Cell culture and transfection

OVCAR3, SKOV3 and CAOV3 cells were cultured in DMEM (Hyclone) containing 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO2 at 37℃. These cells were transfected with shFZD5 lentiviruses (GV112/hU6- MCS-CMV-Puromycin, Genechem, China) to stably knockdown FZD5 expression. Puromycin (2 µg/ml, Sigma) was used to select cells at 48 h after infection. The target sequence for shFZD5-1 is 5′-CGGCATCTTCACGCTGCTCTA-3′, for shFZD5-2 is 5′-GGCCACCTTCCTCATCGACAT-3′, and for control is 5′-TTCTCCGAACGTGTCACGT-3′. FZD5-knockdowned cells was transfected with ALDH1A1 overexpression plasmid (GV219/CMV-MCS-SV40-Neomycin, Genechem, China) according to the manufacturer’s instructions.

Western blot

Cells are lysed using RIPA lysis buffer containing 1% PMSF for 1 h on ice. The cell lysates were centrifuged at 12,000×g for 40 min at 4℃. The protein concentration of the supernatant was determined using a BCA assay. Total protein lysate (30 µg) was separated by gel electrophoresis on a 12% SDS-PAGE gel and transferred to a PVDF membrane. The membranes were blocked in Tris-buffered saline-Tween 20 (TBST) containing 5% skimmed milk for 2 h at room temperature. The membranes were incubated with primary antibodies at 4℃ overnight. Subsequently, the membranes were washed 3 times with TBST and then incubated with secondary antibodies for 2 h at room temperature. The chemiluminescence (ECL) detection system (Tanon 5200, Shanghai, China) was applied for the imaging of the target protein expression. The following primary antibodies were used: FZD5 (Cell Signaling Technology, #5266, USA, 1:1000), E-cadherin (Cell Signaling Technology, #3195, USA, 1:1000), ALDH1A1 (Santa Cruz, sc-374,076, USA, 1:500), BRCA1 (Cell Signaling Technology, #9010, USA, 1:1000), RAD51 (Abcam, ab133534, UK, 1:1000), active β-catenin (Cell Signaling Technology, #8814, USA, 1:1000), Akt (Cell Signaling Technology, #9272, USA, 1:1000), pAkt (Cell Signaling Technology, #9271, USA, 1:1000), pErk1/2 (Cell Signaling Technology, #4370, USA, 1:1000), and pJnk1/2 (Cell Signaling Technology, #9251, USA, 1:1000).

Immunohistochemistry

25 OC tissues and 7 normal ovarian tissues were collected from Tumor hospital of China medical university with the informed consent of the patients. The use of the specimens for research was approved by Institutional Research Ethics Committee of Tumor hospital of China Medical University (KY20230905). The tissues were fixed in 4% paraformaldehyde, embedded in paraffin and then sliced into 4 μm sections. Xylene and gradient alcohol were used to deparaffinize and hydrate, respectively. 3% H2O2 was used to eliminate endogenous peroxidase activity. Sections were incubated with anti-FZD5 antibody (Abcam, ab75234, UK, 1:200) overnight at 4 °C and corresponding second antibody at 37 °C for 30 min. Subsequently, sections were stained with DAB. Finally, sections were examined under a microscope (Leica, USA, DM2500 LED).

Survival analysis

Correlation of FZD5 with overall survival (OS) and progression-free survival (PFS) in OC was analyzed using GSE26193 database at Kaplan-Meier Plotter website (https://kmplot.com/analysis/). Correlation of ALDH1A1 with OS and PFS in OC was analyzed using GSE26712 database at Kaplan-Meier Plotter website.

Immunofluorescence

Cells were inoculated on slides in 24-well plates at 40–50% confluence and grown in an incubator at 37℃ for 24 h, fixed with paraformaldehyde for 20 min and washed with PBS three times. Next, cells were permeabilized with 0.5% Triton X-100 for 5 min at room temperature, blocked with 5% donkey serum for 1 h at room temperature and incubated with primary antibody at 4℃ overnight; the following primary antibodies were used: E-cadherin (Cell Signaling Technology, #3195, USA, 1:200), EPCAM (Immunoway, YM6053, USA, 1:100), γH2AX antibody (Cell Signaling Technology, #9718, USA, 1:400), and active β-catenin (Cell Signaling Technology, #8814, USA, 1:800). Subsequently, slides were incubated with fluorescent-labeled secondary antibody for 2 h. Nuclei were stained with DAPI in the dark and visualized using a laser scanning confocal microscope.

Phalloidin staining

Cells were fixed with 4% paraformaldehyde at room temperature for 10 min. Slides were washed three times with PBS, and incubated with 0.5% Triton X-100, then treated with TRITC-conjugated Phalloidin solution (YEASEN, Shanghai, China) at room temperature for 30 min. Nuclei were stained with DAPI. Cell morphology was visualized by laser scanning confocal microscopy.

Correlation analysis of gene expression

Gene expression at mRNA levels was investigated in a panel of human OC cell lines using data from the CCLE database. Correlation analysis of gene expression levels, including those of FZD5, CDH1, EPCAM, ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, and ALDH1L2, was performed using the Pearson statistic.

