2.1 Cell lines and cell culture
Human ovarian cancer cell lines (HOC7、OVCAR8) were obtained from MDACC characterized Cell line Core Facility. Human ovarian cell lines (A2780, ES2, SKOV3, OVCAR3, Caov3, OV90, TOV-112D, TOV-21G) were obtained from the American Type Culture Collection (ATCC). ID8 is a mouse ovarian cancer cell line derived from C57BL/6, which was a gift by Professor K. Roby (Department of Anatomy and Cell Biology, University of Kansas, U.S.A).
Cell lines were all passaged less than 30 times and were cultured under 37℃,5% CO2 incubator. 3D cell was cultured in the same condition with PDO models.
2.2 Antibodies and compounds
Olaparib(S1060)、AZD5153(S8344) were bought from Sellek. IdU (1336001) and CIdU(C6891)were from sigma. CELLTiter GLO 3D (G9682) were from PROMEGA. Components added in the PDO culture medium are all bought from BD Biosciences. GAPDH (A19056), β-TUBULIN (AC008), α-TUBULIN (A6830), RFC4 (A5485), SMC1A(A4693) antibodies were from Abclonal. PTEN (ab267787), RFC3 (ab182143), P-SMC1A (ab75768), RAD51 (ab133534), P-CHK317 (ab226929), PI3K(ab40776), BRCA1(ab238983), BRCA2(ab239375) antibodies were from Abcam. Brd4 (#83375), γH2AX (#80312, #9718), RPA32 (#52448), P-RPA32 (#83745) antibodies were from Cell Signaling Technology (CST).
2.3 Clinical Specimens
All primary ovarian cancer tissues are anonymized and obtained from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, in accordance with the Declaration of Helsinki. All the operations were approved by the Ethics or Institutional Review Board.
2.4 Establishment of PDX model
PDX (Patient-derived xenografts) models were obtained by subcutaneously transplanting fresh tumor tissue into nude mice. 6-8 weeks-old female BALB/C nu-mice were purchased from Beijing HFK Bioscience and raise in specific pathogen-free conditions. All manipulations were performed under the guidance of the Animal Laboratory of Tongji Hospital. Solid tumor xenografts are passaged with the same technique after established.
2.5 Establishment of PDO model
PDO (Patient-derived organoids) models were established by using patient tissues from Tongji Hospital. Fresh tumor tissue can be stored in DMEM/F12 (1% PS) under 4 ℃ within 12 hours. The tumor tissue was minced and filtered through 70um (Falcon, #352360) and 40um (Falcon, #352340) strainer, to get a suspension of multicell-spheroid, which has a diameter between 40-100um. After treated by red cell lysis buffer and washed by PBS, the multicell-spheroid was resuspended by Matrigel (Corning, #356231). The suspension was planted into 6 or 96 well plates according to the following experiments. For example, 10 µL Matrigel with 5000 organoids per well was used for short drug screening. The Matrigel needs to solidify at 37℃ for 30 minutes, then the culture medium was added to the plates.
PDO was cultured in DMEM/F12 with Glutanmax 1×, HEPES 1×, R-spondin 100 ng/ml, Noggin 100 ng/ml, EGF 50 ng/ml, FGF10 10 ng/ml, FGF2 10 ng/ml, B27 50×, Nicotinamide 10 mmol/ml, N-Acegglytene 25 mmol/ml, ProstaglandinE2 1 umol/ml, SB02190 10 umolg/ml, A8301 500 nmol/ml and Y27632 10 µmol/ml.
2.6 Generation of PARP inhibitor resistant cells
A2780、HOC7、ID8 cells were cultured with an increased concentration of Olaparib. After 3-4 months of treatment, these cells can grow rapidly in the presence of 10uM Olaparib. Cells were cultured in the absence of Olaparib for 1 month. Before use, IC50 was calculated again to confirm its drug resistance.
