Cells and virus
Three human CSCC cell lines (C33A, SiHa, and CaSki) were purchased from the American Type Culture Collection (ATCC). All cells were maintained in 5% CO2 at 37 ℃ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (All from Thermo Fisher Scientific).
CVB3/2035A strain (GenBank Accession No. KY286529.1) was isolated in 2008 from a throat swab of a patient with mild hand, foot and mouth disease at the Centers for Disease Control and Prevention in Xiamen, China. The virus was then propagated in Hela cells (ATCC). Virus titers were determined in Hela cells using the TCID50 assay according to the Reed-Muench method.
In vitro viral infectivity assay
C33A, SiHa, or CaSki cells were seeded in 96-well plates in DMEM without FBS and then infected with 10-fold serial dilutions of CVB3/2035A (50 µL/well in quadruplicate, from MOI = 10 to MOI = 0.01). After incubation in 5% CO2 at 37 ℃ for 72 h, cytotoxicity was assessed using a Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s instructions (MCE, HY-K0301).
Flow cytometry analysis
C33A, SiHa, or CaSki cells were incubated with rabbit polyclonal antibodies against CAR (Abcam, ab100811), followed by incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibodies (Sigma, AP132F). The cells were then washed, centrifuged, resuspended in PBS, and analyzed for receptor expression using a flow cytometer (BD Bioscience, BD FACSAria™ III). The data were analyzed using FlowJo software version 10.8.1.
In vivo anti-tumor studies using subcutaneous xenografts in nude mice
All animal procedures were conducted under specific pathogen-free (SPF) conditions and in accordance with the approved animal use protocols of Xiamen University Laboratory Animal Center. For xenografts in BALB/c nude mice, 5 × 106 cells (C33A, SiHa, and CaSki) were subcutaneously injected into the right flanks of 6-8-week-old female BALB/c nude mice (GemPharmatech, China). When tumors reached diameters of approximately 6 mm, the mice were inoculated with CVB3/2035A and/or PTX. To evaluate the effectiveness of CVB3/2035A, mice received intratumoral (i.t.) or intravenous (i.v.) injections of five consecutive doses (1 × 108 TCID50 per dose) of CVB3/2035A at two-day intervals. The same volume of PBS was administered i.t. as a negative control. To evaluate the combined effect of CVB3/2035A and PTX, mice were either mock-treated or received i.t. injections of CVB3/2035A (1 × 108 TCID50), intraperitoneal (i.p.) injections of PTX (5 mg/kg [17]; diluted in solution containing 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline [18]), or a combination administered five times consecutively at two-day intervals. Tumor sizes were measured with calipers every other day for 20 days. The tumor volume was calculated as length×width×width/2 and expressed as means ± SEM.
Sample collection
Seven human CSCC tissue samples were collected from patients at Zhongshan Hospital of Xiamen University during surgical resection procedures (Table S1). Three normal cervical tissues were obtained from patients who underwent a hysterectomy performed for benign uterine diseases. None of the CSCC patients received any treatment before surgery. We utilized four CSCC tissue samples and three normal cervical tissues for the histoculture drug response assay, while the remaining three CSCC tissue samples were employed for organoid derivation. Informed consent for this study was obtained from each patient. This study received approval from both the hospital’s Institutional Review Board and the Research Ethics Committee of Xiamen University. Experiments were carried out in strict accordance with the guidelines and regulations of Xiamen University.
Histoculture drug response assay
Four CSCC tissue samples and three normal cervical tissues were used in the histoculture drug response assay (HDRA) with slight modifications to a previous protocol [19]. Briefly, collagen gel sponges were cut into 1 cm cubes and placed in the wells of a 24-well plate, followed by the addition of 1 mL DMEM medium containing 20% FBS and 10% penicillin/streptomycin solution. The tissues were minced into approximately 1-mm diameter pieces and randomly placed on the collagen gel sponges in the 24-well plate. After an overnight culture at 37 ℃, the tissues were either exposed to 1 × 107 TCID50 of CVB3/2035A per well or left untreated. Following a 72-hour incubation, 100 µL of 0.06% collagenase (type IV; Sigma) in DMEM was added to each well, and the plates were incubated at 37 ℃ for another 4 h. Subsequently, the tissues and supernatants from each well were harvested and centrifuged at 5,000 g for 10 min (mins) at 4 ℃. The resultant pellets from each well were resuspended in 100 µL DMEM medium, and cell viability was assessed using the CCK-8 assay in 96-well plates. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 – A/B) × 100, where A represents the mean absorbance of the treated wells per 1 g of tumor, and B represents the mean absorbance of the control wells per 1 g of tumor.
