Cell culture and animal care
MIA PaCa-2 and PANC1 human pancreatic cancer cell lines were purchased from the JCRB Cell Bank (Osaka, Japan). The original stocks of the cell lines were mycoplasma-, bacteria-, and fungi-free. MIA PaCa-2 and PANC-1 cells were cultured at 37 °C in 5% CO2 in MEM (Sigma Aldrich, UK) and RPMI-1640 (Wako, Japan, respectively). Media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Immunodeficient male BALB/c nude mice at 6 weeks of age were obtained from CLEA Corporation (CLEA, Inc., Tokyo, Japan). They were maintained in a pathogen-free animal care system at 21–25 °C with 40–70% humidity. Food and water were provided ad libitum. All animal experiments were monitored, approved, and performed in accordance with the Kobe University Animal Experimentation Regulations (approval number: P160801).
X-ray irradiation
An MBR-1505R2 X-ray generator (Hitachi, Tokyo, Japan) at a voltage of 150 kV and a current of 5 mA with a 1-mm thick aluminum filter (0.5 Gy/min at the target) was utilized. For the in vivo study, mice were intraperitoneally anesthetized using somnopentyl (0.1 mg/g body weight), and then tightly fixed to expose the tumor tissue, while the remaining body parts were covered with lead shields during the radiation process, as previously described [36].
Preparation of nanoparticles
TiOxNPs were prepared according to previously described methods [37]. Briefly, nanoparticles were synthesized from TiO2NPs by immersion in a 6% H2O2 solution, and surfaces were coated using polyacrylic acid to prevent aggregation of the bare TiOxNPs under physiological conditions. TiOxNPs synthesis was confirmed via dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) and by transmission electron microscopy (TEM) using a JEM-2100 F instrument (JEOL, Tokyo, Japan) as described previously [33].
To evaluate the uptake of TiOxNPs by adherent and sphere MIA PaCa-2 and PANC-1 cells, adherent cells were cultured in 6-wells plate overnight, followed by the addition of 400 μg/mL TiOxNPs and PBS as a control. One hour later, cells were washed twice with PBS, trypsinized, suspended in serum-free media without phenol red, and centrifuged. Centrifuged cells were washed twice with Fluorescence-activated cell sorting (FACS) buffer (PBS with 0.01% bovine serum albumin) and stained. Finally, cells were resuspended in 500 μL FACS buffer, and forward scattering (FSC), side scattering (SSC), and fluorescence signals were analyzed by flow cytometry (BD FACSVerse™, BD Biosciences, USA). Sphere cells were first dissociated with 500 μL accutase, then incubated with 400 μg/mL TiOxNPs for one hour, followed by centrifugation and preparation for FSC and SSC.
For TEM evaluation, TiOxNP-treated cells and spheres were washed three times with PBS and fixed in 2.5% paraformaldehyde and 0.5% glutaraldehyde for 8 h. They were then fixed in 1% osmium tetroxide for 2 h and dehydrated using ethanol solution. Then, cells were embedded in Epon (Polysciences Inc.), sectioned, and visualized using a JEM-2100 F instrument (JEOL, Tokyo, Japan) as described previously [38].
For dark field images, single-cell suspensions were obtained from adherent and sphere cells as described above. Nuclei were counterstained using Hoechst stain, and samples were viewed under a dark field to detect the white nanoparticles. Combined images were analyzed using a Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).
Sphere formation assay
MIA PaCa-2 and PANC-1 cells were pre-treated with 200 or 400 μg/mL TiOxNPs for one hour followed by 2 or 5 Gy radiation treatment. Next, cells were trypsinized, counted, and plated in ultra-low attachment 96-wells plates (EZ-BindShut II, AGC Techno Glass, Shizuoka, Japan) at a density of 1000 cells/well. The cells were maintained in serum-free alpha MEM supplemented with B27 (Life Technologies), 10 ng/mL rhEGF (PeproTech), and 10 ng/mL rhbFGF (PeproTech), and then mixed with 1% methylcellulose. The number of spheres over 20 μM was evaluated after 10–12 days using BZ analysis software on a Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).
For the second passage, spheres were dissociated with 500 μL accutase for 5 to 10 days at 37 °C until a single cell suspension was obtained, followed by incubation with 200 or 400 μg/mL TiOxNPs for one hour followed by 2 or 5 Gy radiation treatment. Cells were then cultured and analyzed after 10–12 days.
