3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and DOX (D1515) were purchased from Sigma-Aldrich (St Louis, MO, USA). Rabbit anti-LC3 (12135–1-AP) was purchased from Proteintech (Chicago, IL, USA), rabbit anti-MMP-2 (ab37150) and rabbit anti-MMP-9 (ab38898) were purchased from Abcam (Cambridge, MA, USA), rabbit anti-Shh (bs-1544R), rabbit anti-Gli-1 (bs-1206R) and rabbit anti-Beclin 1 (bs-1353R) were purchased from Beijing Bioss Biosynthesis Biotechnology (Beijing, China), and mouse anti-β-actin (sc-47,778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell lines and culture
Human OSCC cell lines SAS, HSC-4, and HSC-3 and normal HT293 cells were purchased from the Human Science Research Resources Bank (Osaka, Japan) and the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Exponentially growing cells were used in the experiments.
Animals and tumor model
Male, 4-week-old BALB/c nude mice were purchased from SLAC Laboratory Animal Company (Shanghai, China) (N = 24). SAS cell suspensions (1 × 106 cells/ml) were subcutaneously injected into the right-side flanks of mice. When the tumor volume reached 100 mm3 in size (about 7 days after inoculation), the tumor-bearing mice were randomly divided into four groups. Tumor diameters were measured with calipers and calculated using the following formula: V = π/6 × L × S
2, where L and S are the long and short diameters of the tumors. All animal protocols were approved by the Laboratory Animal Committee of the Harbin Medical University.
Archived paraffin-embedded OSCC and matched adjacent normal tissues were obtained from 74 patients who had undergone surgical excision at the Harbin Medical University Stomatological Hospital between January 2006 and December 2011. Patient clinical characteristics have been described previously . All patients gave informed consent, and the study was approved by the Research Ethics Committee of Harbin Medical University (Harbin, China).
The ultrasonic generator and power amplifier used in this study were designed and manufactured by the Harbin Institute of Technology (Harbin, China). The ultrasonic setup is described in our previous publication  (Additional file 1: Figure S1). For in vitro experiments, the sound pressure level distribution was simulated by finite element simulation using COMSOL as shown in Additional file 1: Figure S2A & S2B. The ultrasonic transducer (diameter: 40 mm; center frequency: 1.1 MHz; duty factor: 20%; repetition frequency: 100 Hz) was made of a PZT disk attached to a 2.5 cm thick aluminum block serving as the acoustic transmission medium. The LIUS intensities in the culture plate were also measured using a PT0907110 needle-type hydrophone (0.2 cm active element size, 1–10 MHz bandwidth) (Beijing, China). The peak acoustic pressure was 2.6 MPa and the temporal intensity distribution from the center of the transducer along the direction perpendicular to the transducer is shown in Additional file 1: Figure S2a & S2b. For the in vivo experiments, the sound pressure level distribution is shown in Additional file 1: Figure S2C & S2D. The ultrasonic transducer (diameter: 30 mm; center frequency: 1.1 MHz; duty factor: 20%; repetition frequency: 100 Hz) was attached to a tapered aluminum buffer head with a 5 mm diameter front surface that was positioned directly in contact with the skin at the tumor site using an acoustic couplant. The temperature increase in the solution was less than 2 °C in all experiments.
DOX and ultrasonic treatment of cells in vitro and in vivo
For both in vitro and in vivo experiments, four treatment groups were used: 1) non-treatment (Control); 2) DOX; 3) low intensity ultrasound (LIUS); and 4) DOX in combination with ultrasound (DOX + LIUS).
For the in vitro experiments, cells in the DOX and DOX + LIUS groups were incubated with DOX in the dark. For the control and LIUS groups, an equivalent amount of media was added in place of DOX. After 4 h incubation, the cells in the LIUS and DOX + LIUS groups were exposed to ultrasound (1.1 MHz, 1 W/cm2, 20% duty cycle) for 3 min in the dark. After the treatment, the cells were collected for further analyses.
For the in vivo experiments, DOX solution was intraperitoneally injected into tumor-bearing mice in the DOX and DOX + LIUS groups at a dosage of 3 mg/kg, 2 times/week. For comparison, 0.9% normal saline solution was injected into tumor-bearing mice in the control and LIUS groups. Tumors were irradiated by ultrasound (1.1 MHz, 1 W/cm2, 20% duty cycle) for 5 min in the dark. The treatment was repeated twice a week for 2 weeks. Tumor diameters and mouse body weights in each group were measured for 14 days. For the prognosis of animals (N = 20), the mice were observed daily and sacrificed if they lost 15% of their body weight. The solid tumors removed from different groups were processed for western blotting, immunohistochemistry and Masson’s trichrome staining.
