TiO2 nanoparticles promote tumor metastasis by eliciting pro-metastatic extracellular vesicles | Journal of Nanobiotechnology


TiO2 NPs were obtained from Deke Daojin Science (Beijing, China). The primary sizes and shapes of the NPs were observed by Hitachi H-7500 transmission electron microscope (TEM, Jeol, Japan).

Cell cultures

Human umbilical vein endothelial cells (HUVECs) were freshly isolated from umbilical cords and cultured in endothelial cell growth medium (ECGM) (Promocell, Germany) supplemented with 5% FBS, 5 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, 0.5 ng/mL vascular endothelial growth factor, 20 ng/mL insulin-like growth factor, and 0.2 μg/mL hydrocortisone.

Human breast cancer cells MDA-MB-231-Luc (American Type Culture Collection) and MCF-7 (American Type Culture Collection) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. All cells were cultured according to the instructions.

EVs isolation and characterization

EVs were isolated from tumor cells-derived conditioned media by sequential ultracentrifugation. MDA-MB-231 cells were cultured in DMEM supplemented with 10% EV-depleted FBS for 48 h. Then the conditioned media were collected, centrifuged at 500g for 5 min, 2000g for 10 min, and 16,800g for 20 min at 4 °C to remove dead cells and debris. The supernatants were passed through a 0.22 μm filter device (Merck, Germany), and then ultracentrifuged at 130,000g for 70 min at 4 °C. The pellets were washed with PBS, followed by ultracentrifugation at 130,000g for 70 min again. The resulting EVs were dissolved in PBS and stored at − 80 °C.

The prepared EVs were confirmed using transmission electron microscopy (TEM) and western blot. For TEM, purified EVs were placed on the carbon-coated 200 mesh grids to dry. The grids were then stained with 2% uranyl acetate for 30 s and observed using a Hitachi H-7500 TEM (Jeol, Japan).

The protein content of prepared EVs were measured using the BCA protein assay reagent kit (Beyotime, China). The size and concentration were measured by Nanoparticle Tracking Analysis (NTA) (Nanosight NS300; Malvern Instruments, UK). For EVs labeling, purified EVs were labeled with PKH67 Green dye (Merck, Germany) according to manufacturer’s instructions. Then, the labeled EVs were collected by ultracentrifugation and resuspended in PBS.

Immunofluorescence staining

Cells were fixed with pre-cooled 4% paraformaldehyde for 10 min and washed with PBS. The cells were then incubated with Triton X-100, and blocking buffer in PBS containing 5% BSA was added for 30 min. Subsequently, the cells were probed overnight with primary antibodies, followed by 1 h incubation with Alexa Fluor-conjugated secondary antibody. The following primary antibodies were used: CD31 (Proteintech, 11,265-1-AP, 1:200 dilution), VMF (Proteintech, 27,186-1-AP, 1:50 dilution), VE-cadherin (CST, 2158S, 1:1000 dilution), ZO-1 (Cell Signaling, #8193, 1:1000 dilution), S1PR1 (Proteintech, 55,133-1-AP, 1:200 dilution), LC3 (Proteintech, 14,600-1-AP, 1:500 dilution). DAPI (Beyotime, China) was used to stain the nuclei without light for 15 min. To visualize the actin filaments, cells were fixed, permeabilized and stained with TRITC phalloidin (Yeasen, China). The stained samples were observed by a confocal laser scanning microscope (IX5-RFACA; Olympus, Japan).

RNA interference and plasmids

The miR-301a-3p inhibitor, mimics and negative control (NC) were synthesized by GenePharma (Shanghai, China). The sequences of inhibitor and miRNA mimics referred above were listed in Additional file 1: Table S1. The vectors overexpressing miR-301a-3p and inhibiting miR-301a-3p were constructed and produced. The cells including 293 T cells, HUVECs, and MDA-MB-231 cells were transfected above mentioned plasmids with Lipofectamine™ 3000 (Invitrogen, USA) and were used for further experiments 24 h–48 h, respectively.