Cell proliferation assay
At 6-well plates, MIA PaCa-2 and PANC-1 cells (5 × 105 cells/well) were pretreated with 200 or 400 μg/mL TiOxNPs for one hour followed by exposure to 2 or 5 Gy radiation treatment, and incubation at 37 °C for 48 h. Living cells were stained with trypan blue and counted after 24 and 48 h using a Countess II automated cell counter (Invitrogen Life Technologies, USA). For spheres, cells were first dissociated, and single cell suspensions were obtained, where 2 × 104 cells were treated with 200 or 400 μg/mL TiOxNPs for one hour followed by exposure to 2 or 5 Gy radiation doses. Cells were plated in 96-wells plate in serum-free medium containing sphere-forming growth factors. Living cells were stained with trypan blue and counted after 24 and 48 h using a Countess II automated cell counter (Invitrogen Life Technologies).
Measurement of cell viability
MIA PaCa-2 and PANC-1 cells pre-treated for one hour with 200 or 400 μg/mL TiOxNPs and exposed to 2 or 5 Gy radiation treatment were cultured at a density of 1.5 × 105 cells/well and incubated for 48 h. Fresh medium was added to each well with 10% WST-1 solution (Takara-Bio, Japan) and incubated at 37 °C for 1 h. Absorbance was measured at 420–480 nm using an EnSpire multimode microplate reader (PerkinElmer, USA). For spheres, cells were first dissociated and single cell suspensions were obtained as described in “Cell Proliferation Assay.” Cells were plated in 96-well plates in serum-free medium containing sphere-forming growth factors. WST-1 solution was then added to each well as mentioned above, and cell viability was measured using an EnSpire multimode microplate reader.
Wound healing assay
MIA PaCa-2 and PANC-1 cells were plated into 6-wells plate until 70–80% confluence. An artificial wound was created using a 200 μL pipette tip. Cells were then treated with 200 or 400 μg/mL TiOxNPs in serum-free media for one hour, followed by exposure to 2 or 5 Gy radiation. Images of the healing process were captures at 0, 24, and 48 h using a Biorevo BZ-9000 microscope. The wound area was calibrated and measured using ImageJ.
Cell migration and invasion assays
MIA PaCa-2 and PANC-1 were treated as per “Cell Proliferation Assay.” For the invasion assay, 2.5 × 105 cells were suspended in 200 μL serum-free media in a 24-well plate, and an 8 μm pore-sized millicell cell culture insert (Sigma Aldrich, UK) containing 100 mL of Matrigel (Corning, USA) was added to each well. Serum-containing media (750 μL) was added to the bottom of each well. The cells were incubated at 37 °C overnight. The next day, the media were removed, and cells were washed twice with PBS followed by fixation with formaldehyde (3.7% in PBS). Cells were then washed twice and permeabilized with 10% methanol for 20 min at room temperature. Cells were washed twice with PBS and dried for 30 min, followed by staining with Giemsa stain. Cells were then maintained in dark conditions for 15 min and washed twice with PBS. The upper layer of the insert was wiped off using a cotton swab. Invading cells were imaged using a Biorevo BZ-9000 microscope, and were calibrated and counted using Image J. For spheres, cells were dissociated and treated as mentioned above, and the invasion assay was carried out as described in this section. The migration assay was performed in a similar manner, but the insert was not covered with Matrigel.
Apoptosis assay
The apoptosis assay was performed using the FITC Annexin V Apoptosis Detection Kit with PI (BioLegend, USA), following manufacturer instructions. In brief, MIA PaCa-2 and PANC-1 spheres were dissociated with 500 μL of accutase and incubated with 200 μg/mL TiOxNPs for one hour, followed by 5 Gy radiation treatment. Cells (1 × 106 cells seeded in 6-wells plate in serum-free media for 48 h) were trypsinized, centrifuged, and washed twice with PBS, then suspended in 1 mL of annexin-binding buffer. A total of 100 μL was transferred a new tube, where 5 μL Annexin and 10 μL propidium iodide were added. The mixture was incubated for 15 min at RT in the dark. Annexin-binding buffer (400 μL) was added to each well. The stained cells were analyzed by flow cytometry (BD FACSVerse™, BD Biosciences, USA) to determine the number of apoptotic cells. Annexin and propidium iodide staining were represented by FITC and PI fluorescence intensities, respectively.
TUNEL assay
The tumor sections were cut and stained with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) using the In Situ Cell Death Detection Kit (Roche, Indianapolis, USA) according to manufacturer instructions. Tissue specimens were incubated with TUNEL at 37 °C for 60 min in the dark and counterstained with 4,6-diamidino-2-phenylindole (DAPI) at room temperature in the dark for 15 min. The specimens were mounted with an anti-fluorescence quenching solution and observed under a BZ-9000 fluorescence microscope. The percentage of TUNEL-positive cells was calculated by dividing the number of TUNEL-positive cells by the total number of cells.
