Upregulation of E-cadherin in bronchoalveolar lavage fluid-derived exosomes in patients with lung cancer

Patients

We collected BALF samples from patients with lung cancer from November 2018 to December 2018. The Human and Animal Ethics Review Committee of the First Hospital of Jilin University approved this study. All BALF samples were collected during diagnostic bronchoscopy procedures.

Bronchoalveolar lavage fluid (BALF)

BALF was performed using a flexible bronchofiberscope after administration of local anesthesia with lidocaine. We instilled 50 mL fractions of sterile saline into the right middle lobe or the left lung segment. BALF was retrieved using gentle syringe suctioning and transferred into sterile containers prior to storage at 4°C.

Exosome isolation

The BALF was centrifuged using a Beckman Coulter Avanti-J-26XPI centrifuge at 3000 rpm at 4°C for five minutes to remove detached cells. The supernatant was recovered and sequentially centrifuged as follows: 500 rcf/10 minutes, 12 000 rcf/20 minutes and 100 000 rcf/90 minutes (× 2).

ELISA

BALF and exosomes solution were added to a Human E-Cadherin SimpleStep ELISA Kit (ab233611), according to the manufacturer's instructions. The concentrations of E-cadherin from two types of BALF were measured in duplicate and interpolated from the E-cadherin standard curves and corrected for sample dilution. The interpolated dilution factor corrected values were then plotted (mean ± SD, n = 3).

Transmission electron microscopy

Using a 100 mesh sample-loaded copper mesh, 20 μL of the freshly obtained exosome solution was diluted with the same volume of PBS, dropped onto the copper mesh, left at room temperature for one minute, and then air-dried at room temperature. Then, we used a dropper to take 20 μL of 3% (w/v) sodium phosphotungstate solution and this solution was dropped onto the copper grid for one minute and excess liquid gently absorbed using filter paper. After we placed the copper mesh under a transmission electron microscope for evacuation, we observed and photographed the morphology of the exosomes.

Protein concentration determination

Exosomes were lysed by repeated freezing and thawing in liquid nitrogen on at least three occasions and the lysate then centrifuged at 13 000 rpm using Thermo Scientific Sorvall Legend Micro17/21. The supernatant was added to a BCA protein Assay Kit (P0012), according to the manufacturer's instructions.

Western blot (WB)

Exosomes were lysed with buffer containing 260 mM Tris–HCl, pH 6.8, 0.8% SDS (w/v), and 40% glycerol, supplemented with protease inhibitors: 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 mM phenylmethyl sulfonyl. Equal amounts of protein (30 μg) were resolved by SDS-PAGE for blotting using anti-CD63 (1:200, santa), CD81 (1:1000, santa), TSG101 (1:1000, proteintech), and E-cadherin (1:1000, proteintech). HRP-conjugated anti-rabbit IgG antibody were used as secondary antibodies (1:5000, proteintech).

Migration assay

A total of 500 000 cells were seeded per well in 24 well plates in triplicate. After 24 hours, the medium was replaced by medium without FBS and maintained overnight. A wound was then made in the monolayer with a pipette tip, and the medium was replaced with serum-free media and exosomes from each group. Pictures of the wounds were taken at 0 and 24 hours using an Olympus IMT-2 microscope. Wound closure was measured using ImageJ software. Results represent the migrated distance between 0 and 24 hours, expressed as a percentage relative to A549.

Invasion assay

A total of 8 μm pore inserts were covered with 50 μL Matrigel (356 231, Corning) at a final concentration of 1.25 mg/mL. Matrigel was incubated for one hour at 37°C. Once the Matrigel was polymerized over the Matrigel layer, 30 000 cells with exosomes from each group were seeded in 100 μL medium without FBS. Complete medium (supplemented with FBS, which stimulates the cells to cross the Matrigel layer) was added to the well under the insert, covering the bottom of the insert. Twenty-four hours after seeding, cells were fixed with absolute ethanol. The top of the insert was cleaned with a cotton bud to remove the Matrigel and any cells that did not cross the layer. Images were taken of the entire bottom of the insert where the invading cells were located, using an Olympus IMT-2 microscope (amplification 20X) and X80 software.

