Patients and tissue samples
After obtaining informed consent from each participant, tissue microarrays (TMAs) containing para-carcinoma tissues (n = 57), OC tissues (n = 57) and peritoneal metastasis tissues (n = 57) collected from patients with OC at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine between 2008 and 2019 were produced. Tumor types of OC patients could be classified in epithelial tumors, sex cord stromal tumors, germ cell tumors, soft tissue tumors, unclassified type and metastatic secondary tumors . The epithelial OC tumor types were selected in our study. Overall survival (OS) was measured from the date of initial surgery to the date of death from any cause or the date of the most recent follow-up. Our study was approved by the Ethics Committee of Xinhua Hospital, and each clinical investigation was conducted according to the principles outlined in the Declaration of Helsinki.
Fourteen patient samples were used to conduct single-cell sequencing. The samples included peripheral blood, para-carcinoma tissues, OC tissues, peritoneal metastasis tissues, ascites and lymph nodes. Fresh para-carcinoma ovarian tissue, OC tissue and peritoneal metastasis tissue samples from patients were cut into small pieces and then digested using a MACS Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, NRW, Germany). We used marker genes CD45 and EPCAM to separate the epithelial cells from stromal cells. Cells were negative for CD45 and strongly expressed EPCAM were identified as epithelial cells [34, 35]. Cells were filtered through a seventy-millimeter cell-Strainer (BD Biosciences, San Jose, CA, USA) and centrifuged at 1200 rpm for five minutes. Then, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec, Bergisch Gladbach, NRW, Germany) to lyse red blood cells. Single cells were processed using a GemCode Gel Bead, Chip and Library Kit (10 × Genomics, Pleasanton, CA, USA) for droplet-based single-cell RNA sequencing. A total of 6000 cells were loaded for each sample. Single-cell RNA-sequencing data were quantified with Cell Ranger Single-Cell Software Suite 2.3 (10 × Genomics, Pleasanton, CA, USA).
Cell lines and culture
The human OC cell lines Skov3-ip1 and Hey were obtained from the American Type Culture Collection (ATCC, Gaithersburg, Maryland, USA). All cells were cultured in high-glucose DMEM (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA), penicillin (100 U/ml) and streptomycin (100 ng/ml) and were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
Plasmid construction and viral infection
To generate stable cell lines, lentiviruses harboring control shRNA (negative control, NC) and specific shRNAs against STC1 (STC1-sh1/sh2), FOXC2 (FOXC2-sh1/sh2) or ITGB6 (ITGB6-sh1/sh2) (Genomeditech, Shanghai, China) were transduced into OC cells according to the manufacturers’ instructions. The sequences of NC were as follows: 5’-TTCTCCGAACGTGTCACGT-3’. The sequences of the shRNAs against STC1 were as follows: 5’-GCTGGTGATCAGTGCTTCTGC-3’ (sh1) and 5’-GCCTCAACAGTGCTCTACAGG-3’ (sh2). The sequences of the shRNAs against FOXC2 were as follows: 5’-GCGAGCAGAATTACTACCGGG-3’ (sh1) and 5’-GCTTCAGCGTGGAGAACATCA-3’ (sh2). The sequences of the shRNAs against ITGB6 were as follows: 5’-GCAACTTTAGACTGGGCTTCG-3’ (sh1) and 5’-GCCAACCCTTGCAGTAGTATT-3’ (sh2). Briefly, the OC cell lines Skov3-ip1 and Hey were infected by incubating cells with medium containing virus and 1 ng/ml polybrene (Sigma, ST LOUIS, MO, USA) for 24 h. Then, stably transduced cells were screened using puromycin (Sigma) at a final concentration of 2 mg/ml for 3 days. Plasmid DNA was transfected into cells with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA).
RNA sequencing data analysis
RNA sequencing assays were performed as previously described . Briefly, total RNA (1 ug) was isolated from OC tissues and peritoneal metastasis tissues and incubated with VAHTS mRNA Capture Beads (Vazyme, Nanjing, China) to enrich polyA + RNA before construction of RNA libraries. Computational analysis of RNA sequencing data was performed with the Ensemble Human Genome Assembly (Genome Reference Consortium GRCh38) as the reference genome. The expression levels for each gene transcript were estimated as the number of reads per kilobase of exon model per million mapped reads (RPKM). Gene set enrichment analysis (GSEA) were used for gene functional annotation. Hallmark collections were acquired from the Molecular Signatures Database (MSigDB), and were analyzed by GSEA with GSEA software (version: 4.0.3). A total of 5000 permutations were performed, based on the gene set, to determine p-values. Pathways with p < 0.01 and false discovery rate (FDR) < 0.25 were considered significant. “Signal to noise” was used as a metric for ranking the genes.
