Cell culture and chemicals reagents

The human ovarian cancer cell lines used for the experiments, including ES2 and OVISE, were obtained from the Shanghai Key Laboratory of Female Reproduction Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University. ES2 cells were cultured in McCoy’s 5 A medium (BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum (Excellbio, Suzhou, China) and 1% Penicillin-Streptomycin (NCM Biotech, Suzhou, China), OVISE cells were cultured in RPMI1640 medium (BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin, then maintained in an incubator with 5% CO2 at a constant temperature of 37 ℃.

Compound WX390 was obtained from Shanghai Jiatan Pharmaceutical Technology Co., Ltd (Shanghai, China). Cisplatin (CDDP) was from Selleck Chemicals (Houston, Texas, USA). Primary antibodies targeting p-AKT, AKT, p-mTOR and p-GSK3B were purchased from CST (Beverly, USA). Primary antibodies targeting mTOR, P62, LC3, Beclin1, Lamp2 and GSK3B were purchased from Proteintech (Wuhan, China). Primary antibody targeting GAPDH, Ki67, Bax and Bcl2 were purchased from Servicebio (Wuhan, China).

Cell viability detection

Cells were seeded in 96-well plates at a density of 5 × 103 and then treated with WX390 for 48 h. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8 kit, NCM Biotech, Suzhou, China), and OD value was detected at 450 nm.

Synergy determination with SynergyFinder

ES2 and OVISE cells were inoculated into 96-well plates at a density of 5 × 103 per well, and the following treatments were performed. The single drug (WX390, CDDP) or the combination (WX390 and CDDP) was analyzed according to the dose specified in the above cytotoxicity test. For ES2 cells, each drug (WX390: 0.032, 0.16, 0.8, 4 and 20 µM; CDDP: 0.39, 0.78, 1.56, 3.12, 6.25, 12.5 and 25 µM) was treated with a constant dilution ratio for 48 h; for OVISE cells, each drug (WX390: 0.032, 0.16, 0.8, 4 and 20 µM; CDDP: 0.78, 1.56, 3.12, 6.25, 12.5, 25 and 50 µM) was treated with a constant dilution ratio for 48 h. The cell viability was detected by CCK-8 kit. The drug synergistic score was calculated by “inhibition index” through response surface model and zero interaction titer (ZIP) calculation method using online SynergyFinder software (https://synergyfinder.fimm.fi) [25]. A ZIP Synergy score greater than 10 is considered a synergistic effect (red area). At the same time, a heat map of the drug combination reaction was drawn to further evaluate the drug combination.

Clonogenic assay

To test the colony forming ability of the different treatments, cells were inoculated into 6-well plates with 800 cells per well and incubated for 24 h. The cell lines were treated with different concentrations of drugs and the cells were incubated for 14 days. The culture medium was discarded, washed twice with Phosphate Buffered Saline (PBS) (BasalMedia, Shanghai, China), fixed with 4% paraformaldehyde (Servicebio, Wuhan, China), stained with 0.1% crystal violet staining solution (Servicebio, Wuhan, China), washed with PBS and then dried at room temperature. The colony formation rate was calculated.

Wound healing assay

The cells were inoculated into 12-well plates and incubated overnight. After 48 h of treatment with different drugs, scrape the wound with a 100µL pipette and replace the medium with a fresh medium without bovine fetal serum. Images of the migration state at the same location in the hole were taken at 0 h and 48 h using a light microscope. The wound area was calculated by ImageJ software. The rate of cell migration was obtained using wound healing area.

Cell apoptosis assessment

The cells were inoculated in a 6-well plate. Cells (including cells in the culture supernatant) were collected 48 h after drug treatment. The cells were re-suspended with 1x Binding Buffer, 5 µL Annexin V-APC and 10 µL propidium iodide (Liankebio, Hangzhou, China) were added to each tube, gently mixed in a vortex, and incubated at room temperature for 5 min away from light. Apoptosis was evaluated by flow cytometry, and FlowJo software was used to analyze the results.

