Patients and specimens
The clinical CRC and paired normal tissues were obtained from CRC patients (n = 8) in the Second Affiliated Hospital of Zhejiang University School of Medicine. This project was approved by the ethical committee of the Second Affiliated Hospital of Zhejiang University School of Medicine. Informed consent was obtained from all patients.
The human colorectal cancer cell line SW620 and Human umbilical vein endothelial cell (HUVEC) were obtained from the American Type Culture Collection (Rockville, MD, USA). The ANGPTL1 stably overexpressed cell line (SW620-ANGPTL1) and its control cell line (SW620‐Ctrl) were established in the previous study . The Immortalized Mouse Kupffer Cell line (ImKC) was purchased from MilliporeSigma (USA). SW620 cells and HUVECs were cultured in RMPI-1640 medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Gibco, Brazil). ImKC were cultured in DMEM medium (Gibco, Carlsbad, CA) supplemented with 10% FBS (Gibco, Brazil).
Exosome collection and characterization
Exosomes were collected by sequential ultracentrifugation. CRC cells were cultured in the exosome-depleted (160,000 × g, 16 hours) complete medium for 72 hours. The supernatants were collected and centrifugate at 500 × g for 10 min to remove cell contamination, then at 3,000 × g for 20 min to remove apoptotic bodies and large cell debris, followed by centrifugation at 12,000 × g for 20 min to remove large microvesicles. Next, exosomes were collected by 100,000 × g centrifugation for 70 min (Beckman Ti70). The exosome pellet was resuspended in 20 mL of phosphate-buﬀered saline (PBS) and collected by 100,000 × g ultracentrifugation for 70 min (Beckman Ti70). Exosome preparation was verified by Transmission electron microscopy (TEM). Exosome size was measured by dynamic light scattering (DLS) analysis using Zetasizer Nano ZSE (Malvern Panalytical, Shanghai, China).
For tissue-derived exosome collection, the CRC tumors and paired normal tissues were cut into 1mm × 1mm pieces and cultured in 15mL of FBS-free RMPI-1640 medium for 24 hours. Then the supernatant was harvested for further isolation of exosomes.
Exosome treatment and labeling
Purified exosomes were injected into the mouse retro-orbital venous sinus in a total volume of 100µL PBS. For in vivo education experiments, mice received 5µg of exosomes every other day for 21 days. Retro-orbital injection of PBS was used in control groups. For in vitro education, exosomes (10µg/mL) were added into the culture medium (CM) of ImKC for 3, 6, 12, 24 hours. For exosome-tracking experiments, exosomes were labeled using PKH67 membrane dye (Sigma, Shanghai, China), followed by 100,000 × g ultracentrifugation for 70min, and labeled exosomes were resuspended in PBS. In experiments involving the evaluation of exosome incorporation, labeled exosomes were injected retro-orbitally into the mice or added into the CM of ImKC 24 hours before immunofluorescence analysis for exosome cells.
To analyze the role of exosomal ANGPTL1 in CRC liver metastasis, we pre-educated the 6–8-week-old scid-beige mice (SLAC Laboratory Animal Co. Ltd., Shanghai, China) retro-orbitally with exosomes for 21 days (5ug/100ul, every other day). Then, 2 × 106 SW620 cells were injected into the mice spleen as described in the previous study [29, 30]. A small animal IVIS Lumina Imaging System (Caliper Life Sciences, Hopkinton, MA) was used for liver lesion monitor. All mice were sacrificed at one month, and the livers were harvested for Hematoxylin and eosin (H&E) staining and analysis of metastases. For liver macrophage elimination, liposome clodronate was injected via the tail vein in a dose of 0.2 mL/20-25g as a tool to suppress macrophage function by inducing apoptosis . Liposomes containing PBS were injected as a control. All animal experiments were approved by the Institutional Ethics Committee of the Second Affiliated Hospital Zhejiang University School of Medicine.
In vivo vascular permeability assay
After pre-education with exosomes for 21 days, mice were injected with the recombinant mouse MMP9 (rmMMP9; 50 ug/kg body weight; R&D) intravenously. The rmMMP9 was preactivated using 1 mM aminophenylmercuricacetate (AMPA, Sigma, Shanghai, China) for 2 hours at 37°C. One hour after rmMMP9 injection, FITC-Dextran (~70KD; 100 mg/kg; Sigma, Shanghai, China) was injected through the tail vein. After one hour, mice were sacrificed and perfused with PBS to remove the excess dye. Liver tissues were embedded in Tissue-Tek O.C.T. Compound (Sakura; Torrance, CA, USA) to make frozen blocks for sectioning and immunofluorescent staining. Stained sections were viewed and photographed with a fluorescence microscope. The intensity of fluorescence was measured using ImageJ software (ImageJ software v1.8.0)
HUVECs (2 × 104) were seeded on transwell filters (0.4μm pore size; Corning, Shanghai, China). After reaching confluence, HUVECs were treated with CM from ImKC educated by PBS, Ctrl-Exo, or ANGPTL1-Exo (with or without rmMMP9, 100ng/mL) for 48 hours. Then, FITC-Dextran (1mg/mL) was added to the top well. 40μL medium in the bottom well was taken for fluorescence measurement every 30 minutes using a SpectraMax microplate reader (SpectraMax i3, Molecular Devices, USA) at 488nm excitation and 520nm emission. The fluorescence intensity represents the passage of FITC-Dextran, which represents the the HUVECs layer permeability.
