Cell cultures and reagents
Two representative cervical adenocarcinoma cell lines, HeLa and HCA-1 were obtained from the Japanese Collection of Research Bioresources (JCRB). A cervical squamous cell carcinoma cell line, SiHa was kindly provided by Prof. Mitomu Kioi, Yokohama City University Graduate School of Medicine (Yokohama, Japan). HeLa (HPV-18 positive) and SiHa (HPV-16 positive) cells contain wild-type p53, while HCA-1 cells express mutated p53 (R273C). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Fujifilm Wako Chemicals, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Cytiva, Marlborough, MA, USA), 100 U/mL of penicillin, and 100 mg/mL of streptomycin and then incubated at 37 ℃ in a humidified 5% CO2 atmosphere. CDDP and paclitaxel (PTX) were purchased from Fujifilm Wako Chemicals and Sigma-Aldrich (St. Louis, MO, USA), respectively.
HeLa (1 × 104 cells per well) and HCA-1 (2 × 104 cells per well) cells were seeded in ultra-low attachment (ULA) 96-well U-bottom plates (#MS-9096U; Sumitomo Bakelite, Tokyo, Japan), cultured for 24 h at 37 ℃ in a humidified 5% CO2 atmosphere, and formed into a 3D structure. All tests were performed after confirming the formation of single spheroids in each well. Cultures were maintained by replacing 50% of the medium by every two days.
Cell culture in collagen gel
HeLa cells and spheroids were mixed with 30 mL of cold neutralized collagen type-I gel (Cellmatrix; Nitta Gelatin, Osaka, Japan) containing 1 × minimum essential medium (MEM) and placed on a 6-well plate. After the collagen containing the cells and spheroids had solidified, 2 mL of DMEM with 10% FBS was added to each well.
Sphere cultures of hCSCs
HeLa cells were suspended as single cells and placed in the ULA 96-well U-bottom plates containing a serum-free DMEM/F12 medium with 20 ng/mL of epidermal growth factor (EGF), 20 ng/ml of basic fibroblast growth factor (bFGF), and 0.4% bovine serum albumin (BSA, Sigma–Aldrich). Cultures were maintained by replacing 50% of the medium by every two days.
In the microtubes, the cells and spheroids were irradiated with X-rays using an X-ray irradiation device (#MBR-1520R-4; Hitachi Power Solutions, Hitachi, Japan). The X-ray generator was operated at 150 kVp and 20 mA with 0.5 mm Al and 0.3 Cu filters. The dose rate of X-rays was approximately 2.1 Gy/min. Some cells and spheroids in 6-well plates were also irradiated with C-ion scanning beams with an initial energy of 140–200 MeV/n and a spread-out Bragg peak (SOBP) width of 6 cm generated by the ion-beam Radiation Oncology Center in Kanagawa (i-ROCK, Kanagawa Cancer Center, Yokohama, Japan). Some of the irradiations were performed using C-ion passive beams accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institutes for Quantum and Radiological Science and Technology (QST, Chiba, Japan). The initial energy of the C-ion beam was 290 MeV/u and the width of the SOBP was 6 cm. The isocenter planes were matched to the center of the SOBP.
Clonogenic survival assay and crystal violet assay
HeLa cells were irradiated with 0, 2, 4, and 6 Gy of X-rays or 0, 1, 2, and 3 Gy of C-ion beams. Cells were then immediately seeded in flat-bottomed 6-well plates at a predetermined number of cells (X-ray: 200 cells for 0 Gy, 400 cells for 2 Gy, 800 cells for 4 Gy, and 1600 cells for 6 Gy; C-ion beam: 200 cells for 0 Gy, 400 cells for 1 Gy, 800 cells for 2 Gy, and 1600 cells for 3 Gy) and cultured for 10–14 days . Thereafter, cells were washed, fixed with methanol, and stained with 0.5% crystal violet (Kanto Chemical, Tokyo, Japan). After washing and drying the plates overnight, the colonies were counted. The survival curves were fitted to a linear-quadratic (LQ) model: S = exp (− αD − βD2) for the X-ray and linear models: S = exp (− aD) for the C-ion beam. S is the survival fraction, and D is the dose expressed in Gray . Statistical analyses were performed using GraphPad Prism 9.3.1 (GraphPad Software, La Jolla, CA, USA).
HCA-1 cells were seeded in 96-well plates at a density of 1 × 103 cells per well and cultured for 10–14 days. Viability was evaluated using a crystal violet assay . Cells were washed, fixed with 4% glutaraldehyde, and stained with 0.5% crystal violet. After washing and drying plates, stained cells were lysed with 100 µL of 1% sodium dodecyl sulfate (SDS), and the absorbance was measured at 570 nm using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Cell viability was determined using a Cell Titer-Glo assay (#G9683; Promega, Madison, WI, USA) or a Cell Counting Kit-8 (CCK-8) colorimetric assay (#CK04, Dojindo Molecular Technologies, Kumamoto, Japan). For the Cell Titer-Glo assay, cells were added 25 µL of Cell Titer-Glo 3D were added to the cells in each well, followed by incubation for 30 min at room temperature. Luminescence was measured using a SpectraMax L Plate Reader (Molecular Devices, San Jose, CA, USA). For the CCK-8 assay, a CCK-8 solution was added to each well and the absorbance was measured at 450 nm using an iMark microplate reader after 2 h. Data were normalized to the control wells for each cell culture.
