Cell lines and cultures
Vero (African green monkey kidney) was purchased from the American Type Culture Collection (ATCC). Human BC cell lines including MDA-MB-231, MDA-MB-468 and BT549 were obtained from the Cell Bank of Type Culture Collection Chinese Academy of Sciences. The cells were cultured in cell culture flasks (Corning) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) or RPMI 1640 medium (BT549). These media were supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco), 1% L-glutamine, and 1% penicillin/streptomycin (Gibco). The cultures were incubated in a humidified atmosphere with 5% CO2 at 37 °C.
Amplification of measles virus vaccine strain
The virus stock was generated by infecting VERO cells cultured in Opti-MEM (Gibco) at a multiplicity of infection (MOI) of 0.1. About 24–36 h later, after the cells had fused into syncytia, the freeze-thaw procedure was repeated three times on dry ice. The samples were centrifuged (4 °C, 4500 g, 10 min), and the supernatants were collected. The virus stocks were quantified using a plaque-forming unit (PFU) assay on VERO cells.
Detection of CD46 and Nectin-4 expression levels
A total of 1 × 106 cells were analyzed by flow cytometry (BD Biosciences) following the protocols of the antibody identification kit. Briefly, after culturing for 48 h, the BC cells were digested with 0.25% trypsin without EDTA (Gibco, 15090-046; 1mL/T25) for 5 min. The cells were centrifuged at 800 g for 3 min at 25 °C, then resuspended and washed twice with sterile PBS. The BC cells were blocked with 2% BSA (in 1× PBS) on ice for 40 min, then 10 µl/sample of PE-Mouse anti-human IgG (BD 555787) was added, with PE mouse anti-human CD46 antibody (BD 564252) or PE mouse anti-human Nectin-4 antibody (BD 564252). The samples were gently mixed with a pipette and incubated on ice in the dark for 40 min. Then the cells were centrifuged again under the same conditions, washed twice with PBS, and resuspended in 600 µl PBS. Flow cytometry analysis was performed using the PE channel.
Cell viability assays
5 × 103 cells /well of BC cells seeded in 96-well plates were infected with multiple MOIs (0, 0.1, 0.5, 1, 5, 10) of rMeV-Hu191, and cell viability was quantified every 24 h with a Cell Counting Kit-8 (CCK8, TargetMol). For assessment, cells were cultured with 100 µl working medium consisting of 90 µl DMEM and 10 µl reagent, for 30 min at 37 °C, and the absorbance at 450 nm was measured using a multifunctional microporous plate detector (TECAN-SPARK).
Cytotoxicity of rMeV-Hu191 in vitro
The BC cells (2 × 105 cells/well) seeded in 6-well plates were infected with rMeV-Hu191 at multiple MOIs (0, 0,1, 0.5, 1, 5, and 10). After 96 h post-infection, the medium was removed and the cells were fixed with 4% paraformaldehyde at room temperature for 2 h. Paraformaldehyde was discarded, and the remaining cells were stained with 0.1% crystal violet for 7 min, rinsed with water, and photographed under a scanner (Canon).
Cell death analysis by flow cytometry
The BC cells (2 × 105 cells/well) seeded in 6-well plates were exposed to rMeV-Hu191 at an MOI of 0.1, with or without 50 µM Z-VAD-FMK (A1902, ApexBio), and then harvested for 48 and 72 h. To assess apoptosis, flow cytometry was conducted using an annexin V-fluorescein isothiocyanate (annexin V-FITC) apoptosis detection kit from BD Bioscience following the manufacturer’s protocol. Briefly, following rinsing with PBS, 1 × 105 cells were resuspended in 100 µl of binding buffer, and then treated with 5 µl each of propidium iodide (PI) and annexin V-FITC for 15 min in the dark at room temperature. Analysis was performed with a flow cytometer (BD).
