Tissue specimens
In our study, 153 benign ovarian tumor tissues, 187 primary lesions of epithelial ovarian cancer, and 154 metastatic specimens from greater omenta were collected after acquisition of the informed consent in Xinhua Hospital (Shanghai, China). Tissue samples were gained between 2008 and 2017. Overall survival (OS) was calculated as the interval from surgery initiation to death for whatever reason or final time for follow-up. Progression-free survival (PFS) was measured from surgery initiation to disease relapse or progression.
Ovarian cancer organoids generation
Ovarian cancer organoids were gained from human high-grade serous ovarian carcinoma (HGSOC). Organoids generation and culture were performed as previously described[42]. Fresh cancer tissues were promptly carried to laboratory and dipped in Advanced Dulbecco's Modified Eagle's Medium (DMEM)/F12. Tissues were diced into approximately 2-3 mm sections and subsequently digested in 37°C for 1 hour.
OCT4 knockdown cell lines construction and culture
Ovarian cancer SKOV3ip1 and OVCA433 cells were cultured in DMEM with 10% FBS included (Gibco, Life Technologies). To generate stable cell lines, lentiviruses harboring scrambled shRNA (Vector) or two kinds of specific shRNAs against OCT4 (OCT4-sh1/2) were transduced into above ovarian cancer cells. The shRNA sequences of OCT4 were as follows: 5’- CACACCAGTTATCAATCTCCC-3’ (sh1); 5’- CCATTCGGGATTCAAGAACCT- 3’ (sh2). The silencing effect of OCT4 was confirmed by follow-up immunoblotting.
Wound healing assay
Firstly, cells were seeded on 6-well plates with 9 × 105cells/well as confluent monolayers. Subsequently, scratch wound healing assay was performed by pipette tips. After incubating for 48 h, we calculated the migrated area using microscopy.
Transwell assays of migration and invasion
Transwell migration and invasion assays were performed as previously described[43].
5 × 104 cells were planted into the upper chamber plates and cultured with serum-free DMEM medium. The lower chamber was added with DMEM containing10% FBS. Non-migrated or non-invasive cells were removed by cotton buds after 48 h, and the migrated or invasive cells were stained with crystal violet. Stained cells were counted in five random fields per well under a light microscope (Nikon, Japan).
Tube formation
Firstly, 24-well plates were coated with 300 μL matrigel (BD Biosciences). Subsequently, 2*104 cells were suspended in 200 μL medium. 6 h later, we investigated and measured tube formation using an microscope (Olympus, Japan).
Immunoprecipitation
Pierce Crosslink Immunoprecipitation kit was utilized to perform immunoprecipitation (IP) following the manufacturer's protocol. In short, lysates of SKOV3ip1 ovarian cancer cells were generated via lysis buffer and then tested by BCA Protein Assay Kit (Beyotime, China). In the subsequent procedures, 1 mg total cell lysates were pre-cleared and blended with 10 µg antibody or IgG, and co-incubated with A/G agarose beads at 4℃ overnight. Finally, immunoblotting assay was carried out to examine the target proteins.
Mass spectrometry
Firstly, cell sediment was collected by centrifugation. And then, protein was extracted by 8 mol/L urea lysate, which was dissolved in 100 mmol/L ammonium bicarbonate. Through Bicinchoninic Acid (BCA) assay, protein concentration was determined and 300 µg protein was used for the following mass spectrometry (MS) assay. After processes of reduction reaction, purification and enzymatic hydrolysis, samples were desalted via the desalination column, and finally tested by mass spectrometer (Thermo Fisher, USA).
Immunoblotting
After protein quantification via BCA assay 20 µg protein was utilized for immunoblotting assay. Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and proteins were transferred to polyvinylidene fluoride (PVDF) membrane. Next, primary antibodies were added to the membrane and incubated for 2 hours at Room temperature (RT). In our study, primary antibodies against OCT4 (ab181557), LRPPRC (ab97505), p-VEGFR2 (Tyr1214, ab131241), VEGFR2 (CST 9698), p-VEGFR2 (Tyr1175, CST 2478), p-AKT (Ser473, CST 9018) and β-actin (CST3700) were used. Then, the membrane was washed for 3 times, and incubated with corresponding secondary antibodies. Finally, chemiluminescence kit (Millipore, USA) was applied for visualization of protein bands.
RNA sequencing
After total RNA was extracted by TRIzol (Thermo Fisher, USA), 1 mg RNA was used for subsequent RNA sequencing analysis. The method was detailed described as reported[44].
Immunohistochemistry assay
Paraffin-embedded tissue was sliced into 3 µm thin sections for immunohistochemistry (IHC) analysis. Antibodies of OCT4 (ab181557, Abcam) and LRPPRC (ab97505, Abcam) were applied and subsequently incubated with corresponding secondary antibodies at 37°C for 1 h. Histochemistry score (H-score) and positive ratio were utilized to assess the expression level as described formerly[43].
Quantitative real time PCR
Total RNA was centrifugated and extracted from ovarian cancer cells and tissues with TRIzol reagent (Invitrogen, US) in order to perform quantitative real time PCR (qRT-PCR). Primers in our study were exhibited in Supplemental Table 1.
Immunofluorescence assay
Ovarian cancer cells or tissues were fixed with 4% paraformaldehyde at room temperature (RT) for 15 min and then incubated with 0.3% Triton X-100. After blocking of 5% goat serum (Life Technologies, US) at RT, cells or tissues were treated with appropriate amount of antibody overnight at 4 ℃. In the next step, primary antibody was cleared up by phosphate buffered saline (PBS) for 3 times, and then corresponding secondary antibody was added to the cells or tissues. 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies, US) was utilized for nuclear staining. Ultimately, the stained cells were visualized using a Leica SP5 confocal fluorescence microscope.
Nude mice model
BALB/c nude mice tumor associated experiments protocols were authorized and approved by Ethics Committee at Xinhua hospital. In this study, a total of 60 female mice (aged 4 weeks) were used. The models of subcutaneous and intraperitoneal ovarian cancer mice were generated by injection of 5 × 106 SKOV3ip1 cells. Subcutaneous tumor volume was regularly measured and calculated twice a week by vernier caliper. Intraperitoneal tumor volume of the ovarian cancer mode was observed by luminescence imaging techniques.
Zebrafish model
In our study, transgenic enhanced green fluorescent protein (EGFP) expressing zebrafish model was used for assessing angiogenesis in vivo. OCT4 morpholino plasmid and/or LRPPRC cDNA were microinjected into 1-cell zebrafish embryos. After 24 hours post fertilization (Hpf), fluorescence microscopy was used to detect the blood vessel formation in zebrafish model. For subsequent examination of VEGFA mRNA of zebrafish embryos, qRT-PCR assay was performed following the procedure above.
Statistical analysis
Data in our study were analyzed by GraphPad Prism 8.0 and SPSS 24.0 software. Student’s t test was used for statistical analyses. The Kaplan-Meier survival analysis was performed to assess OS and PFS. p value less than 0.05 was considered as statistical significance.