EMT‐cancer cells‐derived exosomal miR‐27b‐3p promotes circulating tumour cells‐mediated metastasis by modulating vascular permeability in colorectal cancer

Abstract Background Metastasis is the main cause of death in colorectal cancer (CRC). Circulating tumour cells (CTCs) are regarded as the precursor cells of metastasis. The CTCs, which underwent epithelial‐mesenchymal transition (EMT), are associated with metastasis and responsible for poor prognosis. EMT cancer cells modulate endothelial permeability in the invasive front and facilitate cancer cell intravasation, resulting in CTCs‐mediated distant metastasis. Exosomes derived from cancer cells are key mediators of cancer‐host intercommunication. However, the mechanism by which EMT‐tumour cells‐derived exosomes modulate vascular permeability and promote CTCs generation has remained unclear. Methods Exosomes isolation and purification were conducted by ultra‐centrifugation. Exosomal miRNA was identified by sequencing followed by quantitative PCR. In vitro co‐culture assay experiments were conducted to evaluate the effect of exosomal miR‐27b‐3p on the permeability of blood vessel endothelium. Dual‐luciferase reporter assay, chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP) were performed to investigate the underlying mechanism by which miR‐27b‐3p is packaged into exosomes. A mouse model was established to determine the role of exosomal miR‐27b‐3p in blood vessel permeability modulation in vivo. Results We found that EMT‐CRC cells attenuate the blood vessel barrier by transferring miR‐27b‐3p to human umbilical vein endothelial cells (HUVECs) in exosomes. Mechanically, miR‐27b‐3p atteuated the expression of vascular endothelial cadherin (VE‐Cad) and p120 at the post‐transcriptional level by binding to 3′‐untranslated region of VE‐Cad and p120 directly. The packaging of miR‐27b‐3p into exosomes was induced by heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which activated by STAT3. Clinically, miR‐27b‐3p up‐regulated in CRC tissues. Plasma exosomal miR‐27b‐3p was positively correlated with malignant progression and CTC count in CRC patients. Our study reveals a novel mechanism by which EMT‐CRC cells promote metastasis, increasing blood vessel permeability and facilitating the generation of CTCs. Conclusion Exosomal miR‐27b‐3p secreted by EMT‐CRC cells increases blood vessel permeability and facilitates the generation of CTCs. Exosomal miR‐27b‐3p may become a promising biomarker for CRC metastasis.


The sequences of the primers for qRT-PCR
exosomes were resuspended in 1 mL Diluent C mixed with 4 μLPKH67 and then co-incubated for 5 mins at room temperature. 2 ml 0.5% BSA/PBS was added to bind excess PKH67. Furthermore, the labeled exosomes were washed with PBS at 100,000 g for 1 hour, and the collecting exosomes were applied for uptake experiment.. For cell treatment, total 2 µg of exosomes were added into 2 × 105 recipient cells, incubated for 72 h.

Animal experiment
All procedures for animal experiments were carried out according to the guidelines Hubei Key Laboratory of Tumor Biological Behaviors and approved by the Institutional Animal Care and Ethical Committee in Zhong nan Hospital of Wuhan University.
For orthotropic metastasis assay, 5 × 106 growing CRC cells were subcutaneously injected on the right flank region of 4-6 weeks old female BALB/c nude mice. Once the xenografts established (within 14 days), the xenograft tumor were dissect under asepsis, and separated the fibrotic tissue from tumor, excised and minced tumor tissue into 1 mm3 pieces. Under anesthetization, both the cecum and ascending colon of the nude mice were exteriorized and the 1 mm3 pieces of xenograft were implanted sub-serosally. After suture, the bowel was returned into abdomen. For in vivo permeability assay, rhodamine-dextran(100mg/kg) was injected into the mice via tail vein. To detect the circulating tumor cells and exosomal miR-27b-3p in the blood of tumor-bearing nude mice, 1ml blood was collected in tubes with EDTA by cardiac puncture and centrifugation to isolate cells and plasma. The cells were resuspended by PBS for further CTC detection. Exosomes from plasma were further isolated by using exoRNeasy Serum/Plasma MaxiKits (QIAGEN, Germany) with the manufacturer's protocol. The RNA was extracted from exosome by using Trizol reagent and qRT-PCR were applied to quantify the miR-27b-3p level in exosome. The paraffin-embedded lungs and the livers were serial sectioned and stained with hematoxylin-eosin (HE), under microscope, metastatic nodules were screened to evaluate metastasis.
Tube formation assay, angiogenesis assay, and endothelial permeability assay.
For tube formation analysis, Matrigel was placed in a German ibidi plate (approximately 10 microliters per well) and incubated at 37 ° C for 30 minutes to polymerize Matrigel. Treated HUVECs were seeded into Matrigel-coated wells. The ibidi plates were then incubated at 37 ° C. in a humidified atmosphere of 5% CO 2. Tube formation was observed with a microscope at 12 hours. The angiogenic capacity was determined by measuring the number of tubes. The experiment was repeated 3 times. For 3D Vascular Sprouting Assays, treated HUVEC cells were suspended in M199 medium containing 0.25% (w / v) methylcellulose and seeded in non-adhesive round bottom 96-well plates (Greiner, Frickenhausen, Germany) in. All suspended cells were concentrated on the non-adherent bottom surface to form a single spheroid (in vitro angiogenesis: 500 cells per spheroid). All suspended cells aggregate to form a single spheroid (in vitro angiogenesis: 500 cells per spheroid). The spheroids were cultured for 24 hours and used for budding experiments. Matrigel Matrigel was placed in a German ibidi plate (approximately 10 microliters per well) and incubated at 37 ° C for 30 minutes to polymerize Matrigel. Individual HUVEC spheroids were individually seeded into matrigel-coated wells. The ibidi plates were then incubated at 37 ° C. in a humidified atmosphere of 5% CO 2. After 5 days, the vascular structure was imaged. Count at least 50 ellipsoids in each experiment and repeat the experiment 3 times. For Permeability assay, A single layer of treated HUVEC was grown on a 0.3 cm2 polyethylene terephthalate ultrafilter (pore size 0.4 μm; BD Biosciences; Franklin Lakes, NJ).
EVOM2 voltmeter 17 (World Precision Instruments; Sarasota, FL) was used to calculate the unit area resistance. The permeability of the treated HUVEC monolayer membrane (average pore size 0.4 μm; BD Biosciences) was evaluated by the transfer of rhodamine B isothiocyanate-dextran (average MW ~ 70,000; Sigma). Specifically, rhodamine-dextran was added to the top well at a concentration of 20 mg / ml, excited at 544 nm and emitted at 590 nm using a SpectraMax