Western blot
Proteins were extracted using RIPA lysis buffer (Beyotime Biotechnology) and quantified using a BCA detection kit (Beyotime Biotechnology) according to the manufacturer’s instructions. Equal amounts of protein were loaded onto SDS-PAGE gels and electrophoretically transferred onto PVDF membranes (Bio-Rad). The membranes were then blocked with 5% nonfat milk in Tris-buffered saline for 1 h at room temperature. The membranes were then incubated with specific primary antibodies overnight at 4 °C, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein signals were visualized using an enhanced chemiluminescence reagent (Biological Industries) and captured using a chemiluminescent detection system (Gene X). Reagents for Western blot analysis: anti-caspase-3 (CST, 9665 S, 1:1000), anti-caspase-7 (CST, 9492, 1:1000), anti-PARP (CST, 9542 S, 1:1000), anti-β-actin (CST, 4970 S, 1:5000), anti-CDK4 (Abcam, ab108357, 1:2000), anti-cyclin D1 (Abcam, ab134175, 1:2000), anti-β-tubulin (CST, 2146 S, 1:10000), anti-measles nucleoprotein (anti-MV-N, Abcam, ab106292, 1:5000), anti-GAPDH (CST, 5174 S, 1:5000).
5-Ethynyl-2′-deoxyuridine (EdU) cell proliferation assays
2 × 105 cells/well of BC cells seeded in 6-well plates were infected with rMeV-Hu191. Cell proliferation was measured using a BeyoClick™ EdU-594 Cell Proliferation Assay Kit (Beyotime Biotechnology, #C0078S). 20 µM pre-warmed 2X EdU working solution was added, and cells were incubated for 4 h. Cells were then fixed with 1 mL of 4% paraformaldehyde for 15 min, washed three times, and permeabilized with 0.3% Triton X-100 in PBS for 10 min. After two washes, the Click reaction solution was added (0.5 mL per well) and incubated in the dark for 30 min. Nuclei were stained with 1X Hoechst33342 solution for 10 min, followed by three washes. Fluorescence detection was performed using a Zeiss Observer Z1 fluorescence microscope, using the DAPI channel for Hoechst 33,342 and the RFP channel for EdU-labeled cells.
Immunofluorescence
The cells were planted in glass coverslip-bottomed chambers (Corning), and treated with vehicle or oncolytic measles at an MOI of 0.5 for 36 h. Following two rinses with PBS, the cells were fixed in 4% paraformaldehyde at 25 °C for 20 min and subsequently permeabilized with 1% Triton X-100 in PBS at 25 °C for 10 min. The primary antibodies used were anti-MV-N (Abcam, ab106292, 1:500) and anti-Ki67 (Abcam, ab15580, 1:500). The cells were washed with PBS three times and incubated with a fluorescent secondary antibody at room temperature for 1 h. After washed with PBS, the cells were stained with DAPI (Beyotime Biotechnology, C1002) for 10 min. Then, the cells were observed under Zeiss Observer Z1 fluorescence microscopy.
Senescence-associated β-galactosidase (SA-β-gal) staining
The BC cells (2 × 105 cells/well) seeded in 6-well plates, were placed with glass crawlers in advance, and then were infected with rMeV-Hu191 at an MOI of 0.1. At 24–36 h post-treatment, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min, washed with PBS twice, and incubated at 37 °C with fresh SA-β-gal stain solution (Beyotime Biotechnology) for 12 h. After staining, the cells were washed in PBS and then compressed between glass slides for observation under a light microscope (Zeiss Observer Z1).
Total intracellular ROS detected by fluorescence microscopy
Intracellular ROS signaling was tested by an ROS assay kit (Beyotime Biotechnology, #S0033). After treated with rMeV-Hu91 at an MOI of 0.5 for 36 h, the cells were incubated with 10 µM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH‐DA) for 20 min at 37 °C and then were washed with DMEM three times. Then, the fluorescence distribution was detected by a Zeiss Observer Z1 fluorescence microscope.
Flow cytometry for intracellular ROS measurement
The treatment of rMeV-Hu191 was described as above. After being infected with rMeV-Hu191 for 24–36 h, the BC cells were dispersed into single cells by 0.25% Trypsin-EDTA (Gibco) and incubated with 10 µM DCFH-DA for 20 min at 37 °C. Then, the fluorescence intensity and proportion of positive cells were measured in the FITC channel by a flow cytometer (Navios, Beckman Coulter, USA), and the results were analyzed using FlowJo 10.0 software (Tree Star Inc, USA).