2.7 Alkaline single-cell agarose gel electrophoresis (Comet) assay
Alkaline comet assay was performed by following the manufacturer’s instructions of Trevigin’s Comet Assay Kit (#4250-050-K). Cells were suspended in Low melting agarose and mounted on comet slides as instructed. After the gel was solidified, the comet slide was incubated in lysis solution for 1 hour at 4℃ and the freshly prepared unwinding solution for 20 minutes at room temperature in a dark place. Electrophoresis was performed under 21V for 25 minutes in the freshly prepared electrophoresis solution. Slides can be stained with SYBR Green I to analyze the comet tail, which stands for DNA strand breakage. Average damage from three independent experiments was calculated.
2.8 DNA fiber assay
Cells were labeled with 25uM CIdU for 30 minutes, washed by PBS for 3 times, and then labeled with 250uM IdU for 45 minutes. After labeling, cells were collected, resuspended to 5*10^5 cells/ml in ice-cold PBS. 7ul freshly prepared spreading buffer was mixed with 2ul cell suspension and pipetted onto a microscope slide. By carefully tilt the slides at 25-60 degrees, the stream of DNA was allowed to travel slowly down the slide. Then the slides were airdried and fixed in methanol/acetic acid (3:1) for 10 minutes. Slides were then washed with ddH2O, denatured in 2N HCL for 30 minutes, and block with 5% BSA-PBS. After that, the slides were incubated with 1:150 rat anti-BrdU (Abcam, ab6326) and 1: 50 mouse anti-BrdU (BD Biosciences, #347580) antibody for 3 hours at room temperature. Then the slides were rinsed and incubated in 1:150 anti-Rat AlexaFluor 488 antibody and 1:150 anti-Mouse AlexaFluor 568 antibody for 1 hour at room temperature. The results were obtained by using Zeiss Laser Scanning Confocal Microscope 880.
2.9 Fluorescence in situ hybridization (FISH) assay
The slides were pretreated with xylene and gradient ethanol to dewaxing and hydration, boiled, digest with 200µL pepsin solution, and then put into 2xSSC at room temperature for 3 minutes. Dehydration with gradient ethanol for 2 times and dried at room temperature. Samples and probes were hybridized in an environment protected from light. Then the slides were washed and counterstained.
The probes(LBP Guangzhou,# F.01005-01)can hybrid with chromosome 10 centromere (green signal) and PTEN gene (red signal). The normal cell contained 2 red and 2 green signals. A PTEN amplificated signal mode contains more red signals.
2.10 Metaphase spread assay
Cells were exposed to colchicine (100 ng/ml) (Sellek, S2284) for 3 hours, collected and resuspension in hypotonic solution (0.075M KCl) for 30 minutes at 37°C incubator. Cells were then fixed in methanol: acetic acid (3:1) at 4℃ for 30 minutes and repeat for 3 times. Then the fixed cells were dropped on precooled slides and put into a 65℃ incubator to air-dried. After cooled down, the slides were stained in 3% Giemsa and coded for blind analysis. A total of 25 metaphases was analyzed from each sample to detect the presence of chromosomal fragments.
2.11 NCI60, CCLE and GSCALite
Gene expression profiles (Gene transcript level z score) for correlations analysis in NCI60 human tumor cell lines were obtained using the web-based tool provided by CellMiner (http://discover.nci.nih.gov/cellminer/).
Gene mutation data of cell lines was collected from Cancer Cell Line Encyclopedia (CCLE) (https://portals.broadinstitute.org/ccle/data/browseData?conversationPropagation=begin).
The correlation between gene expression and drug reaction of human tumor cell lines were obtained by using the web-based tool provided by GSCALite (http://bioinfo.life.hust.edu.cn/web/GSCALite/). The gene expression and drug reaction data were collected by GSCALite from CCLE, CTRP and GDSC.