Quantitative RT-PCR

Total RNAs were extracted from cells or EVs using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instruction. Thereafter, extracted RNA was reverse transcribed by using PrimeScript RT Master Mix kit for cDNA (TaKaRa, Japan). qRT-PCR was then performed by using SYBR Green PCR Master Mix (TaKaRa, Japan) on Applied Bio-systems 7500 Fast Real-Time RCR System (Applied Biosystems, USA). Each sample was performed in triplicate and the relative transcription levels of target genes were normalized to GAPDH or U6 (for miRNA). The sequences of all indicated primers are presented in Additional file 1: Table S1.

Western blot assay

The cells and EVs lysates were extracted in RIPA lysis buffer (Beyotime, China). The protein content in the supernatant was quantified by BCA protein assay kit (Beyotime, China). Subsequently, equal amounts of denatured proteins were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, USA). After blocking with 5% nonfat dry milk for 1 h, the membranes were probed overnight at 4 °C with diluted primary antibodies. The following primary antibodies were used: Alix (Proteintech, 12,422-1-AP, 1:1000 dilution), CD9 (Proteintech, 20,597-1-AP, 1:1000 dilution), S1PR1 (Proteintech, 55,133-1-AP, 1:2000 dilution), LC3 (Proteintech, 14,600-1-AP, 1:2500 dilution), β-actin (Proteintech, 20,536-1-AP, 1:1000 dilution). After washing with TBST buffer, the membranes were further reprobed with IRDye-conjugated secondary antibodies (1:5000) for 1 h at room temperature. The membranes were analyzed using an Odyssey infrared imaging system (LI-COR Biosciences, USA).

Cell viability assay

Cell viability was determined by the CCK-8 assay (Beyotime, China) according to the manufacturer’s instructions. Briefly, 5 × 103 cells were seeded in 96-well plates and treated with EVs of different concentrations. After 24 h, 10 μl CCK-8 solution was supplemented to each well for another 2 h and absorbance at 450 nm was measured using a microplate reader (ELx-800; BioTek Instruments, USA).

Transwell permeability assay

The integrity of endothelial cell barrier was determined by quantifying the amount of FITC-dextran (MW = 70 kDa, Merck, Germany) that passed through the endothelial monolayers. Briefly, 2 × 104 HUVECs were grown on the top well of the transwell filters (0.4 μm pore; Corning Costar, USA) until the confluent monolayer was obtained. The cells were exposed to EVs (20 µg/mL) for 1 h, and then FITC-dextran (1 mg/mL) was supplemented. After EVs treatment, the FITC-dextran could be transferred to the lower compartment of transwell. FITC fluorescence signals were measured using a microplate reader (ELx-800; BioTek Instruments, USA) at 520 nm. The fluorescence signal of the treatment groups was normalized to the control group, and the leakage degree was given.

Transendothelial invasion assay was performed to study the effects of endothelial cell leakage on tumor cell migration. 2 × 104 HUVECs were first seeded on the upper well of the transwell insert (8 μm pore; Corning Costar, USA) until confluent. Then, the HUVECs were treated with EVs (20 µg/mL) for 1 h. Thereafter, 1 × 104 Cell-Tracker Green labeled MDA-MB-231 cells, MCF-7 cells, or Cell-Tracker Red labeled A549 cells resuspended in serum-free media were placed inside the upper chamber, respectively. Complete DMEM media containing 10% FBS was placed into the bottom chamber. After 24 h, non-migrated cells in the upper chamber of transwell inserts were removed with cotton buds and washed with PBS. Then, the tumor cells that invaded through HUVEC monolayers and stuck to the lower surface of the membrane were fixed with 4% paraformaldehyde and calculated under a inverted fluorescence microscope (IXplore Pro; Olympus, Japan) at least three random visual fields per well.

Angiogenesis assay

For tube formation assay, matrigel matrix (100 μL/well) was plated in 48-well plate and incubated at 37 °C for 30 min to allow the matrigel to polymerize. The EVs-treated HUVECs were seeded on the matrigel-coated wells and then incubated for 6 h–12 h. The tube formation ability was determined by measuring the number of tubes with inverted microscope (IXplore Pro; Olympus, Japan).