Cell cycle analysis

The cells were inoculated in a 6-well plate. After 48 h of drugs treatment, cells were treated with cell cycle assay kit (Liankebio, Hangzhou, China) and cell cycle progression was detected by flow cytometry.

EdU proliferation assay

For EdU assays, cells were inoculated in 12-well plates and treated with different drugs for 48 h. The results were evaluated using an Edu test kit (Beyotime, Shanghai, China) and observed using fluorescence microscopy.

Western blot analysis

After washing the cells with cold PBS, total proteins were extracted using a RIPA buffer containing protease and phosphatase inhibitors (Beyotime, Shanghai, China). The protein concentration was quantified using BCA protein assay kit (Beyotime, Shanghai, China). The protein loading buffer was added to the lysate, boiled at 100℃ and stored at − 80 ℃. Each protein sample of 30 µg was separated by SDS-PAGE gel and then transferred to NC membrane. The membrane was closed with TBST of 5% skim milk at room temperature for 1 h and then incubated with primary antibody at 4 ℃ overnight. Finally, the membrane was incubated with the corresponding secondary antibodies at room temperature for 1 h. Western Blot was detected by enhanced chemiluminescence (NCM Biotech, Suzhou, China).

Construction of patient-derived xenografts (PDX)

The animal study protocol was approved by the Institutional Review Board of Department of Laboratory Animal Science Fudan University (protocol code: 201904008Z). Female immunodeficient NCG mice (NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt) aged 5 weeks were purchased from GemPharmatech CO., Ltd (Jiangsu, China). The NCG mice were all raised free of specific pathogens (SPF). To construct the PDX model, fresh patient specimens were collected, cut into 2 × 2 × 2 mm fragments, and then implanted under the skin of NCG mice, with 4–5 fragments per mouse. When the tumor size reached 1500 mm [3], it was resected for cohort expansion. The animal experiment was randomly divided into four groups (control, WX390, CDDP and combined therapy of WX390 and CDDP) with five animals in each group. WX390 was given intragastric at 0.2 mg/kg once a day; CDDP was administered intraperitoneally at 3 mg/kg twice weekly. The baseline and clinicopathological information of the patients included in the construction of the PDX model is shown in Table S1.

Immunohistochemistry and H&E staining

PDX tissues and major organs of mice (heart, liver, spleen, lungs, and kidneys) were collected, fixed with 4% paraformaldehyde, tissue treated, and paraffin embedded. The above sections were stained with hematoxylin and eosin. For IHC, the tissue sections were treated blocked and incubated overnight with primary antibodies as follows: Ki67, Bax, Bcl2. The stained sections were imaged under an inverted phase contrast microscope.

Reactive oxygen species assay kit (DCFH-DA)

Cells were seeded in six-well plates, incubated overnight, and treated with the corresponding drugs for 48 h. DCFH-DA (Beyotime, Shanghai, China) was diluted with serum-free culture solution at 1:1000, and the collected cells were suspended in the diluted DCFH-DA and incubated in a cell incubator at 37 ℃ for 20 min. The cells were washed with serum-free cell culture solution three times and detected by flow cytometry.

RNA-seq data processing

Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reversely transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). The purified double-stranded cDNA fragments were end repaired, A base added, and ligated to Illumina sequencing adapters. The ligation reaction was purified with the AMPure XP Beads (1.0X). And polymerase chain reaction (PCR) amplified. The resulting cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

Data analysis and statistics

All statistical analyses were performed using Prism 10.1.0 (GraphPad, La Jolla, USA) and SynergyFinder software [25]. Using R (version 4.2.1) to analyze RNA-seq data. All experiments and analyses were performed in triplicate and the result were presented as mean ± standard deviation (SD). The differences between different groups were analyzed using Student’s t-test, unpaired t-test and one-way ANOVA. Statistical significance was *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.