2.12 ChIP-Seq Analysis
ChIP-seq data for human cell lines from PMID 27803105, PMID 29491412 and GSM2090919, GSM 2090922 was collected from Cistrome (http://cistrome.org/db/#/) and analyzed by UCSC genome browser (http://genome.ucsc.edu/).
2.13 Crispr screening and mass spectrum data
Crispr screening data of Olaparib acquired resistant cell lines was downloaded from PMID29973717 and the mass-spectrum data (ID 013196) was downloaded from proteome-central(http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD013196). The pathway enrichment of the data was done by using web-based tool provided by DAVID (https://david.ncifcrf.gov/summary.jsp).
Total RNA (1 µg) was reversely transcribed into cDNA with the Hiscript Q-RT SuperMix for qPCR (Vazyme Biotech). According to the manufacture instructions. Real-Time PCR Master Mixes kit (Life Technologies) was used for the thermocycling reaction in a BioRad CFX96 Real-Time system. The mRNA levels analysis was carried out in triplicate and normalized by GAPDH. Primers sequences of PTEN were as listed.
2.15 Virus transfection protocol
In a six-well culture plate, cells are cultured at 50%-70% confluency in an antibiotic-free growth medium supplemented with FBS. The mixture was gently mixed and incubated for 30min at room temperature. Use the antibiotic-free growth medium to wash the cells twice. Add 0.2ml virus to well for each transfection. Add 40 ul Transfection Reagent Complex to well, covering the entire layer and gently swirling the plate. Incubate the cells at 37℃ in a CO2 incubator for 6 hours. Add 1ml normal growth medium containing 2 times FBS and antibiotics into each well, and incubate for 18-24 hours in 37℃, 5% CO2 incubator. Use puromycin to select stably transfected cells. PTEN shRNA and PTEN overexpression virus are bought from Genechem.
2.16 Western Blot assay
Cells were lysed with RIPA buffer (Servicebio, G2002-100) containing protease and phosphatase inhibitor cocktail (Servicebio, G2002-100). The lysates were centrifuged at 12000rpm 4℃ for 20 minutes to collect supernatants. After determining the protein content, the cell lysates were separated by 10% SDS-PAGE and electro-transferred onto 0.45um PVDF membranes. The membranes were blocked with 5% BSA-TBST at room temperature and then incubated with primary antibodies at 4℃ overnight. Secondary antibody (Antgene) was incubated for 1 hour at room temperature. Bands were visualized by using WesternBright ECL Kit (Advansta, 190113-13).
Immunohistochemistry was performed as previously described11. The primary antibody included Ki67(Abcam,1:500), RAD51(Abcam,1:500), γH2AX(Abcam,1:500).
Tumor cell staining was assigned a score using a semi-quantitative grading system: 0, 0–5% tumor-cell staining; 1, 5–25% tumor-cell staining; 2, 26–50% tumor-cell staining; 3,51–75% tumor-cell staining; and 4, >75% tumor-cell staining. Staining intensity was assigned a score using a semi-quantitative four-category grading system: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Every core was assessed individually and the mean of three readings was calculated for every case. The tumor cell staining score was determined separately by two independent experts simultaneously under the same conditions. In rare cases, discordant scores were reevaluated and scored based on consensus opinion.
2.18 In vivo small animal imaging technology
C57BL/6 mice were purchased from Beijing HFK Bioscience and raise in specific pathogen-free conditions. ID8 cells are planted intraperitoneally into mice.
Mice were anesthetized intraperitoneally with 4% chloral hydrate (g / ml) at a dose of 150ul / 20g body weight. 5mg of the fluorescent substrate was injected intraperitoneally for 10 minutes. The anesthetized and injected mice were placed in the instrument. The images were collected by the small animal imaging instrument (SIEMENS Inveon), and the same low and high values of fluorescence signals were set for each group.
2.19 Statistical analysis
GraphPad Prism 8 and IBM SPSS statistic 26.0 were used for statistical analysis. P < 0.05 was considered to indicate a significant difference.