Patient background
Patient A was 49 years old and underwent total hysterectomy and bilateral ovarian and fallopian tube resection for atypical endometrial hyperplasia; the epithelium of the ovarian surface layer was extracted via a fine surgical cut using a scalpel. Patient B, aged 57 years, developed breast cancer, and genetic testing revealed a BRCA1 mutation (C.405_406 del (P.Arg 136 Thrfs*5)) and was diagnosed with hereditary breast/ovarian cancer (HBOC). Patient C developed breast cancer at the age of 53 years, and genetic testing revealed a BRCA2 mutation (C.1813 del (P.lle 605 Thrfs*9)) and was diagnosed with HBOC. In Patients B and C, the ovarian epithelium was collected via abrasion from surgical specimens using risk-reducing salpingo-oophorectomy (RRSO). In none of these cases of ovarian or fallopian tube carcinoma, the serous tubal intraepithelial carcinoma (STIC) was grossly or histopathologically observed in hematoxylin and eosin (HE)-stained specimens with paraffin block embedding.
Establishment of primary cell lines
The surface of the excised ovarian tissue was cut into small pieces with a scalpel, and 10 mL of 1% collagenase + Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 with l-glutamine and sodium pyruvate (Product Number: 045-30665) was added to a 15 mL tube. The tube was incubated horizontally in a 37°C incubator (100 rpm) for approximately 24 h. Subsequently, the mixture was filtered twice into a 50 mL tube using a 70 µm strainer (Product Number: 352350). The mixture was then centrifuged at 1,300 rpm for 8 min, and the supernatant was discarded. The cells were washed with 2 mL of DMEM/Ham’s F12, and then 8 mL of DMEM/F12 was added; the mixture was transferred to a 15 mL tube and filtered into a 50 mL tube using a 40 µm (Product Number: 352340) strainer. The cells were seeded in a 60 mm dish with DMEM/Ham’s F12 + 20% fetal bovine serum (FBS) + 1.0 penicillin–streptomycin and incubated in a CO2 incubator at 37°C. The next day, blood cells were removed by washing with phosphate-buffered saline (PBS), and the cells were seeded at a density of 2.0 × 105 cells/well in a 24-well plate. On the following day, the cells were infected with an SeV vector carrying three immortalization genes (hTERT, Bmi1, and SV40T). The SeV vectors were used for gene loading and vector production by ID Pharma, Inc. Finally, the infected cells were subjected to long-term passaging.
SeV vector infection
The cells were seeded (2 × 105 cells) in 24-well plates. The next day, the cells were infected with SeV-hTERT (with simultaneous enhanced green fluorescent protein (EGFP) loading), SeV-Bmi1 (with simultaneous orange fluorescent protein (OFP) loading), and SeV-SV40T in a safety cabinet (the amount of virus used for infection is not disclosed owing to a pending patent).
Flow cytometry
The cell count was 2.5 × 105 cells/100 mL PBS + 1% FBS/well. Immortalized cells derived from human tissues (Ovn Passage + 20, Ov BRCA1 Passage + 5, Ov BRCA1 Passage + 20, Ov BRCA2 Passage + 20) were used along with positive control cells (colon cancer cell line HCT116, ovarian clear cell carcinoma cell line TU-OC-1 [11], and ovarian serous adenocarcinoma cell line TU-OS-6). Flow cytometry was performed with accutase treatment for 1–3 min when the confluency of all cell lines reached approximately 70% (Online Resource 1). The survival rate was approximately 90% for all cell lines.
The primary antibodies (1 mg/100 mL/well) used were anti-EpCAM antibody (hZAK3A101; original stock: 1.105 mg/mL; 20210505) and negative control antibody (hIgG; original stock: 1 mg/mL; Invitrogen; 12000C; S1257958). The cells were incubated at 4°C for 1 h and then washed twice with PBS + 1% FBS (200 mL/well). The secondary antibody anti-hIgG antibody Alexa 488 (original stock: 1.5 mg/mL; Jackson ImmunoResearch, 109-545-190) was added (0.075 mg/30 mL/well) to the cells, and the mixture was incubated at 4°C for 1 h and then washed twice with PBS + 1% FBS (200 mL/well).
Cloning of ovarian cells
The established cell lines were seeded at low concentrations in a 100 mm dish. The cells were observed for more than 40 days, and the area where colonies formed from single cells was observed using an all-in-one microscope to confirm that both green fluorescent protein (GFP) and OFP were detected. The cells were selected under a microscope and seeded in 96-well plates. This step was repeated multiple times to pass on more than 50 cloned cells, scaling up from 96-well plates to 24-well plates, 12-well plates, 6-well plates, 60 mm dishes, and 100 mm dishes, in that order. Cells that could be passed on were designated as cloning cells. The cloned cells were defined as those that could be passaged.
Chromosome analysis
Each cell line was seeded in a 60 mm dish, treated synchronously with MAS (Genial Genetic Solutions), and then fixed using the Carnoy’s fixation method. The fixed cells were spread on slides and stained with quinacrine mustard (QH; Sigma–Aldrich, Hoechst 33258; Sigma–Aldrich) for multicolor fluorescence in situ hybridization (mFISH) (24XCyte: MetaSystems Inc., Altlußheim, Germany), according to the manufacturer’s instructions (MetaSystems). Metaphase images were captured with an AxioImagerZ2 fluorescence microscope (Carl Zeiss GmbH, Jena, Germany) and analyzed using the Ikaros software program (MetaSystems) and ISIS software program. Chromosome karyotyping of metaphase was performed to determine the chromosome number and karyotypic structural aberrations.
Human transcriptome sequencing analysis
Total RNA was extracted using the RNeasy Mini Kit (Qiagen; cat. No. 74104, 74106), and quality control was performed using the Agilent 2200 TapeStation to confirm that there was at least 2.0 µg of RNA. Analysis was performed using NovaSeq 6000 (Illumina). Libraries were prepared using the TruSeq stranded mRNA Library Prep Kit with a library length of 332–346 bp. The read length was 101 bp and was analyzed using the multiplex method. Cluster analysis was performed using the k-means method by classifying clusters into five categories according to the slope of the expression variation ratio.
Real-time polymerase chain reaction
Total RNA was extracted from each cell line using the RNeasy Mini Kit (Qiagen; Cat. Nos. 74104 and 74106). DNase treatment was performed using deoxyribonuclease (RT Grade, Cat. No. 313–03161) and the RNaseOUT™ Recombinant Ribonuclease Inhibitor (Cat. No. 10777019).
A high-capacity cDNA Reverse Transcription Kit (Applied Biosystems; Cat. No. 4368814) was used for cDNA synthesis. Real-time polymerase chain reaction (PCR) was performed using the StepOnePlus Real-Time PCR system (Applied Biosystems) and TaqMan Fast Advanced Master Mix (Applied Biosystems) according to the manufacturer’s protocol.
Transcript levels were normalized to those of beta-actin (ACTB). The PCR conditions were as follows: 50°C for 2 min, 95°C for 20 s, followed by 40 cycles at 95°C for 1 s and 60°C for 20 s. Each reaction was performed in triplicate. The PCR primer sequences are shown in Online Resource 2. PCR primers that showed more than 2-fold gene expression in Ov BRCA1 and Ov BRCA2 compared with that in Ovn upon human transcriptome sequencing analysis were selected.
Ethics statement
All patients provided written informed consent following the institutional guidelines. All methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Ethics Committee of Tottori University (IRB number: 20A179).