Quantitative RT-PCR (qRT-PCR)
RNA was extracted from tissue homogenates using a GenMagSpin Viral DNA/RNA Kit (GenMag Bio, China). Real-time PCR analysis was performed with the primers (forward, 5’-TCCTCCGGCCCCTGA-3’; reverse, 5’-AATTGTCACCATAAGCAGCCA-3’; and probe, 5’-FAM-CGGAACCGACTACTTTGGGTGTCCGT-BHQ1-3’) using a One-Step RT-PCR Kit (GenMag Bio, China) and Roche 96 system according to the manufacturer’s protocol. The thermal conditions were set to 50 ℃ for 10 min, 95 ℃ for 10 min, followed by 45 cycles of 95 ℃ for 15 s (secs) and 55 ℃ for 50 s. Relative cDNA level was calculated using a standard curve of Ct (cycle threshold) values generated from the dilution series of pMD18-CVB3/2035A plasmid containing the target gene.
Immunohistochemistry assay
The expression of CAR in four CSCC tumors and three normal tissues used in HDRA was assessed by immunohistochemistry (IHC). Tumor sections (5 μm) underwent heat-induced antigen retrieval using citrate buffer, followed by endogenous peroxidase quenching using hydrogen peroxide. The sections were then incubated with rabbit anti-CAR antibodies (Abcam, ab100811) for one hour at room temperature. Subsequently, IHC staining was performed using an Ultrasensitive TMS-P kit (Fuzhou Maixin Biotechnology Development Co., Ltd., China) and a DAB detection kit (streptavidin-biotin; Fuzhou Maixin Biotechnology Development Co., Ltd., China) according to the manufacturer’s instructions. Finally, the sections were counterstained with hematoxylin, dehydrated, and cover-slipped. CAR staining was quantified using ImageJ (version 1.0) and presented as relative optical density normalized to sections incubated with PBS.
Establishment and culture of CSCC organoids
Three CSCC tissue samples were washed and disinfected in DPBS containing 10% penicillin/streptomycin for 5 min. Subsequently, the tissues were sectioned into small pieces, with some used for immunofluorescence analysis and the remainder for organoid derivation. A tissue dissociation solution was prepared by mixing 10 mL of AdDF+++ (Advanced DMEM/F12 supplemented with 1×Glutamax, 10 mM HEPES, and penicillin-streptomycin, all from Thermo Fisher Scientific), 10 µM RHO/ROCK pathway inhibitor (TargetMol, Y-27632), and 1 mg/mL type I and type IV collagenase (Gibco, 17100017; Gibco, 17104019). The tissue samples intended for organoid derivation were finely chopped in this dissociation solution and then dissociated on an orbital shaker at 37 °C for one hour. Following centrifugation, trypsin digestion was performed for 30 min. Upon completion of the digestion process, the sample was filtered through a 70 μm cell strainer. After an additional round of centrifugation, red blood cells were removed from the sample using a red blood cell lysis buffer (Solarbio, R1010). Then, 10 mL of AdDF+++ was added, and the suspension was centrifuged at 500 g for 3 min.
The cell pellets were resuspended in cold basement membrane extract (BME; R&D Systems, 3536-005-02). Next, 120 µL drops of the BME cell suspension were allowed to solidify on pre-warmed 6-well culture plates at 37 °C for 30 min. Following this, organoid growth medium [AdDF+++ supplemented with 100 ng/mL Noggin (R&D Systems, 6057-NG-100), 500ng/mL RSPO1 (R&D Systems, 4645-RS-250), 1× B27 supplement (GIBCO, 175044), 2.5 mM nicotinamide (Sigma, N0636), 1.25 mM n-Acetylcystein (Sigma, A9165), 10 µM ROCK inhibitor (TargetMol, Y-27632), 500 nM A83-01 (Tocris, 2939), 10 µM forskolin (Bio-Techne, 1099), 25 ng/ml FGF7 (SinoBiological, 10210-H07E), 100 ng/ml FGF10 (SinoBiological, 10573-HNAE), and 1 µM p38 inhibitor SB202190 (Sigma, S7067)] was prepared as previously described [2] and 4 mL added to each well. The plate was placed in a 37 °C/5% CO2 incubator and medium was refreshed every 2 to 3 days. Growth was monitored every 2 days using the Opera Phenix High-Content Screening System (PerkinElmer).