Immunohistochemical (IHC) assay
The TMAs were produced by Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine. IHC staining was performed on 7-mm-thick TMA sections. The sections were deparaffinized and rehydrated in a descending ethanol series. After antigen retrieval, the sections were incubated with 3% hydrogen peroxide for 20 min. Tissue slides were then incubated for 12 h at 4 °C with primary antibodies specific for the following proteins: STC1 (ab229477, Abcam, 1:100 dilution), FOXC2 (AF6989-SP, R&D, 1:100 dilution) and ITGB6 (10,176–2-AP, Proteintech, 1:100 dilution). The next day, after incubation with a horseradish peroxidase-labeled secondary antibody for 30 min at room temperature, the sections were rinsed three times with phosphate buffer saline (PBS) for 5 min each. Approximately 5 ml of diaminobenzidine (DAB) was added for 3–15 min to induce color development. The sections were then rinsed thoroughly with tap and were then counterstained, dehydrated, cleared with xylene, covered with neutral resin, and observed under a microscope. The antibodies used are listed in Supplementary Table S1 (The primary antibodies).
Untargeted lipidomics and proteomics
Lipids from cells were extracted in a methanol: MTBE: chloroform (MMC) mixture (40/30/30, v/v/v), containing the antioxidant BHT (10 μg/100 ml). Each sample was then vortexed and placed in a shaker at room temperature for 30 min (950 rpm). Samples were then centrifuged to pellet proteins (10 min, 8000 rpm), and the supernatant saved for analysis in fresh Eppendorf tubes. A volume of two μl of each sample was injected into the LC–MS instrument. All Dionex Ultimate 3000 series modules (Thermo Fisher Scientific, Waltham, MA, USA) and a Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) were used. Liquid chromatographic separation was performed at 45 °C using a Kinetex F5 reversed-phase column (Phenomenex Inc.), at a flow rate of 0.65 ml/min. The mobile phases consisted of 5 mM ammonium formate and 0.1% formic acid in water and 5 mM ammonium formate and 0.1% formic acid in isopropanol. Mass spectrometry analysis was performed first with positive/negative ion switching method in full MS scan mode. Preliminary untargeted data analysis with Lipostar software (Molecular Discovery Ltd., UK) allowed automatic generation of inclusion lists for masses of interest (i.e., potential lipids). Finally, MS/MS spectra were uploaded in the Lipostar session to improve lipid identification, and the same software was used for multivariate statistical data analysis.
Transmission electron microscopy (TEM)
For TEM observation, cells subjected to different treatments were fixed with 5% glutaraldehyde (in PBS buffer) at 4 ℃ for 12 h. After fixation, cells were washed with PBS, and fixed again for 2 h at 4 °C in aqueous 1% (w/v) osmium tetroxide and then embedded in Epon. Observation and imaging were carried out with an electron microscope (JEM-2000EX TEM, JEOL, Tokyo, Japan).
Oil Red O (ORO) Staining
After 8 days of differentiation, cells in different groups were stained with ORO (#O0625, Sigma, USA). Staining was performed according to the manufacturer's instructions. To quantify the staining of fat droplets, 100% isopropanol was used to dissolve the lipid droplets, and the absorbance was measured at 510 nm.