Small interfering (siRNA) transfection
Mouse MMP9 siRNA was purchased from Tsingke biological technology (Hangzhou, China). The MMP9 siRNA and scramble siRNA were added into the ImKC 24 hours after exosome pre-education. The siRNA transfection was performed with Lipofectamine 3000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The siRNA sequences were as follows: MMP9 siRNA, 5′‐CAAGACAAAGCCUAUUUCUTT‐3′ (sense), 5′‐AGAAAUAGGCUUUGUCUUGTT‐3′ (antisense).
Total RNAs were isolated from cells and mouse livers with TRIzol reagent (Invitrogen, USA) and evaluated using NanoDrop 2000 spectrophotometer (Thermo Scientific, Pittsburgh, PA, USA). qRT-PCR was conducted using a standard SYBR-Green PCR kit protocol (YEASEN, Shanghai, China) with a 7500 Fast Real-Time PCR System (Life Technologies, Shanghai, China). The primers were synthesized by Tsingke biological technology (Hangzhou, China). The sequences of all primers are listed in Table 1.
Total RNA of ImKC cells educated by PBS or exosomes for 24 hours in vitro was extracted and subjected to BGI (Huada Genomics Institute Co. Ltd, Guangzhou, China) for RNA sequencing. The DEGseq R package was used to analyze differentially expressed genes based on the conditions of a fold change (FC) ≥ 1 and Q-values ≤ 0.001. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to explore the significant pathways. All mRNA sequencing data were uploaded to NCBI (accession number PRJNA656088).
Western blot and ELISA
The tissue lysates were prepared using RIPA lysis buffer, while the cell and exosome lysates were prepared using 3× Loading Buﬀer. All samples were performed as depicted previously . Exosome proteins were detected with the antibodies as follows: Alix (1:1000, Cell Signaling Technology, Beverly, MA, USA), Calnexin (1:1000, Cell Signaling Technology, Beverly, MA, USA), CD9 (1:500, Santa Cruz Biotechnology, Dallas, Texas, USA), ANGPTL1 (1:500, Santa Cruz Biotechnology, Dallas, Texas, USA), TSG101 (1:1000, Abcam, Shanghai, China), ANGPTL1 (1:1000, R&D Systems, Minneapolis, MN, USA). ImKC proteins were detected with the antibodies as follows: STAT3 (1:1000, Cell Signaling Technology, Beverly, MA, USA), phospho-STAT3 (Y705) (1:1000, Cell Signaling Technology, Beverly, MA, USA), JAK2 (1:1000, Cell Signaling Technology, Beverly, MA, USA), phospho-JAK2 (Y1008) (1:1000, Cell Signaling Technology, Beverly, MA, USA), GAPDH (1: 2500, Huabio, Hangzhou, China). HUVECs proteins were detected using the antibodies listed as follows: Claudin-5 (1:1000, Abcam, Shanghai, China), ZO-1 (1:1000, Proteintech Group, Rosemont, USA). All samples were further incubated with the peroxidase-conjugated secondary antibody (1:5000, Huabio, Hangzhou, China). Bands were visualized using enhanced chemiluminescence reagents (YEASEN, Shanghai, China) and scanned via a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Quantitative analysis of WB was performed with ImageJ software (ImageJ v1.8.0).
In order to detect the protein level in the CM of ImKC through ELISA, cells were cultured in the presence of exosomes (10 µg/mL) for 24 hours before supernatants were collected, and the MMP9 level was measured by mouse MMP9 ELISA kit (R&D Systems, Minneapolis, MN, USA).
For histological analysis, tissues were dissected and fixed in a mix of 2% Paraformaldehyde (PFA) and 20% sucrose solution overnight, followed by embedding and section. Then 6 µm O.C.T. tissue cryosections were stained with antibodies against F4/80 (1:100, eBioscience, Shanghai, China), αSMA (1:500, Abcam, Shanghai, China), CD31 (1:200, Abcam, Shanghai, China), MMP9 (1:200, Abcam, Shanghai, China). Secondary antibodies conjugated to Alexa Fluor 488, 555, or 594 were used (1:500, Abcam, Shanghai, China). Fluorescent images were obtained using a Zeiss LSM 710 laser confocal microscope (Carl Zeiss, Germany) and analyzed using Zen software (ZEN 3.0).
For ZO-1 detection in HUVECs, cell immunofluorescence was conducted as described previously . Cells were incubated with primary antibody ZO-1 (1:400, Proteintech Group, Rosemont, USA) at 4°C overnight. Nuclei were stained with DAPI for 5 min. Cells were viewed under the Zeiss LSM 710 laser confocal microscope (Carl Zeiss, Germany) and analyzed using Zen software (ZEN 3.0).
All data are presented as the mean ± standard error of the mean (SEM) or mean ± standard deviation of the mean (SD). Statistical analyses were performed with Student’s t-test for comparisons between two groups and with ANOVA for more than two groups using Prism 8.0. P < 0.05 was considered statistically significant.