Medical records collection, immunohistochemistry (IHC) and statistics of stemness gene expression in clinical samples
Eighteen HGSOC patients receiving surgery in the National Taiwan University Hospital from January 2008 to December 2016 were consecutively enrolled in this cohort study. All patients were diagnosed by the validation of clinical properties, imaging examination and pathologic analysis. This investigation was approved by the Research Ethics Committee of the National Taiwan University Hospital (No. 200706002R and 202006036R), and all patients provided informed consent. The baseline demographic, pathological and clinical characteristics of all patients, including age, differentiation type, tumor size, and tumor-node-metastasis (TNM) stage, were collected. The TNM stage was assessed according to the 7th edition of the American Joint Committee on Cancer (AJCC) cancer staging manual.
For immunohistochemical staining, tissues were fixed with formalin, and paraffin-embedded Sects. (5 μm thick) were stained with a Ventana BenchMark XT automated slide-staining system (Ventana Medical Systems, Inc., Roche, Tucson, Arizona, USA). The antibodies used in this study are listed in Additional file 1: Table S1. OCT4, SOX2, and NANOG expression in tumor tissues was quantified with a histological score (HSCORE) ranging from 0 (no staining) to 3 (maximal staining), which was calculated to assess IHC, and the computational formula was as follows: HSCORE = Σpi(i), where “pi” represents the percentage of positive cell counts in total cell counts, and “i” represents the intensity. An HSCORE of 0.05 was considered as the threshold which can distinguish high expression and low expression of the protein expression in IHC staining. Data are presented as the mean ± standard deviation (SD), and statistical analyses were carried out using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Kaplan–Meier analysis was performed, and the log-rank test was used to investigate the correlation between OCT4, SOX2, and NANOG expression and overall survival (OS). A p value ≤ 0.05 was considered to indicate significance.
Cell lines and cell culture
The human OC cell line OVCAR-3 was provided by Dr. Wen-Fang Cheng (Graduate Institute of Oncology, College of Medicine, National Taiwan University). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS), penicillin (100 Units/ml) and streptomycin (PS, 100 µg/ml) (All form Gibco); and the cultures were maintained in a humidified incubator at 37 °C with 5% CO2. The human embryonic stem cell (hESC) line H9 and the iPSC line generated from human normal peripheral blood mononuclear cells (iPBMCF) were maintained on mitomycin-c-treated mouse embryonic fibroblast cells (MEFs) with DMEM/F12 medium supplemented with 20% KnockOut Serum Replacement (KOSR), 10 ng/mL bFGF, 0.1 mM nonessential amino acids (NEAAs), 1 mM glutamine (all from Gibco), and 0.1 mM β-mercaptoethanol (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA). Cultures were maintained in a humidified incubator at 37 °C with 5% CO2. The medium was changed daily; and the hESCs and hiPSCs were passaged via microdissection once a week (split ratio 1:3). In addition, Array Comparative Genomic Hybridization (aCGH) were performed for genome-wide screening to confirm the 45, XX karyotype of OVCAR-3 and iOVCAR-OSKM, and the consistent detectable genetic alteration in these two cells (Additional file 11: Figure S10).
Generation of iOCICs utilizing the Sendai virus (SeV) reprogramming system
The CytoTune™-iPS 2.0 Sendai Reprogramming Kit was used for the generation of iOCICs via transduction of Yamanaka factors following the manufacturer's protocol (Invitrogen). The TRA-1–60 Alexa Fluor™ 488 Conjugate Live Imaging Kit (Invitrogen) was used to select Tra-1–60-positive colonies of mitomycin-c-treated MEFs. These cells were cultured in Primate ES cell medium (ESRC) (Reprocell, Beltsville, MD, USA) containing 10 ng/ml bFGF (Sigma–Aldrich, St. Louis, MO, USA). Cultures were maintained in a humidified incubator at 37 °C with 5% CO2; and the medium was changed daily. iOCICs were passaged via microdissection once every week or two weeks.