Immunofluorescence (IF) assay
Cells or frozen sections of OC tissues were fixed with 4% paraformaldehyde for fifteen minutes, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 5% goat serum (Life Technologies, USA) for 1 h at room temperature. Then, sections were incubated with primary antibodies for 12 h at 4 °C. Secondary antibodies were added, and staining with DAPI (Life Technologies, USA) was then performed. Stained sections were imaged using a Leica SP5 confocal fluorescence microscope. Primary antibodies specific for the following proteins were used in the IF experiments: STC1 (sc-293435, Santa Cruz Biotechnology, 1:100 dilution, Dallas, Texas, USA), FOXC2 (23,066–1-AP, Proteintech, 1:100 dilution, Wuhan, Hubei, China), ITGB6 (19,695–1-AP, Proteintech, 1:100 dilution, Wuhan, Hubei, China), UCP1 (AF8292, Beyotime, 1:100 dilution, Shanghai, China), TOM20 (11,802–1-AP, Proteintech, 1:100 dilution, Wuhan, Hubei, China), perilipin1 (ab172907, Abcam, 1:100 dilution, Cambridge, UK). All antibodies are listed in Supplementary Table S1 (The primary antibodies). The secondary antibodies were FITC- or Texas red-conjugated donkey F(ab)2 fragments specific for mouse IgG or rabbit IgG (Jackson ImmunoResearch Laboratories).
Western blotting (WB) assay
WB analysis was performed as previously described . Briefly, cells were harvested and lysed in RIPA buffer in the presence of protease inhibitor cocktail (Pierce, WA, USA) and a protein phosphatase inhibitor (Beyotime, Shanghai, China). One hundred micrograms of protein was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Tanon, Shanghai, China). Membranes were blocked with 5% bovine serum albumin (BSA) for 2 h and were then incubated for 12 h with primary antibodies at 4 °C. Membranes were probed with antibodies specific for STC1 (20,621–1-AP, Proteintech, 1:1000 dilution, Wuhan, Hubei, China), FOXC2 (23,066–1-AP, Proteintech, 1:1000 dilution, Wuhan, Hubei, China), p-FAK (8556S, CST, 1:1000 dilution, Boston, MA, US), p-AKT (86,444–1-Ig, Proteintech, 1:1000 dilution, Wuhan, Hubei, China), p-PI3K (17366 s, CST, 1:1000 dilution, Boston, USA), PI3K (20,584–1-AP, Proteintech, 1:1000 dilution, Wuhan, Hubei, China), FAK (12,636–1-AP, Proteintech, 1:1000 dilution, Wuhan, Hubei, Chian), AKT (10,176–2-AP, Proteintech, 1:1000 dilution, Wuhan, Hubei, China), ITGAV (27,096–1-AP, Proteintech, 1:1000 dilution, Wuhan, Hubei, China), ITGB6 (19,695–1-AP, Proteintech, 1:1000 dilution, Wuhan, Hubei, China), ITGB7(A5873, Abclonal, 1:1000 dilution, Wuhan, Hubei, China). GAPDH (10,494–1-AP, Proteintech, 1:5000 dilution, Wuhan, Hubei, China) was used as the internal control. Goat anti-mouse or goat anti-rabbit horseradish- peroxidase-conjugated IgG was used as the secondary antibody (Beyotime, Shanghai, China) at a 1:1000 dilution. Membranes were incubated with the secondary antibody for 1 h at room temperature, and immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ, USA) according to the manufacturer’s instructions. Three independent experiments were performed for final analyses.
Cell proliferation assay
Cell proliferation assay was tested with a CCK-8 kit (Beyotime, Shanghai, China, Cat# C0039). The different groups of cells were seeded in 96-well plates at a density of 2000 cells per well. After culturing for 0, 24, 48, 72, 96, and 120 h, 200 μl of medium containing CCK-8 reagent was prepared, added to the wells, and incubated for two hours. The absorbance values were measured at a wavelength of 450 nm, with a microplate reader (BioTeke, Beijing, China).
Cell migration and invasion assay
Starved cells (5 × 105 for Skov3-ip1 cells, 3 × 105 for Hey cells) were plated in the upper compartment of Transwell chambers in 250 μl of serum-free medium. Complete medium containing 10% FBS was added to the bottom compartment. Plates were incubated in 5% CO2 at 37 °C for 12 h. Then, the cells remaining in the upper compartment were removed with a cotton swab, and the migrated cells were fixed with paraformaldehyde and stained with crystal violet. The invasion experiment required the membrane in the upper compartment to be coated with Matrigel before cell seeding. Images acquired, and some of the stained cells were counted under a microscope.
Colony formation assay
Cells were seeded in six-well plates at a density of 500 cells per well. Cells were incubated at 37 °C in 5% CO2 and allowed to grow for at least 10 days, the medium was then discarded. Colonies were counted after being fixed with paraformaldehyde (4%) for 15 min and were stained with crystal violet (Beyotime, Shanghai, China) for 30 min.
