2.1 Patients and samples
We collected serum samples from 101 squamous cervical cancer (SCC) patients who underwent surgery at the Obstetrics and Gynecology Hospital of Fudan University between December 2013 and October 2014. All SCC specimens were confirmed by pathology. Patients who received radiotherapy/chemotherapy before surgery or had any other diseases were excluded. Thirty control serum samples were collected from healthy participants who underwent routine physical examination. All specimens were immediately stored at -80°C in our tissue bank until use. The clinicopathological characteristics of the patients, including age, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor size, lymphovascular invasion, stromal invasion depth, lymph node metastasis, parametrial invasion, margin, are detailed in Supplementary Table 1. Overall survival (OS) was calculated from the date of surgery to the date of death or the end of follow-up. Recurrence-free survival (RFS) was defined as the time interval between the date of surgery to the date of recurrence or the end of follow-up.
2.2 Cells and cultures
Five human CC cell lines (HPV 16-positive SiHa and CaSki cells, HPV 18-positive HeLa and SW756 cells, and HPV-negative C33A cells) were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were authenticated using short tandem repeat (STR) profiling. All mycoplasma-free cells were cultured at 37°C in a humidified incubator with 5% CO2 and grown in Eagle's minimum essential medium (EMEM; Siha, HeLa and C33A cells), RPMI-1640 medium (Caski cells), or Leibovitz's L-15 medium (SW756 cells) (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml).
2.3 Establishment of stable knockdown (KD) or overexpression (OE) cell lines
For stable KD of the E6 oncogene, E6-shRNAs were constructed by targeting the sequences of HPV 16 with KD-1: 5’-GGTCGATGTATGTCTTGTTGC-3’; KD-2: 5’-GGGAATCCATATGCTGTATGT-3’; and KD-3: 5’-GCTGCAAACAACTATACATGA-3’; and those of HPV 18 with KD-1: 5’-GGTGCCAGAAACCGTTGAATC-3’; KD-2: 5’-ACCCTACAAGCTACCTGATCT-3’; and KD-3: 5’-GACTCCAACGACGCAGAGAAA-3’. Lentiviruses encoding E6-KD or a negative control (NC) were generated by Hanyin Co. (Shanghai, China). To stably overexpress Wnt7b, the full-length Wnt7b gene was synthesized and cloned into a lentiviral vector (named Wnt7b-OE); the control viruses were named Wnt7b-NC. After infection with the lentiviruses and selection for 2 weeks using puromycin, stable cell lines were established. The efficiency of KD or OE was confirmed by qRT-PCR.
2.4 Quantitative real-time polymerase chain reaction (qRT-PCR)
RNA extraction and qRT-PCR experiments were performed as previously described [31]. The primer sequences of Wnt1, Wnt2, Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt7b, Wnt10b, Wnt11, HPV16, HPV18 and GAPDH are listed in Supplementary Table 2. The relative expression of each gene was calculated based on the corresponding Ct values, normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Fold changes in the expression of each gene were calculated by a comparative Ct method using the 2-ΔΔCt relative quantification method.
2.5 Western blotting
We performed Western blotting as previously described [32]. Primary antibodies against Wnt7b, β-catenin, CD9, CD63 and TSG101 were purchased from Abcam (Cambridge, UK). Antibodies against Flag and Hsp70 were purchased from Cell Signaling Technology (Massachusetts, US).
2.6 Isolation and verification of EVs
MISEV2018 recommendations were followed for EV isolation. After centrifuging serum free cell culture supernatants or serum samples, we used the total EV isolation kit (catalogue no. EXOTC50A-1; System Biosciences, Palo Alto, California) to isolate EVs from the cell supernatants (30ml/48hr) and used the ExoQuick EV Precipitation Solution Kit (catalog no. EXOQ5A-1, System Biosciences, Palo Alto, CA, USA) to isolate EVs from the serum samples (5ml) according to the protocol. The concentration of EVs was tested by a BCA assay. The EVs were stored at -80°C until use.
Three methods including transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting were used to verify the isolated EVs as previously described [33]. Briefly, EV suspension samples were observed under a Hitachi H-7650 transmission electron microscope (Hitachi, Ltd., Tokyo, Japan) for the TEM analysis. Additionally, resuspended EVs were detected using a NanoSight NS300 instrument (Malvern Instruments Ltd., Worcestershire, UK) to test the size distribution of the EVs. Moreover, known EV biomarkers were tested by Western blotting for CD9, TSG101, Hsp70 and CD63.