RNA isolation and reverse-transcription polymerase chain reaction (RT–PCR)
Total RNA was extracted with TRIzol reagent (Invitrogen) and treated with DNaseI (Biorad) to eliminate the contamination of genomic DNA and isolated using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. A 1 µg aliquot of total RNA was reverse transcribed to single-strand cDNA by using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Polymerase chain reaction (PCR) was performed using the ProFlex™ 96-well PCR system (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA). Elimination of the exogenous reprogramming SeV vector was determined using SeV-specific primers. Endogenous gene expression, including pluripotent genes (OCT3/4, SOX2, KLF4, c-MYC, and NANOG), epithelial-mesenchymal transition (EMT)-related genes (SNAIL, SLUG, TWIST, FIBRONECTIN, VIMENTIN, N-CADHERIN, and E-CADHERIN), and a housekeeping gene, GAPDH, was amplified. The oligonucleotide primer sequences are listed in Additional file 1: Table S2. The thermal cycling conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of amplification (95 °C for 30 s, 55–60 °C for 1 min, and 72 °C for 1 min) and a final extension at 72 °C for 10 min. PCR products were analyzed by 1.8% agarose gel electrophoresis.
Quantitative reverse transcription polymerase chain reaction (qRT–PCR)
The mRNA expression level of OVCAR-3 and iOVCAR-3-OSKM was assessed. Total RNA was extracted with TRIzol reagent (Invitrogen) and treated with DNaseI (Biorad) to eliminate the contamination of genomic DNA and isolated using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. A 1 µg aliquot of total RNA was reverse transcribed to single-strand cDNA by using the RevertAid H. qRT–PCR was performed using FastStart Universal SYBR Green Master Mix (Applied Biosystems) and was analyzed with a StepOne Plus™ Real-time PCR system (Thermo Fisher Scientific). Each sample contained 50 ng cDNA in final volume 10ul. The thermal cycling conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of amplification (95 °C for 3 s, 55 °C for 30 s, and 72 °C for 30 s) and a final extension at 72 °C for 10 min. The relative mRNA expression levels of endogenous pluripotent genes (OCT3/4, SOX2, KLF4, c-MYC, and NANOG), previously reported OCIC markers (CD133, CD117, CD24, and CD44), and chemoresistance-related genes (ABCG2 and ALDH1) were assessed using the oligonucleotide primers listed in Additional file 1: Table S2.
Immunofluorescence (IF) staining
Briefly, the cells were fixed with 4% paraformaldehyde (Sigma–Aldrich), permeabilized with 0.2% Triton-100 (Thermo Fisher Scientific), blocked with 3% BSA (Sigma–Aldrich) and incubated with primary antibodies against pluripotent proteins (OCT3/4, TRA-1–60, NANOG, and CD133) and OC lineage differentiation-related proteins (CK7 and CA125) at 4 °C overnight. Cells were then incubated with appropriate secondary antibodies (1:250 diluted in Dulbecco’s phosphate-buffered saline [DPBS], Corning, Corning, New York, USA) at room temperature for 1 h. The antibodies used in this study are listed in Additional file 1: Table S1. Nuclei were stained with Hoechst 33,342 (Thermo Fisher Scientific) stain solution (diluted in DPBS, Corning, Corning, NY, USA) for 5 min. Fluorescence images were captured with an inverted fluorescence microscope (ECLIPSE TE2000-U, Nikon, Tokyo, Japan).
Flow cytometry (FCM)
Cells were trypsinized into single cells and fixed and permeabilized using the BD Cytofix/Cytoperm Kit (BD Pharmingen™, San Jose, CA, USA). A single-cell suspension of cultured cells was immunostained with primary antibodies against pluripotent proteins (OCT3/4, TRA1-60, and NANOG), previously reported OCIC markers (CD133, CD117, CD24, and CD44), chemoresistance-related proteins (ABCG2 and ALDH1), OC lineage differentiation-related markers (CK7 and CA125), and appropriate secondary antibodies. After washing, the cells were resuspended in DPBS (Corning) containing 2% FBS (Gibco). The samples were analyzed with a FACS Aria II instrument (BD Pharmingen™). The percentages of OCT3/4-, TRA1-60-, NANOG-, CD133-, CD117-, CD24-, CD44-, ABCG2-, ALDH1-, CK7-, and CA125-positive cells were recorded. The antibodies used in this study are listed in Additional file 1: Table S1.
In vitro differentiation
A differentiation procedure was used to determine the in vitro differentiation of iOVCAR-3-OSKM cells. Briefly, iOVCAR-3-OSKM cells were detached with Accutase solution (Gibco) and seeded onto 0.4% pluronic acid precoated ultralow attachment 6-well plates (Corning) with the treatment of 10 μM of Rock inhibitor (Y27632) (Sigma–Aldrich) and cultured with ESRC (Reprocell, Beltsville) containing 10 ng/ml bFGF (Sigma–Aldrich) for 7 days. After 7 days, the cells were collected and transferred onto 0.1% gelatin-coated plates and cultured in DMEM with 10% FBS (Gibco) for 14 days. The cells were fixed on day 14 with 4% paraformaldehyde (Sigma–Aldrich). Antibodies specific for the OC lineage markers CK-7 and CA125 were used for IF. Appropriate secondary antibodies were used for detection. The antibodies used in this study are listed in Additional file 1: Table S2.