Colony formation assay
MIA PaCa-2 and PANC-1 spheres were detached with 500 μL of accutase and incubated with 200 or 400 μg/mL TiOxNPs for one hour followed by 2 or 5 Gy radiation treatment. Cells (1 × 103 cells) were plated in 6-wells plate. Ten days later, the growing colonies were stained with Giemsa stain and counted using ImageJ.
Tumor growth analysis
MIA PaCa-2 cells (5 × 106) were inoculated subcutaneously into the flank region of BALB/c nude mice. Once the tumor volume reached approximately 100–200 mm3 using the formula L × W2/2, where L is the longest axis, and W is the shortest axis of the tumor, mice were divided into four groups: the control group (treated with PBS), the 5 Gy group (treated with 5 Gy X-ray irradiation), the TiOxNPs group (treated with TiOxNPs suspension at a concentration of 3 mg/mL), and the TiOxNPs combined with 5 Gy group (treated with TiOxNPs suspension at a concentration of 3 mg/mL and 5 Gy X-ray irradiation). To inject MIA PaCa-2 spheres, spheres were first dissociated to obtain a single cell suspension, and then 1.5 × 106 cells were injected as mentioned above. Tumor volume and body weight were measured every two–three days for 30 days after treatment. On day 30, all mice were euthanized for tumor tissue collection. The Kaplan-Meier method was used to estimate the survival rate for each group. The number of mice censored during the experiment was recorded for each group. Censoring parameters were determined based on the presence of the following three factors: tumor volume > 20 × 20 mm, tumor ulceration, and lack of animal response.
Pre-treated 1 × 107 and 1 × 106 adherent MIA PaCa-2 and dissociated MIA PaCa-2 spheres, respectively, with 5 Gy X-ray irradiation and/or 400 μg/mL TiOxNPs were injected subcutaneously into the flank region of BALB/c nude mice. Tumor volume, tumor weight, and body weight were measured every two to three days for 60 days. On day 60, all the mice were euthanized. Kaplan-Meier analysis was used to measure the onset of tumor outgrowth once size reached approximately 50 mm3.
To evaluate body toxicity and the efficacy of TiOxNP treatment against tumor growth, 1.5 × 106 dissociated MIA PaCa-2 sphere cells were inoculated subcutaneously into the flank region of BALB/c nude mice. Once the tumor volume reached approximately 100–200 mm3, the mice were divided into four groups, as described in “Tumor Growth Analysis”. TiOxNPs at a concentration of 25 mg/kg body weight were injected intravenously into the lateral tail vein. One hour later, the mice were irradiated with a dose of 5 Gy. Tumor volume and body weight were measured every two to three days for 30 days post-treatment. On day 30, all mice were euthanized to collect serum and lung, liver, spleen, heart, and kidney tissues. Kaplan-Meier analysis was performed according to previously described factors. The organ index was determined by measuring the organ-to-body weight ratio.
Biochemical parameters
Blood samples were obtained from mice 30 days after intravenous injection of TiOxNPs to evaluate nanoparticle toxicity. Serum samples were collected from all groups and prepared for analysis of the following parameters: glutamic-oxaloacetic transaminase, glutamic-pyruvate transaminase, alkaline phosphatase, and creatinine, according to the manufacturer instructions (Wako, Japan).
Histopathology and immunohistochemistry
For histopathological observation, tumor, lung, liver, spleen, heart, and kidney samples were promptly excised and fixed in 4% paraformaldehyde in PBS. Paraffin sections (5 mm) were prepared and stained with hematoxylin and eosin. For immunohistochemistry (IHC), paraffin sections were stained with antibodies against p-H2AX (#9718; Cell Signaling Technology), c-caspase-3 (#9664; Cell Signaling Technology), ki67 (ab16667; Abcam), PCNA (sc-56; Santa Cruz Biotechnology), snail and slug (ab180714; Abcam), and vimentin (#5741; Cell Signaling Technology). Mayer’s hematoxylin stain was used for nuclei counterstaining (Muto Pure Chemicals Co., Tokyo, Japan).