Cell viability and apoptosis assay
The three human OSCC lines were seeded on detachable 96-well plates and subjected to different treatments for the four groups in each cell line. Then, all groups were cultured for 24 h. Cell viability was quantified by the MTT assay.
In the LIUS and DOX + LIUS groups, the apoptotic cells were detected using Hoechst 33,258 (Sigma-Aldrich) according to the manufacturer’s instructions. Nuclei were visualized by fluorescence microscopy (Olympus, BX51, Japan) with an excitation wavelength of 355 nm and emission wavelength of 465 nm.
All OSCC cells were fixed in methanol for about 30 min. Then the cells were blocked with 1% BSA for 20 min and incubated with primary antibody (LC3: 1:50; Beclin 1: 1:100) at 4 °C overnight. After rinsing in PBS, the cells were incubated in secondary antibody for 1 h followed by counterstaining with DAPI. The cells were examined by fluorescence microscopy (Olympus, BX5, USA).
Scanning electron microscopy (SEM)
After exposure to different intensities of LIUS, cells were fixed in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.2–7.4) for 24 h. The cells were then immobilized in 1% osmium tetroxide (OsO4), washed with PBS, dehydrated with graded alcohol, displaced, and dried at a critical point. A thin layer of gold was evaporated onto the surface before observation under a scanning electron microscope (S-3400 N, Hitachi, Japan).
Transmission electron microscopy (TEM)
Cells were fixed in 2.5% glutaraldehyde overnight. After washing with PBS, the samples were dehydrated with graded alcohol and embedded in Epon812 for 72 h at 60 °C. Ultra-thin sections were cut, stained with uranium acetate and lead citrate, and then observed under a transmission electronic microscope (Hitachi, Tokyo, Japan).
In vitro migration/invasion activities were assessed using a BD BioCoat™ Matrigel™ Invasion Chamber (BD Biosciences, San Jose, CA, USA). Invasion assays were performed with Falcon cell culture inserts containing 8-μm pore size polyethylene terephthalate membranes with a thin layer of matrigel-reconstituted basement membrane. The cancer cells were seeded into the upper chamber (2 × 104 cells/well). After 24 h incubation at 37 °C, non-invading cells were removed from the upper surfaces of the membranes by scrubbing with cotton-tipped swabs. Invading cells were fixed with methanol, stained with Giemsa and counted under a microscope. In vitro migration assays were performed according to the same procedure but using PET membranes that were not coated with matrigel.
Cell migration and invasion were quantified by counting the number of cells in 10 visual fields on the lower surface of each filter using phase-contrast microscopy.
Cells and tissues were lysed in RIPA buffer, separated by 10% SDS-PAGE, and transferred to nitrocellulose membranes. After blocking with 5% non-fat dried milk, membranes were incubated with primary antibodies (LC3: 1:500; Beclin 1: 1:500; MMP-2: 1:1000; MMP-9: 1:1000; Shh: 1:100; Gli-1: 1:100) at 4 °C overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Signals were detected using an enzymatic Chemiluminescence Kit (Pierce, Rochford, IL, USA). β-actin (1:500) was used as an internal control.
Analysis and evaluation of immunohistochemical results were performed as previously described . The concentrations of primary antibody were 1:100 for Shh and Gli-1, and 1:500 for MMP-2 and MMP-9.
Masson’s trichrome staining was used to observe deposited collagenous fibers [22, 26]. Collagen fibers stained blue and muscle fibers stained red. The extent of collagenous fiber expression was assessed per unit area.
Intracellular DOX measurement
The cells of DOX and DOX + LIUS groups were pretreated with 0.2 μg/ml for 3 h prior to sonication. For observation of cellular uptake of DOX using fluorescent microscopy (Olympus, BX5, USA), the cells were fixed in 4% paraformaldehyde for 10 min. The cells were then permeated with 0.1% Triton X-100 for 5 min. For quantification of intracellular DOX concentrations, the cells in 10 randomly selected fields of view (magnification ×200) were counted, and the fluorescence intensity index was used as a measure of cell DOX uptake.
Data are expressed as means ± standard deviations (SD) of three independent experiments. The data were analyzed by one-way ANOVA. Survival analysis was estimated using the Kaplan-Meier method and compared using the log-rank test. The Spearman’s rank correlation coefficient test was used to examine correlations between the expression of collagen fibers, MMP-2, MMP-9, Shh and Gli-1. Results were considered significant when p < 0.05.