A total of 105 cells were transferred to ultralow attachment multiwell plates (Corning) in ESRC (Reprocell, Beltsville, MD, USA) containing 10 ng/ml bFGF, 10 mg/ml human insulin, 100 mg/ml BSA (all from Sigma–Aldrich) and 100 mg/ml human transferrin (Roche, Basel, Switzerland); and incubated as described above for 14 days. The numbers of spheres were manually counted using an inverted microscope (ECLIPSE TE2000-U, Nikon, Tokyo, Japan).
For animal experiments, to decide an appropriate cell dose which could identify the tumorigenicity of OVCAR-3 and iOVCAR-3-OSKM, ten 6-week-old female immunodeficient NOD.CB17-Prkdcscid/NcrCrl mice (LASCO, Taiwan) were randomized into two groups with five mice each (Group 1: injected with OVCAR-3 and Group 2: injected with iOVCAR-3-OSKM).We used sequential tenfold dilutions of cells; a total of 107, 106, 105, 104, and 103 OVCAR-3 or iOVCAR3-OSKM cells in 100 µl serum-free DPBS (Corning) were subcutaneously injected into both dorsal flanks of two groups of NOD-SCID mice (LASCO, Taiwan). The mice underwent continuous observation after subcutaneous inoculation. Four months after tumor cell injection, all mice were anesthetized with isoflurane before sacrifice, and subcutaneous tumors were surgically excised. Tumor incidence was counted and tumor volume was measured and calculated as volume = 0.5 × L × W2 (L: length, W: width). Each animal served as an experimental unit and none of the data collected was excluded.
To further clarify the tumorigenicity between OVCAR-3 and iOVCAR-3-OSKM. We performed a repeat animal experiment. Sixteen 6-week-old female immunodeficient NOD.CB17-Prkdcscid/NcrCrl mice (LASCO, Taiwan) were randomized into two groups with eight mice each (Group 1: injected with OVCAR-3 and Group 2: injected with iOVCAR-3-OSKM). A total 103 OVCAR-3 or iOVCAR-3-OSKM cells were subcutaneously injected into both dorsal flanks of two groups of NOD-SCID mice. Four months after tumor cell injection, all mice were anesthetized with isoflurane before sacrifice, and subcutaneous tumors were surgically excised. Tumor incidence was counted and tumor volume was measured and calculated as volume = 0.5 × L × W2 (L: length, W: width). Each animal served as an experimental unit and none of the data collected was excluded.
Researchers were not blinded to both groups, as monitoring tumor mass was part of humane animal study endpoints. Mice in this study were housed 4–5/cage and acclimated to the temperature-controlled (22 ± 1 °C) vivarium with a 14:10 light:dark cycle. Rodent chow (Teklad 7912) and water were available ad libitum throughout the study and cotton nestlets and plastic huts were provided for nesting. The experiments were reviewed and approved by the Animal Ethics and Research Committee of National Taiwan University (No. 2016206) and conducted following institutional guidelines.
MTT chemoresistance assay
The cell viabilities of iOVCAR-3-OSKM cells and parental cancer cells after paclitaxel (PTX, Sigma–Aldrich) exposure was measured by MTT colorimetric assays (Roche, Basel, Switzerland). DMSO was used to dissolve paclitaxel. A total of 104 cells were seeded in 96-well plates and cultured for 24 h. Then, the medium was replaced with DMEM containing 0.01–10 µM PTX, and the absorbance at 595 nm was measured using a microtiter plate reader after 72 h-incubation. Cell viability was calculated as the ratio of absorbance values for the treated sample versus the same sample incubated in DMEM without PTX treatment.
Hematoxylin–eosin (H&E) staining and IHC
Mice were sacrificed for histological analyses 10 weeks after subcutaneous transplantation with cells. Tumors were surgically excised and fixed with 4% formalin, followed by paraffin embedding. Then, the tissue specimens were obtained for H&E and IHC staining with anti-human cytokeratin 7 (CK7) rabbit monoclonal antibody or anti-human cancer antigen 125 (CA125) mouse monoclonal antibody using the avidin–biotin immunoperoxidase method. The antibodies used in this study are listed in Additional file 1: Table S1.