ROS evaluation
In a cell-free system, ROS generation in response to TiOxNPs alone (200 or 400 μg/mL), radiation alone (2 or 5 Gy), or combination treatment was examined. OH˙ was evaluated using 3-(p-aminophenyl) fluorescein (APF) (Sekisui Medical Co., Japan), whereas O2˙ was detected by dihydroethidium (DHE) (Molecular Probes, USA). APF and DHE fluorescence intensities were measured using an EnSpire multimode microplate reader (PerkinElmer, USA) at excitation/emission wavelengths of 485/538 nm and 485/590 nm, respectively. H2O2 generation was detected using the BIOXYTECH H2O2–560 reagent according to manufacturer protocol (OXIS International, USA). The absorbance intensity was measured using an EnSpire multimode microplate reader (PerkinElmer, USA) at 560 nm.
ROS generation was determined in MIA PaCa-2 cells and PANC-1 spheres as follows: MIA PaCa-2 and PANC-1 spheres were dissociated using Accutase until a single cell suspension was obtained. Cells were later treated with 200 or 400 μg/mL TiOxNPs for one hour at 37 °C, followed by exposure to 2 or 5 Gy radiation treatment. APF, DHE, and carboxy-2,7-dichlorofluorescein (DCF; H2O2 fluorescence stain detector; Molecular Probes, USA) were added to the cells at concentrations of 10, 100, and 40 μM, respectively, and incubated in the dark for 45 min at 37 °C. Mean fluorescent intensity (MFI) was measured using flow cytometry (BD FACSVerse™, BD Biosciences, USA). APF and DHE levels were also measured using an EnSpire multimode microplate reader (PerkinElmer, USA) at excitation/emission wavelengths of 485/538 and 485/590 nm, respectively.
Mitochondrial membrane potential and mitochondrial mass assay
MIA PaCa-2 and PANC-1 spheres were dissociated using accutase until a single cell suspension was obtained. Cells were later treated with 200 or 400 μg/mL TiOxNPs for one hour at 37 °C, followed by exposure to 2 or 5 Gy radiation treatment. Cells were incubated at 37 °C for 48 h. Then, the cells were washed twice with FACS staining buffer. MitoTracker™ Red CMXRos and MitoTrackerTM Green FM (Invitrogen, USA) were used to detect mitochondrial membrane potential (MMP) and mitochondrial mass, respectively. Each tracker (50 ng/mL) was added and incubated in the dark at 37 °C for 30 min. MMP and mitochondrial mass were measured using flow cytometry (BD FACSVerse™, BD Biosciences, USA) using FL2 and FL1, respectively.
Mitochondrial ROS measurement
The MitoSOX assay was used to measure the generation of ROS, especially superoxide, in the mitochondrial matrix. Single cell suspensions of dissociated MIA PaCa-2 and PANC-1 spheres were prepared and treated as mentioned above. MitoSOX Red Mitochondrial Superoxide Indicator (5 μM; Invitrogen, USA) was added and incubated in the dark at 37 °C for 10 min. Mitochondrial superoxide anion MFI was measured immediately after the indicated treatments using flow cytometry (BD FACSVerse™, BD Biosciences, USA) using FL2.
Western blot analysis
Whole cell lysates of adherent cells, spheres, or tumor tissues were prepared for western blot analysis as previously described [39]. The following primary antibodies were used: nanog (ab109250; Abcam), oct4 (ab109183; Abcam), sox2 (ab92494; Abcam), CD44 (#37259; Cell Signaling Technology), ALDH1A1 (#54135; Cell Signaling Technology), E-cadherin (#3195; Cell Signaling Technology), N-cadherin (#13116; Cell Signaling Technology), p-STAT3 (#4113; Cell Signaling Technology), STAT3 (#8768; Cell Signaling Technology), p-AKT (#4060; Cell Signaling Technology), AKT (#2920; Cell Signaling Technology), p-SRC (#2101; Cell Signaling Technology), SRC (#2110; Cell Signaling Technology), p-ERK (#4370; Cell Signaling Technology), ERK (#9107; Cell Signaling Technology), p-H2AX (#9718; Cell Signaling Technology), c-caspase-3 (#9664; Cell Signaling Technology), p-survivin (NB500–236; Novus Biologicals), snail and slug (ab180714; Abcam), vimentin (#5741; Cell Signaling Technology), and β-actin (#4970; Cell Signaling Technology).
Statistical analysis
Results are presented as mean ± standard deviation. Data were analyzed statistically by Student’s t-test or one- or two-way ANOVA with Tukey comparison test as a post-test. The Kaplan-Meier method with log-rank test was used in the comparison among groups. All data were analyzed using GraphPad Prism 8.0 package (GraphPad Software, USA). Statistical significance was set at P < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.