Statistical analysis

Data is presented as mean value ± standard deviation (mean ± SD) in this study. GraphPad Prism 7.0 (La Jolla, CA, USA) was recruited to conduct statistical analysis. The differences between groups (≤2) were analyzed using a Student's t-test, while a one-way ANOVA was used for three or more (≥3) groups. A P-value less than 0.05 was considered to be statistically significant.

Presence of E-cadherin in BALF

In patients with lung cancer, E-cadherin may facilitate metastasis. To verify the presence of E-cadherin in BALF, we used the ELISA technique. As Figure 1a shows, there was a significant upregulation in the concentration of E-cadherin in the same amount of BALF obtained from patients with lung cancer and control subjects, consisting of patients with the healthy side of pneumonia. Imaging of abnormal soft tissue and postoperative pathology showed that patients with lung cancer had significantly higher concentrations of E-cadherin in their BALF than did the control subjects. We further identified this on the views of the endoscopic and HE stained images and found the tissue obtained by bronchoscopy from cancer groups have typical pathological features as Figure 1b.

Isolation and characterization of exosomes from BALF

Exosomes can carry and transport E-cadherin, thereby promoting cancer metastasis. To verify whether the increased concentration of E-cadherin in BALF obtained from patients with lung cancer was carried and transported by exosomes, we examined exosomes in BALF by transmission electron microscopy (Fig 2a), and observed cup-shaped vesicles with diameters of 20–100 nm and two-layer membranes.

To further characterize the size of the vesicles, as depicted in Figure 2b, we used a zeta potential particle size analyzer. Specifically, we measured the Brownian motion of the particles within the BALF sample using dynamic light scattering techniques. Indirect fitting yielded the particle sizes and distributions. As Figure 2b shows, the particles were uniformly dispersed, distributed mainly at 130–150 nm, with a single-peak diameter and a peak of 141.5 nm. This represented the hydrated vesicles, covalently bonded to the surrounding water molecules. To further verify whether the vesicle structure isolated was exosomes, the presence of exogenous marker proteins CD63, CD81, and TSG101 were detected by WB (Fig 2c). The isolated vesicles exhibited distinct bands at 43 kDa, 22 kDa, and 46 kDa.

Exosomes carrying E-cadherin increase the amount of total E-cadherin in BALF

We measured the protein concentrations on exosomes obtained from patients with lung cancer and controls to test whether exosomes obtained from patients with lung cancer carried more E-cadherin. As Figure 3a,b shows, patients with lung cancer exhibited significantly higher concentrations of E-cadherin on exosomes, compared to the control group by WB and ELISA. Thus, increased E-cadherin content in BALF obtained from patients with lung cancer was attributable to increased exocrine secretion and increased E-cadherin on the exosome surfaces.

Exosomes carrying E-cadherin promote the migration and invasion of A549 cancer cells

To confirm the effect of exosomes carrying E-cadherin, we tested the migratory and invasive capacity of the lung cancer cell line A549 with exosomes isolated from the different groups. Furthermore, the migration ability of the exosomes could be achieved by the invasion assay without matrigel matrix. The cells which had migrated could be counted after 24 hours of seeding as shown at the top of the insert (Fig 4a). At the same time, A549 cells treated in each group were embedded in a Matrigel/collagen matrix and the cell expansion counted. Comparison of the total number of migrating and invading cells revealed that cells from cancer patients mixed with exosomes carried E-cadherin and were significantly more effective at invasion than the cells mixed with exosomes from healthy patients (Fig 4b). A wound healing assay was used to measure cell migration, which is essential for many biological processes including tumor invasion and metastasis. The group treated by exosomes from cancer patients had strong migratory capacity compared to the control group. To confirm the migration ability affected by exosomes from each goup, we performed a Scratch assay to measure the cells treated with exosomes from each group. The analyzed distances treated by exosomes from cancer patients were longer than the control group (Fig 4c). The assays described above illustrate that the exosomes from lung cancer promoted the metastasis of the A549 cancer cells.