Wound healing assay
A wound healing assay was used to measure the cell migration ability of Skov3-ip1 and Hey cells under specific conditions. The different groups of cells were plated in six-well plates and allowed to grow to confluence. A wound was made by scratching the cell monolayers with a 200 μl pipette tip. The indicated cells were deprived of serum for 48 h. Images of migration were acquired at 0 h and 12 h after scraping.
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurement
The ECAR and OCR were measured using a Seahorse XF Glycolysis Stress Test Kit (Agilent Technologies, Palo Alto, CA, USA) and a Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, Palo Alto, CA, USA) and were analyzed using a Seahorse XF24 analyzer (Agilent Technologies, Palo Alto, CA, USA). Briefly, glucose uptake of STC1-sh1/sh2 and NC cells of the two cell lines was detected. These cells were harvested 48 h after seeding, and used to measure the ECAR and OCR. After baseline measurements, for OCR measurement, 1 μmol/L oligomycin, 1 μmol/L FCCP (p‐trifluoromethoxy carbonyl cyanide phenylhydrazone, a reversible inhibitor of oxidative phosphorylation), and 0.5 μmol/L Rote/AA (rotenone plus the mitochondrial complex III inhibitor antimycin A were automatically injected successively. For ECAR measurement, the Seahorse automatically filled each well with 10 mmol/L glucose, 1 μmol/L oligomycin (an oxidative phosphorylation inhibitor), and 50 mmol/L 2‐DG (2‐deoxy‐D‐glucose, a glycolysis inhibitor) successively. Data were analyzed by using Seahorse XF24 Wave software (Agilent Technologies, Palo Alto, CA, USA). The OCR and ECAR values were calculated after normalization to the cell number and were plotted as the mean SD values.
IP experiments were performed as previously described [38, 39]. Briefly, cells were extracted with RIPA lysis buffer containing phosphatase and protease inhibitors. Cell lysates were incubated with 1 μg indicated antibodies and protein A-Sepharose (GE Healthcare). The cell lysates, antibodies and sepharose mix were incubated at 4 °C overnight. Then wash the immunocomplexes four times with lysis buffer and analyzed by WB assays. Antibody used was as follow: STC1 (ab229477, Abcam).
Cell extracts were incubated with 0.5 μg individual antibody and 20 μl protein G beads (GE Healthcare, Pittsburgh, USA). After overnight incubation, beads were washed four times with lysis buffer, separated by SDS-PAGE, and analyzed by immunoblotting.
Chromatin Immunoprecipitation (ChIP) assay
ChIP assays were performed using a Pierce Agarose ChIP Kit (Thermo, #27,177) following the manufacturer’s guidance. Briefly, Skov3-ip1 cells were cross-linked with 1% formaldehyde for 10 min at 37 °C. The cross-linking reaction was quenched with glycine, and cells were lysed in SDS buffer containing protease inhibitor cocktail. Cell lysates were sonicated to shear chromatin into fragments with a length of 200–1000 base pairs and were then subjected to immunoprecipitation with 4 μl of IgG (Cell Signaling Technology), 10 μl of anti-FOXC2 (ab5060, Abcam), or 3 μl of anti-Polymerase II (Imgenex) antibodies. After washing with a series of low- and high-salt washing buffers, immunoprecipitated DNA fragments were decrosslinked at 77 °C under high-salt conditions and purified using a QIAquick PCR purification kit (Qiagen). The amount of DNA was further assessed by qRT-PCR using FOXC2-specific ChIP primers and SYBR Select Master Mix (Applied Biosystems, Grand Island, NY, USA). The primer sequences were as follows: F, 5’- GGCTTGAACACAGATTATTA -3’ and R, 5’- TGAGATATGACAGCTCAGAG -3’.
Total RNA was extracted from cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and it was reverse transcribed using a miScript Reverse Transcription Kit (Qiagen, Dusseldorf, NRW, Germany). The primers used for mRNA quantification are listed in Supplementary Table S2 (The primer sequences of qRT-PCR assay). Quantification was performed with a QuantiTect Probe RT-PCR kit (Qiagen, Dusseldorf, NRW, Germany). The comparative threshold cycle method was used to determine relative gene expression levels.