2.7 The internalization of labelled EVs by the HUVECs
To track the internalization of EVs, EVs were labelled with PKH67 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as previously described [33-34]: EVs resuspended in a buffer provided in the kits were mixed with the PKH67 dyes and were incubated for 5 min at room temperature. Next, the samples were added to PBS supplemented with 5% bovine serum albumin and were ultracentrifuged at 100,000 g for 1 h to remove free dyes. The labelled EVs were co-cultured with HUVECs for 6 h, which were then fixed with 4% paraformaldehyde at 25˚C for 20 mins. The internalization of labelled EVs by the HUVECs was analysed using a fluorescence confocal microscope. All the fluorescence images were captured.
2.8 Cell Counting Kit-8 (CCK8) proliferation assay
We conducted cell proliferation assays using CCK8 (Dojindo, Japan). The cell numbers per well were then determined by testing the absorbance (450 nm) using a 96-well plate reader at the indicated time points.
2.9 Tube formation assay
A tube formation assay was conducted as previously described [33]. Briefly, EV-pretreated cells were incubated with serum-free medium for 12 h and then transferred into 48-well plates precoated with Matrigel. After incubation, tube formation was observed under a microscope. Total tube length was tested by measuring the branches of blood vessels using ImageJ software.
2.10 TOP/FOP Luciferase Reporter Assay
The transcriptional activity of β-catenin was assessed using the TOP/FOP dual-luciferase reporter system (Dual-Glo™ Luciferase Assay System, Promega). The Renilla luciferase plasmid pRLTK (Promega), which controls for transfection efficiency, was cotransfected with β-catenin-responsive firefly luciferase reporter plasmid TopFlash (EMD Millipore) or the negative control FopFlash (EMD Millipore) using the lipofectamine 2000 (Thermo Fisher Scientific). Cells were harvested after 24 h in culture and the luciferase activity was determined by the Luciferase Assay System (Promega) using a Microplate Luminometer (Berthold, Bad Wildbad, Germany).
2.11 Nude mouse model
Female BALB/c athymic nude mice, 4-6 weeks old and weighing 20-22 g, were purchased from Slac Laboratory Animal Co., Ltd. (Shanghai, China). The care for animals was in accordance with institution guidelines. SiHa cells (5 × 106) were subcutaneously injected into mice. Twenty-four mice were then injected with EVs or PBS and divided into four groups: (1) PBS group, (2) EV/NC group, (3) EV/E6-KD group, and (4) EV/E6-KD+Wnt7b-OE group. To further investigate the role of Wnt7b/β-catenin signaling, another 18 mice were divided into three groups: (1) EV/E6-KD group, (2) EV/E6-KD+Wnt7b-OE group, and (3) EV/E6-KD+Wnt7b-OE+FH535 group. After four injections with 0.2 mL PBS containing 50 µg EVs, all the mice were euthanized, and the tumors were collected.
2.12 Immunohistochemistry (IHC)
IHC assay was conducted as previously described [33]: Briefly, the density of blood vessels was detected using CD31 staining. Xenograft tumours were fixed, embedded in paraffin and sectioned into 4-μm thick slices. After deparaffinization and rehydration, sections were blocked and incubated with a CD31 antibody (Abcam, USA). The quantitative analysis was performed on five randomly selected, independent fields per tumour.
2.13 Statistical analysis
Statistical analysis was performed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Independent t-tests were used to analyze continuous data, and the chi-square test or Fisher’s exact test was used to analyze categorical data. Differences among the groups were analyzed using Student two-tailed t-test or one way analysis of variance (ANOVA). OS and RFS analyses were performed using the Kaplan-Meier method, log-rank test, univariate and multivariate Cox regression models. Based on the results of the multivariate Cox analysis, a predictive nomogram for OS and RFS was established using R version 3.5.3 (https://www.r-project.org/) as described in previous studies [33]. P<0.05 was considered statistically significant.
2.14 Ethics committee approval and patient consent
This study was approved by the Research Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University, China. All samples were obtained with full and informed consent.