Immunofluorescence assay of CSCC organoids and original tumors
For immunofluorescence analysis of uninfected CSCC organoids and original tumors, samples cultured in CellCarrier Ultra microplates (PerkinElmer) were fixed with 4% paraformaldehyde, permeabilized with 3% Triton-X, washed with PBS, blocked with 2% BSA for one hour, and incubated overnight at 4 °C with antibodies against Ki67 (Novus Biologicals, NB110-89717), p53 (Cell Signaling Technology, 2527S), PAX8 (Cell Signaling Technology, 59019S), PanCK (Abcam, ab7753), or CAR (Abcam, ab100811). Subsequently, the samples were washed five times with PBS and incubate for one hour at 37 °C with Alexa Fluor 647-labeled goat-anti-rabbit (Beyotime, A0468) or goat-anti-mouse (Beyotime, A0473) secondary antibodies. The nuclei and cytoskeleton were counterstained with DAPI (Beyotime, C1002) and phalloidin (Thermo Fisher Scientific, A12379), respectively. Immunofluorescence data were acquired and analyzed using the Harmony® High-Content Imaging and Analysis Software (PerkinElmer, version 4.9).
For immunofluorescence analysis of CVB3/2035A-infected organoids, samples were fixed with 4% paraformaldehyde, embedded in O.C.T., sectioned into 5 μm thick frozen sections on glass slides, and dehydrated at -20 °C using a 1:1 methanol and acetone mixture. The sections were then blocked with 2% BSA for one hour, followed by overnight incubation at 4 °C with antibodies against double-stranded RNA (J2; Scicons, 10010200) and CVB3/2035A VP1 (clone L8F12, produced in our lab, unpublished). After rinsing five times with PBS, the sections were incubated with Alexa Fluor 647-labeled goat-anti-mouse secondary antibody (Beyotime, A0473) at room temperature for one hour and subsequently stained with DAPI (Beyotime, C1002). Immunofluorescence imaging was performed using the Leica LAS X Widefield Systems Fluorescence Microscope System.
Infection of CSCC organoids and viral growth kinetics
Cultrex Organoid Harvesting Solution (50 µL Solution /5 µL BME; R&D Systems) was added to the organoids, followed by incubation at 4°C for 60 min to digest the BME. The organoids were then mechanically dissociated by pipetting and filtered through a 100 μm strainer to remove large clusters. Subsequently, the organoids were resuspended in growth medium at a concentration of 5,000 organoids/mL and plated in a volume of 100 µL on 96-well ultra-low attachment plates, according to the previous protocol [20, 21], each containing approximately 5 × 104 cells/ well counted after trypsin digestion. Serial 10-fold dilutions of CVB3/2035A, ranging from 1 × 108 to 1 × 105 TCID50/well, were added in 25 µL volumes. The plates were incubated at 37 °C with 5% CO2 for 72 h. For combinatorial treatment strategies, PTX was included in the medium at a final concentration of 2 µM, as reference in [22]. For the evaluation of viral growth kinetics, supernatants from the infected organoids were harvested at 4, 12, 24, and 48 h post-treatment, and virus titers were determined in Hela cells using the TCID50 assay.
Cell viability assay of CSCC organoids
Seventy-two hours following the addition of CVB3/2035A, either alone or in combination with PTX, cellular ATP levels in the organoids were quantified using the Cell Titer-Glo 3D Cell Viability Assay (Promega), according to the manufacturer’s instructions. Luminescence readings were obtained with the EnSight Multimode Plate Reader (PerkinElmer). These results were normalized against a blank control containing an equal volume of PBS, and the data were analyzed for cell viability using GraphPad Prism 9. In addition, the organoids were stained with DAPI and propidium iodide (PI) to visualize the nuclei and dead cells. Fluorescence imaging was subsequently carried out using the Opera Phenix High-Content Screening System (PerkinElmer) to analyze cell viability.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 9. We used Student’s t-test, one-way ANOVA, two-way ANOVA, and Tukey’s multiple comparisons test for statistical significance. The significance levels were denoted as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.
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