples). Each point represent the mean of the technical measurements. E Mean particle diameter of Low-PPAT EVs (162 nm) and High-PPAT EVs (165 nm). F Total particle concentration (normalized per gram of adipose tissue) of Low- and High-PPAT EVs (n = 4; Data shown as mean ± SD). (G) Median Zeta potential of Low- and High-PPAT EVs (n = 4; Data shown as median with rage). p-value by Mann–Whitney U test (p ≤ 0.05). Uncropped Western blot images are provided in Supplementary Fig. 3

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  Fig. 2. Effects of low- and high-risk PPAT-derived EVs on proliferation and apoptosis in PCa cells. A Cell proliferation in 22Rv1 and LNCaP cells after 24 h stimulation with EVs from low- and high-risk human PPAT samples (N = 4). B Relative expression of CDKN1A after 24 h stimulation with PPAT-EVs (N = 5–6). C Relative expression of P53 after 24 h stimulation with PPAT-derived EVs (N = 6). D-E Representative flow cytometry dot plots showing Annexin V and Zombie NIR staining in 22Rv1 (D) and LNCaP (E). Cells were stimulated with PPAT-derived EVs or 0.5 μM stautosporine 98% (STS) as positive control. F-G Quantification of total, early and late apoptosis in 22Rv1 (F) and LNCaP (G) cells (N = 4). H-J Relative gene expression of BAX, BCL-2, and BAX/BCL-2 ratio in 22Rv1 (H) and LNCaP (J) cells. Non EVs-treated cells were included as controls. Data are shown as mean ± SD. * indicates significant differences between groups using one-way ANOVA followed by Tuckey’s post hoc test (*p < 0.05; **p < 0.01)

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  Fig. 3. Evaluation of migratory and invasive capacity of low- and high-risk PPAT-derived EVs in PCa cells. A Scratch wound assay in 22Rv1 following 24 and 48 h PPAT-derived EVs stimulus. Quantification of wound closure is shown in the graph plot (N = 3/4 independent experiments). B Representative images and quantification of transwell migration and invasion assays in 22Rv1 cells, expressed as the number of migrated or invaded cells per field. C Representative images and quantification of transwell migration and invasion assays in LNCaP cells, expressed as the number of migrated or invaded cells per field. D-E Relative gene expression of TIMP2, and TWIST1 in 22Rv1 (D) and LNCaP (E) cells following stimulation with PPAT-derived EVs. Non EVs-treated cells were included as controls. Data are shown as media ± SD. * indicates significant differences between groups using one-way ANOVA followed by Tukey’s multiple comparison test post hoc test (*p < 0.05). Scale bar represent 200 μM

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  Fig. 4. PI3K/AKT and Wnt/β-catenin signalling pathway activation in PCa cell lines induced by High- and Low-PPAT EVs. A Western blot analysis of phosphorylated AKT (p-AKT), total AKT, β-catenin, phosphorylated GSK3β (p-GSK3β), and total GSK3β in 22Rv1 cells stimulated with Low- and High-PPAT EVs. B Western blot analysis of phosphorylated AKT (p-AKT), total AKT, β-catenin, phosphorylated GSK3β (p-GSK3β), and total GSK3β in LNCaP cells stimulated with Low- and High-PPAT EVs. Non EVs-treated cells were included as controls. A representative western blot is presented (left). The membranes were tested with the corresponding antibodies. Protein levels were normalized to β-actin and expressed relative to control cells (Non EVs). Full-length blot and gel are presented in Supplementary Fig. 5. Data are shown as mean ± SD. * indicates significant differences between groups using one-way ANOVA followed by Tukey’s multiple comparison test post hoc test (*p < 0.05)

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  Fig. 5. Low- and High-PPAT-derived EVs differently influence inflammation and macrophage polarization in THP-1 derived macrophages. A Relative gene expression of inflammatory cytokines (IL-1β, MCP-1, TNF-α, and IL-10) in PMA-differentiated THP-1 cells after 24 h of treatment with Low- and High-PPAT EVs; (B) Relative gene expression of associate-macrophage M1 (CD80, CD86) and M2 (CD206, CD163) markers in PMA-differentiated THP-1 cells after 24 h of treatment with Low- and High-PPAT EVs; (C) M1-to-M2 ratio. In all cases, non EVs-treated cells were included as controls. Data are shown as mean ± SD (N = 4). * indicates significant differences between groups using one-way ANOVA followed by Tukey’s multiple comparison post hoc test (*p < 0.05; **p < 0.01). Tendencies (0.05 < p ≥ 0.1) are indicates with the p value

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  Fig. 6. Impact of Low- and High-PPAT EVs on angiogenesis. A Representative pictures of tube formation capability detected in endothelial cells (HUVEC) treated with Low- and High-PPAT EVs for 6 h. Scale bar represent 200 μM. B Quantification of the number of junctions, segments, nodes, total tube length and mesh area of the tube formation assay. C Relative gene expression analysis of VEGFA, IL-6 and IL8 in HUVEC cells treated with Low- and High-PPAT EVs for 6 h. Non EVs-treated cells were included as controls. Data were shown as mean ± SD. * indicates significant differences between groups using one-way ANOVA followed by Tukey’s multiple comparison post hoc test (*p < 0.05; **p < 0.01). Tendencies (0.05 < p ≥ 0.1) are indicates with the p value