For aortic ring assay, thoracic aortas excised from 4- to 8-week-old mice were removed excessive fat and cut into 1–1.5 mm long cross sections. Matrigel matrix (100 μL/well) was plated in 48-well plate and incubated at 37 °C for 30 min to facilitate matrigel polymerization. Then, rings were gently placed onto the polymerized matrigel layer and covered with an additional matrigel (50 μL) for 30 min at 37 °C. Subsequently, ECGM medium (Promocell, Germany) was added to each well. After 24 h incubation, the aortic rings were incubated with ECGM conditioned medium, which included EVs derived from MDA-MB-231 cells. And the media was replaced every 2 days. The number of sprouts was observed on day 3–5 with inverted microscope and quantified by counting all sprouts from one ring.

Microarray analysis

The miRNA expressions of EVs derived MDA-MB-231 cells, TiO2 treated MDA-MB-231 cells were carried out in Illumina NextSeq 500 (Illumina, USA). After extracting and quantifying total RNA samples, the library was constructed, and the quality of the library was determined using Agilent 2100 Bioanalyzer. Sequencing libraries of different samples were mixed, and single-stranded DNA was generated by denaturing with 0.1 M NaOH. Sequencing was performed for 50 cycles using a Illumina NextSeq 500 sequencer. Then, we used miRDeep2 software to quantify known miRNAs and predict new miRNAs for all trimmed reads. Based on CPM standardized miRNAs, the R software edgeR was used to perform differential expression calculations and screen for differential miRNAs. The top differential miRNA target genes were statistically analyzed, and the GO and path analysis of the target genes were performed.

Luciferase activity assay

The wild-type (wt) and mutant (mt) 3′-UTR segments of S1PR1 were inserted into the luciferase reporter plasmid psiCHECK-2 and amplified, respectively. Co-transfections of S1PR1 3′-UTR plasmids with miR-301a-3p mimics (50 nM) into 293 T cells or HUVECs were accomplished by using Lipofectamine 3000 reagent (Merck, Germany). Cell lysates were harvested 48 h after transfection, and luciferase activity was examined by a dual luciferase reporter assay kit. All assays were performed in triplicate and each experiment was repeated three times.

Animal models

Immune-deficient NOD SCID mice (female, 4 weeks old) were purchased from the Vital River Company (Beijing, China). All animal experiments were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jilin University and approved by the Animal Ethics Committee of Jilin University (Changchun, China). MDA-MB-231-Luc cells (5 × 106) were suspended in 100 μL PBS containing matrigel matrix (5 mg/mL) and transplanted into the left fourth abdominal fat pad at the base of the nipple of the mouse. When the subcutaneous xenograft tumor reached ~ 100 mm3, the tumor-bearing mice were anaesthetized and injected with 5 µg of EVs in PBS or TiO2 NPs via tail vein every other day for a total of ten treatments. After 3 weeks, the mice were administered 15 μg of luciferin intraperitoneally per gram of body weight. And the bioluminescence intensity was observed with the IVIS Spectrum In Vivo Imaging System (PerkinElmer, USA). Then the tissue of mice were harvested for bioluminescence imaging and histology analysis right after being intraperitoneally injected with luciferin.

For in vivo vascular permeability analysis, mice were anaesthetized and injected with 5 µg of EVs in PBS via tail vein every other day for a total of ten treatments. Then 100 μL FITC-dextran (100 mg/kg) was intravenously injected into the tail vein of nude mice. After 2 h, the excessive dye was removed by the transcardiac perfusion, and the lungs of mice were taken for examination. For tail vein metastasis assay, 1 × 105 MDA-MB-231 cells were injected into the tail vein of mice one day after the last EVs administration. After 2 weeks, mice injected with MDA-MB-231 cells were sacrificed and lungs were used to quantify metastatic tumor burden. To evaluate the metastasis, lungs were stained with hematoxylin/eosin (HE) and screened for metastatic nodules. Meanwhile, immunohistochemical staining for endothelial cell marker CD31, and tight junction protein ZO-1, was applied to evaluate the extent of vascular damage caused by EVs treatment.

Statistical analysis

Statistical analyses were performed using GraphPad Prism. The statistical significance was ascertained using one-way ANOVA. Quantitative values of all experiments were expressed as the mean ± SD. P < 0.05 was considered significantly. Statistical significance of the data was summarized as follows: *P < 0.05, **P < 0.01, ***P < 0.001.