Cell proliferation assay

Cells (OVCAR3: 103; SKOV3: 2 × 103; CAOV3: 103) were resuspended in 100 µl medium, seeded into 96-well plates, and incubated for the indicated length of time. Then, cells were treated with 10 µl Cell Counting Kit-8 reagent (CCK8, Dojindo Molecular Technologies, Japan) and incubated at 37℃ for 4 h. Absorbance values at 450 nm were measured at different time points using a microplate reader (Bio-Rad Laboratories, USA).

Colony formation assay

Cells (2 × 103) were seeded and cultured in 3.5 cm culture dishes for 2 weeks until visible colonies formed. Cells were then fixed with 4% paraformaldehyde for 20 min. Colonies were washed with PBS, stained with 1% crystal violet for 20 min, counted, and photographed.

Cell cycle assay

Cells (1 × 106) were collected, washed with PBS, and fixed with 70% ethanol at 4℃ overnight. Then, cells were treated with 500 µl PI/RNaSeA staining solution (KeyGEN BioTECH, China) for 1 h at room temperature in the dark. Samples were analyzed using a FACS Calibur Flow Cytometer (BD, USA).

Real-time PCR

RNAiso Plus (Takara, China) was added to collected cells to extract total RNA, following the standard instructions. After RNA quantification, reverse transcription was performed with 1 µg RNA using a cDNA synthesis Kit (Takara, RR047A, China). Then, real-time PCR was performed using a TB Green™ Premix Ex Taq II kit (Takara, China) and an ABI PRISM 7300 Sequence Detection system (Applied Biosystems, USA). GAPDH was used as an internal control. Gene expression was analyzed by the 2−ΔΔ Ct method. The primers used are listed in Table 1.

Table 1 Primers for real-time PCRALDH+ cell subpopulation assay

Cells (1 × 106) were suspended in 1 ml ALDEFLUOR™ Assay Buffer (STEMCELL Technologies, USA) and 5 µl ALDEFLUOR™ Reagent was added in each tube. Then, the mixture was divided into two equal parts: diethylaminobenzaldehyde was added into one part as negative control. After incubation at 37℃ for 45 min, cells were re-suspended in 500 µl ALDEFLUOR™ Assay Buffer. Samples were analyzed using a FACS Calibur Flow Cytometer (BD, USA).

Extreme limiting dilution analysis

Cells (100, 50, 25, 10/well) were cultured in complete MammoCult™ Human Medium (STEMCELL Technologies, USA) in 96-well ultra-low-attachment plates (Corning, USA) for 7 days. Stemness was evaluated using a tool available at https://bioinf.wehi.edu.au/software/elda/.

HR repair assay

Cells were transiently co-transfected with DR-GFP and I-SceI plasmids (Genechem, China) using Lipofectamine 3000 (Invitrogen), according to the manufacturer’s instructions, then cultured for 3 days. The percentage of GFP+ cells was analyzed using a FACS Calibur Flow Cytometer (BD, USA).

Cytotoxicity assay

After inoculation into 96-well plates at a density of 5000/well overnight, cells were treated with cisplatin or olaparib at different concentrations for 48 h. Then, cells were treated with 10 µl CCK8 reagent (Dojindo, Japan) and incubated at 37℃ for 4 h. Absorbance values at 450 nm were measured using a microplate reader (Bio-Rad Laboratories, USA).

Tumorisphere toxicity assay

Cells (5 × 103) were treated with or without cisplatin (5 µM) in complete MammoCult™ Human Medium (STEMCELL Technologies, USA) in 6-well ultra-low-attachment plates (Corning, USA) for 7 days. Tumor spheres were counted in five randomly selected fields.

Apoptosis assay

Cells (1 × 106) were collected and washed with PBS and an Annexin V-PE/7- AAD Apoptosis Kit (KeyGEN BioTECH, China) used to analyze cell apoptosis. Cells were incubated in 500 µL binding buffer with 5 µL 7-AAD at room temperature in the dark for 15 min. The number of apoptotic cells was measured using a FACS Calibur Flow Cytometer (BD, USA).

Mice and in vivo analysis

Female BALB/c nude mice (5–6 weeks old, 18–20 g) were purchased from Weitong Lihua (Beijing, China) and maintained in the specific pathogen free-grade facility of the Department of Animal Experimentation of China Medical University. All studies involving mice were approved by the Animal Ethics Committee of China Medical University (CMU2021249). After resuspension in 100 µl PBS, 1 × 106 OC cells were injected subcutaneously into the lateral flanks of the mice (n = 5 per group). Tumor volume was calculated using the formula: V = ½ × length × width2. Tumor length and width were measured every 3 days using a Vernier caliper. Four weeks after inoculation, mice were euthanized and the tumors harvested. For in vivo chemo-sensitivity assay, mice were administered cisplatin (5 mg/kg, intraperitoneal injection) for five consecutive days, once the tumor volume reached approximately 50 mm3.

Statistical analysis

Data are presented as mean ± SD and were analyzed using GraphPad Prism 9. Differences were analyzed by two-sided Student’s t-test, or one-way or two-way ANOVA. P < 0.05 was considered significant.