Cells were lysed with an ice-cold lysis buffer containing 25 mM of Tris-HCl pH 7.4), 150 mM of NaCl, 1 mM of EDTA, 1% Triton-X, and a protease inhibitor cocktail (Calbiochem, Merck KgaA). Nuclear and cytoplasmic proteins were prepared using a LysoPure™ Nuclear and Cytoplasmic Extraction Kit (Fujifilm Wako Chemicals). Total, nuclear, and cytoplasmic protein concentrations were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Equal amounts of proteins were denatured by heating at 95 ℃ for 5 min with Laemmli sample buffer, separated by SDS-PAGE (Atto, Tokyo, Japan), and transferred to polyvinylidene fluoride membranes (0.45 μm, Millipore, Merck KgaA). After blocking membranes with 5% skim milk, the blots were incubated for 1 h with the following primary antibodies: anti-E-cadherin (E-cad) (#3195; Cell Signaling Technology (CST), Danvers, MA, USA, 1:500), anti-Integrin β1 (ITGB1) (#sc59827; Santa Cruz Biotechnology (SCB), Dallas, TX, USA, 1:500) anti- hypoxia-inducible factor 1α (HIF1α) (#ab51608; Abcam, Cambridge, UK, 1:500), anti-GAPDH (#sc-32233; SCB, 1:500), and anti-TATA-binding protein (TBP; #22006-1-AP; Proteintech, Rosemont, IL, USA, 1:500). Blots were then incubated with goat anti-rabbit or goat anti-mouse secondary antibodies (CST) and visualized with Amersham ECL Prime (Cytiva), followed by chemiluminescence detection using a ChemiDoc Touch imaging system (Bio-Rad). Protein expression levels were evaluated by densitometry using the ImageJ software (https://imagej.nih.gov/ij/).
Flow cytometric analysis
For cell cycle analysis, cells were harvested, fixed with 70% chilled ethanol, and stained with a FxCycleTMPI/RNase Staining Solution (Invitrogen, Thermo Fisher Scientific).
For stem cell analysis, cells were harvested, stained with anti-CD49f (#313602, BioLegend, San Diego, CA, USA) for 30 min on ice, and then stained with Alexa Fluor 488 anti-mouse IgG (#A11029; Invitrogen, Thermo Fisher Scientific) on ice for another 30 min. Cells were also stained with anti-CD44 variant 9 (CD44v9)-PE (#394404, BioLegend) for 30 min on ice.
After washing, measurements were performed using a FACS Canto II cell analyzer (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo software (Treestar, Ashland, OR, USA).
For analysis of sphere formation, irradiated HeLa cells were harvested 1 day later and seeded into ULA 96-well U-bottom plates at 100 cells per well. After 3 days of incubation, images of spheres were captured using a Nikon Eclipse Ti inverted microscope with NIS-Elements Advanced Research software (Nikon, Tokyo, Japan), and the viability of spheres was measured using Cell Titer-Glo 3D (Promega).
Images of monolayer cells and spheroids were captured using a Nikon Eclipse Ti inverted microscope (Nikon). For hypoxia imaging, spheroids were stained with the hypoxia probe solution LOX-1 (Medical and Biological Laboratories, Nagoya, Japan), which was quenched by oxygen and increased in response to hypoxic conditions [19, 20]. Images were captured using a fluorescence microscope (#BZ-9000; Keyence, Osaka, Japan).
For immunohistochemistry, cells were fixed and stained using standard protocols . After irradiation, cells were fixed with 4% paraformaldehyde (Fujifilm Wako Chemicals), treated with 0.5% Triton X-100 in PBS, and blocked with 10% normal goat serum (Invitrogen, Thermo Fisher Scientific). The primary antibody used was anti-γH2AX (#05-636; Upstate, Merck KGaA). The secondary antibody used was Alexa Fluor 568 goat anti-mouse IgG (#A11004; Invitrogen, Thermo Fisher Scientific). Cell nuclei were stained with Hoechst 33342 (Fujifilm Wako Chemicals). Images were taken using confocal microscopy (#LSM710; Carl Zeiss Microscopy GmbH, Jena, Germany).
Data are presented as the mean of three minimal independent experiments with corresponding error bars for standard deviation (SD), as indicated in the figure legends. Data analysis was performed by one-way ANOVA followed by the Bonferroni test or Tukey’s test using GraphPad Prism 9.3.1. A p-value of less significance was set to indicate statistical significance.