Dye efflux activity analysis
iOVCAR-3-OSKM cells were harvested in ESRC (Reprocell, Beltsville, MD, USA) containing 2% FBS (Gibco) and 1 mM HEPES (Sigma–Aldrich), followed by staining with 2.5 µg/ml Hoechst 33,342 (Thermo Fisher Scientific) with or without coadministration of verapamil (VM) (Sigma–Aldrich) at 0, 50, or 250 µM for 90 min at 37 °C. Tubes were gently inverted every 30 min. After incubation, the cells were washed and resuspended in DPBS containing 2% FBS and 1 mM HEPES. The cells were then counterstained with 2 µg/ml PI (Sigma–Aldrich) to label dead cells, passed through a 35 µm mesh filter, and kept on ice for FCM and sorting. The cells were analyzed and sorted with a FACSAria™ II (BD Bioscience). Hoechst dye was excited with a UV laser (355 nm), and the fluorescence was measured with 670/50 (Hoechst Red) and 450/50 (Hoechst Blue) filters.
Trans-well migration assay
Corning® Transwell® CLS3422 6.5 mm Transwell with 8.0 μm pore polycarbonate membrane inserts (Merck, Sigma–Aldrich, St. Louis, MO, USA) was used for Transwell assays. A total of 105 cells were seeded into the upper layer of cell culture inserts with 100 µl serum-free medium, and medium containing 10% FBS was placed below the permeable membrane. After 24 h, cells that migrated through the membrane were stained and counted.
Briefly, 3 × 105 single cells were suspended in medium containing 2% Matrigel (Corning) and 50 nM 17β‐estradiol (E2) (Merck, Sigma–Aldrich) and were seeded into culture dishes coated with 100% Matrigel, followed by incubation at 37 °C in 5% CO2 for 14 or 28 days. The number (> 20 µm organoids under 100X magnification) and diameter (10 organoids under 200X magnification) of organoids were calculated and statistically analyzed by ImageJ bundled with Java 1.8.0_172 software.
Next-generation sequencing (NGS) analysis
More than 5 × 106 of OVCAR-3, OVCAR-3 SP, and iOVCAR-3-OSKM cells were collected and used for RNA extraction with the QIAGEN RNA Isolation Kit. Purified RNA was quantified at OD260 nm using an ND-1000 spectrophotometer (Nanodrop Technology, USA), quantified through a Bioanalyzer 2100 instrument (Agilent Technology, USA) and the RNA 6000 LabChip Kit (Agilent Technology, USA). All RNA sample preparation procedures were carried out according to Illumina's official protocol. The SureSelect XT HS2 mRNA Library Preparation Kit (Agilent) was used for library construction followed by AMPure XP bead (Beckman Coulter, USA) size selection. The sequence was determined using Illumina's sequencing-by-synthesis (SBS) technology (Illumina, USA). Sequencing data (FASTQ reads) were generated using Welgene Biotech's pipeline based on Illumina's base calling program bcl2fastq v2.20. Briefly, the program was used to convert BCL files from all Illumina sequencing platforms into FASTQ reads. Adaptors are short nucleotide sequences that have to be ligated to every single DNA molecule during library preparation and allow DNA fragments to bind to a flow cell for next-generation sequencing. Removal of adaptor sequences, a process called 'adaptor trimming', or clipping, is one of the first steps in analyzing NGS data. Quality trimming was performed to remove low-quality reads/bases. Lower quality bases from the 3' end were removed using a sliding-window approach as the per base quality gradually dropped toward the 3' end of reads. Both adaptor clipping and sequence quality trimming were performed using Trimmomatic v0.36 with a sliding-window approach. Differential expression analysis was performed using StringTie (StringTir v2.1.4) and DEseq (DEseq v1.39.0) or DEseq2 (DEseq2 v1.28.1) with genome bias detection/correction and Welgene Biotech's in-house pipeline. Functional enrichment of the differentially expressed genes (DEGs) in each experiment was performed using clusterProfiler v3.6. Genes with low expression levels (TPM value < 0.3) in either or both the treated sample and the control sample were excluded. Genes with |log2 FC|> 2 were defined as DEGs. The intersecting DEGs of iOVCAR-3-OSKM/OVCAR-3 and SP/OVCAR-3 with > 5 or < 0.2 were selected and assessed with Gene Ontology (GO) analysis. The heatmap was created by Python 3.10 with seaborn0.11.2.
The results were analyzed and plotted with GraphPad Prism™ software (version 7.0a). All values are expressed as the mean ± SD. Student’s t test was used to compare the means between two groups. A P value (P) < 0.05 was considered to denote statistical significance.