Exosomal ANGPTL1 attenuates colorectal cancer liver metastasis by regulating Kupffer cell secretion pattern and impeding MMP9 induced vascular leakiness

Figures

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  Fig. 1. ANGPTL1 level was downregulated in CRC derived exosomes. a Transmission electron microscopy analysis of TDEs and NDEs. Scale bar, 100 nm. b The number of particles and median diameter was determined by dynamic light scattering (DLS) analysis. Data were shown as mean ± standard deviation of three technical replicates. c Western blot (WB) analysis of exosome biomarkers (Alix, TSG101, and CD9). d, e WB analysis of the ANGPTL1 in tissue-derived exosomes, n = 8, P = 0.03. ANGPTL1 level was downregulated in exosomes derived from CRC tumors than paired normal tissues. Data were analyzed using a ratio paired t-test

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  Fig. 2. Exosomal ANGPTL1 attenuated liver metastasis and impeded vascular leakiness in the liver PMN. a Experimental scheme of exosome education and intrasplenic injection mouse model. b Liver metastasis was monitored by the IVIS Lumina imaging system. Represent images (left) and the fluorescence intensity (right; n = 3) of the mice livers at the fourth-week post tumor injection are shown. c H&E examination (left, arrows indicate tumors) and lesion areas (right; n = 5) of mice livers were used for metastatic burden assessment. Scale bar, 200 um. d In vivo vascular permeability measured by the appearance FITC-Dextran (green) injected by tail vein (left; n = 3). Data were presented as FITC-dextran /DAPI ratio (right). Scale bar, 20 um. Error bar indicates SEM of the mean. Data in panel b and d were analyzed using t-test, in panel c were analyzed using the Mann-Whitney test. *P < 0.05, **P < 0.01, ****P < 0.0001

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  Fig. 3. Exosomal ANGPTL1 was mainly taken up by KCs and regulated KCs secretion pattern. a Immunofluorescence analysis of PKH67-labeled exosome uptaken. Arrows indicate PKH67-labeled exosomes (green) in F4/80 positive KCs (red). b Mice were injected with liposome PBS (up) or liposome clodronate (low) for 2 days. Arrows indicate PKH67-labeled exosomes in F4/80+ KCs (yellow). c In vitro exosome uptake by ImKC at 4 h was observed on confocal microscopy. Phase contrast (left) and merged (right) pictures are shown. d GO enrichment analysis of changing genes between Ctrl-Exo education and ANGPTL1-Exo education group. e List of the 20 most significantly downregulated genes is shown (Q < 0.001). f qRT-PCR verification of the mRNA expression of Mmp9, Lif, Cxcl2, Csf3, Il1a, Ccl5. Scale bar represents 20um. Data are presented as the mean ± SEM of three independent experiments, and analyzed using t-test. *P < 0.05, **P < 0.01, ****P < 0.0001

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  Fig. 4. Exosomal ANGPTL1 dependent MMP9 decrease normalized vascular leakiness induced by CRC derived exosomes. a ELISA analysis of MMP9 level in the culture medium (CM) from ImKC educated by PBS, Ctrl-Exo or ANGPTL1-Exo for 24 h. b, c Permeability of the HUVEC monolayers to FITC–Dextran (70 kDa) after exposure to CM from ImKC educated by PBS, Ctrl-Exo, ANGPTL1-Exo (b), or exposure to CM from ImKC educated by Ctrl-Exo followed by transfection of control siRNA (scramble) or MMP9 siRNA or by ANGPTL1-Exo with extra rmMMP9 (100 ng/mL) (c). The fluorescence in the bottom well was detected every 30 min. d rmMMP9 (50 μg/kg) was injected through the tail vein into the mice educated by ANGPTL1-Exo for 1 h. In vivo vascular permeability was then determined (n = 3). Representative images are shown (left). Data were presented as FITC-dextran /DAPI ratio (right). e, f ZO-1 and Claudin-5 expression in HUVECs treated by ImKC CM were detected through qRT-PCR (e) and WB (f). g Immunofluorescence staining of ZO-1 in HUVECs monolayers treated by ImKC CM. Data are presented as the mean ± SEM of three independent experiments. Data in panel a, d and e were analyzed using t-test, in panel b and c were analyzed…

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  Fig. 5. Exosomal ANGPTL1 decrease MMP9 production in KCs by inhibiting the JAK2-STAT3 signaling pathway. a KEGG pathway enrichment analysis of the changing genes between ImKC educated by Ctrl-Exo and ANGPTL1-Exo. The top 11 significant pathways were shown. b WB validation of the JAK2-STAT3 pathway in IMK educated by PBS, Ctrl-Exo and ANGPTL1-Exo at different times. c WB analysis of STAT3 phosphorylation in liver tissues from mice educated by PBS, Ctrl-Exo and ANGPTL1-Exo for 3 weeks (right). The number under the first band indicates the value of P-STAT3/STAT3 and the data were analyzed using t-test (left). d WB validation of STAT3 activation induced by IL-6 (left) and qRT-PCR analysis of MMP9 expression (right) in ImKC treated by ANGPTL1-Exo with or without IL-6 for 24 h. e Schematic diagram of the role of exosomal ANGPTL1 in CRC liver metastasis. Data are presented as the mean ± SEM of three independent experiments, and analyzed using t-test. *P < 0.05