Detection of mitochondrial membrane potential (MMP)
MMP was detected with a JC-1 Kit (Beyotime Biotechnology, #C2006). In brief, after treatment with or without rMeV-Hu191, the BC cells were harvested with trypsin and suspended in PBS. JC-1 staining solution was obtained by diluting 50 µL JC-1 (200X) with 8 mL ultrapure water, and then 2 mL JC-1 staining buffer (5X) was added. The BC cells were stained with 1 mL JC-1 staining working solution and incubated at 37 °C for 20 min. JC-1 staining buffer (5X) was diluted with distilled water (1:4) to prepare a 1X buffer. After incubation, the supernatant was removed, and cells were washed twice with 1X JC-1 staining buffer. Then 2 mL cell culture medium was added. Finally, each group was analyzed with a Zeiss Observer Z1 fluorescence microscope in the GFP and cyc3 channels. The images were analyzed using ImageJ software (Version 2.0.0).
RNA isolation and reverse transcription quantitative real-time PCR (RT-qPCR)
BC cells (2 × 10⁵ per well) were seeded in 6-well plates and infected with rMeV-Hu191 for 48 h. Total RNA was extracted using TRIzol (Thermo Fisher). Cells were lysed with 1 mL TRIzol per 10 cm², incubated for 5 min, then mixed with 500 µL chloroform per 1 mL TRIzol and centrifuged at 12,000 g for 15 min at 4 °C. The aqueous phase was mixed with 500 µL isopropanol per 1 mL TRIzol and centrifuged at 12,000 g for 10 min at 4 °C. The RNA pellet was washed with 70% ethanol, air-dried, and dissolved in double-distilled water. Then the RNA was reverse-transcribed using Prime ScriptTM RT Master Mix (Takara). Quantitative real-time PCR was performed using TB Green® Premix Ex Taq™ (Takara) on a real-time PCR system (ABI-7500 Step-One Plus). The relative expression was normalized to that of GAPDH by the 2-ΔΔCt method. The primer sequences are listed in Table S1.
Transcriptome sequencing
RNA samples (triplicate) from control or rMev-Hu191–treated MDA-MB-231 (MOI = 0.1 each for 48 h) were isolated using TRIzol. For RNA library preparation, PolyA-selection was used with a target fragment size of 300–500 bp. PCR products were cleaned with AMPure XP beads by mixing 65 µl of beads with 100 µl of PCR product, incubating, and separating on a magnetic rack. Beads were washed with ethanol, air-dried, and RNA was eluted with NF-H2O. Libraries were pooled and sequenced using the Illumina NovaSeq 6000 platform. Data quality was evaluated, and libraries were rebuilt if needed. Data processing included RNA-seq analysis, differential expression analysis with DESeq2 (v1.4.5), and functional enrichment analysis using the BGI Dr.Tom system.
Immunohistochemistry
The tumor tissues were fixed in 4% formalin overnight and embedded in paraffin. Slides were treated with xylene I and II for 15 min each, then rehydrated with ethanol gradients (100%, 85%, 75%) and distilled water. Antigen retrieval was performed by heating slides with citrate buffer (95–99 °C, 10 min), EDTA (95–99 °C, 15 min), or TE buffer (95–99 °C, 18 min), followed by cooling. Pepsin digestion was conducted at 37 °C for 10 min. The tumor slices were stained with the primary anti-Ki67 (Abcam, ab15580, 1:500) at 4 °C overnight. After washed with PBS three times, all slices were incubated with the appropriate concentrations of biotinylated secondary antibodies and imaged using DAB solution. Finally, the slices were observed under a microscope (Zeiss Observer Z1).
Statistical analysis
Each experiment was conducted in triplicate, and three independent experiments were performed. Data analyses were conducted using one-way ANOVA followed by the Bonferroni post hoc test. Pairwise comparisons were performed using a two-tailed unpaired t-test. Survival curves were plotted using Kaplan-Meier analysis with the log-rank Mantel-Cox test. All statistical analyses were performed using GraphPad Prism 9.4. Quantification data were represented as mean ± standard error, with p-value thresholds set at *, 0.05; **, 0.01; ***, 0.001; and ****, 0.0001.
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