Förster resonance energy transfer and fluorescence lifetime imaging (FRET-FLIM)
For the FRET-FLIM experiments, donor proteins (fused to GFP) were expressed by the vector pCMV3-C-GFP Spark, and acceptor proteins (fused to RFP) were expressed by the vector pCMV3-C-RFP Spark. FRET-FLIM experiments were performed with a Leica TCS SMD FLCS confocal microscope. Skov3-ip1 or Hey cells transiently coexpressing the donor and acceptor proteins, as indicated in the figures, were visualized thirty-six hours after transfection. Accumulation of GFP-tagged and RFP-tagged proteins was estimated before measuring the fluorescence lifetime. A tunable WLL set at 489 nm with a pulsed frequency of 40 MHz was used for excitation, and emission was detected using a SMD GFP/RFP filter cube. The fluorescence lifetime shown in the figures corresponding to the average fluorescence lifetime of the donor (τ) was determined and analyzed with PicoQuant SymphoTime software. The lifetime is generally defined as the amplitude-weighted mean value using data from the single (GFP-fused donor protein only) or biexponential (the GFP-fused donor protein interacting with the RFP-fused acceptor protein) fit. Mean lifetimes are presented as the means ± SDs based on more than ten cells from at least three independent experiments. The FRET efficiency was evaluated according to the formula E = 1-τDA/τD. τDA is the average lifetime of the donor in the presence of the acceptor, and τD is the average lifetime of the donor in the absence of the acceptor.
Female athymic nude mice (4–6 weeks old) weighing 14–16 g were purchased from Xinhua Hospital Affiliated to Shanghai JiaoTong University School of Medicine and bred under specific pathogen-free conditions. Prior to the study, the protocols for the treatment of animals were approved by the Medical Animal Care Committee. A total of 5 × 105 Skov3-ip1 or Hey human OC cells transfected with luciferase reporter vectors (Skov3-ip1-Luc/Hey-Luc) in 10 μl of serum-free DMEM were injected into the left ovarian parenchyma of the nude mice. Each group contained five mice. Mice were monitored every five days for tumor growth, and tumor size was measured using a caliper. After four weeks, mice were euthanized, and the left ovary and enterocele in the regions showing clear luciferase signals were excised. The total flux, weight, and the distribution of the tumor were recorded. Tumor tissues were fixed in 4% neutral buffered formalin for frozen slide preparation.
Consecutive tissue sections (thickness, 5 μm) of paraffin-embedded tumor specimens were prepared. Tissue sections were stained with hematoxylin for 5 min and rinsed with running water for 5 min. Next, they were soaked in hydrochloric acid solutions for 5 s, rinsed with running water for another 10 min, and then immersed in ammonia for 5 s. Tissue sections were then rinsed with running water for 10 min, stained with eosin solution for 30 s, and briefly immersed in distilled water. Last, the sections were rapidly dehydrated in graded ethanol (80%, 95%, and 100%), and mounted with neutral gum.
Drug treatment and apoptosis detection
Cells were treated with 50 µg/ml DDP (MCE, Newark, NJ, USA). Apoptosis induced by DDP was assessed via flow cytometry after cells were stained with propidium iodide (PI) and Annexin V (Beyotime, Shanghai, China). Briefly, 1 × 106 cells seeded in 60 mm dishes for 24 h were treated with DDP for 12 h and were then trypsinized and washed twice in ice-cold PBS. A total of 1 × 105 cells were resuspended in 100 µl of binding buffer to which 5 µl of 2 mg/ml Annexin V and 5 µl of 50 µg/ml PI were added. After 15 min of incubation in the dark, cells were evaluated by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) in different channels, and Cell Quest software version 3.3 (BD Biosciences) was used for analysis. All experiments were repeated three times.
Differences between two groups were analyzed using Student’s t test and one-way analysis of variance for normally distributed data and using the Mann–Whitney U test for nonnormally distributed data. A P-values < 0.05 was set as the significance threshold. Before the survival analysis, the mean expression level was used to classify patient samples into two groups. Kaplan–Meier survival analysis was performed to assess the associations of gene expression levels with clinical outcomes, and P-values were calculated with the log-rank test.