Experiments were performed using HUVEC (Human Umbilical Vein Endothelial Cells), FTC-238 (lung metastasis of Follicular Thyroid Carcinoma) and NCI-H727 (Non-Small Cell Lung Carcinoma). HUVECs (catalogue number C2519A, pooled donor) were purchased from Lonza (Walkersville, USA). FTC-238 and NCI-H727 cell lines were supplied by the European Collection of Cell Cultures (ECACC; Salisbury, United Kingdom) as catalogue numbers 94060902 and 94060303 respectively, and were purchased from CellBank Australia (Westmead, Australia). All cell lines have been previously described [18–20].
Cells were maintained according to the manufacturer’s instructions and incubated at 37oC, 5% CO2 and 95% humidity. HUVEC cells were cultured using EGM-2 Endothelial Cell Growth Medium-2 BulletKit™ from Lonza (catalogue number CC-13162). FTC-238 cells were cultured using DMEM:F12 (catalogue number D8437, Sigma-Aldrich, Sydney, Australia), supplemented with 5% FBS (Corning, catalogue 35-076-CV supplied by Fisher Biotec, Wembley, Australia) and 1% Penicillin-Streptomycin (Pen-Strep) (catalogue number 15070063, Sigma-Aldrich). NCI-H727 cells were cultured in RPMI 1640 (catalogue number 11875093, Thermo Fisher, Scorsby Australia), supplemented with 10% FBS and 1% Pen-Strep.
Cell Proliferation Assays were initially optimised in the absence of treatments using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (catalogue number G3582, Promega, Australia). The MTS tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is similar to the MTT compound used in previous studies [16], and is bioreduced by viable cells into a coloured formazan product that can be analysed via colorimetry. Cells are incubated with the reagent for between 1-4 hours, and then absorbance readings taken at 490nm in a 96-well plate reader. The amount of formazan product measured by absorbance is directly proportional to the number of viable cells in the culture [21].
Cell seeding densities and MTS development times were optimised to achieve 80% confluence at 72 hours post-treatment for each cell line, as summarised in Table 1. This level of confluence was chosen in order to avoid any potential effects of contact-dependent cell signalling or contact inhibition, and 72 hours is consistent with the manufacturer’s instructions for cell proliferation studies and other experiments using this assay [21–23] or the MTT assay [16].
Table 1
Cell seeding densities and MTS development times
Cell Line
|
Flask Size
|
Seeding Density
|
MTS development time
|
HUVEC
|
T25cm
|
8,000 cells / flask
|
25min
|
FTC-238
|
T25cm
|
12,000 cells / flask
|
25min
|
NCI-H727
|
T12.5cm
|
50,000 cells / flask
|
15min
|
HUVEC and FTC-238 cells from stock cultures were plated into T25cm2 Tissue culture flasks (Sigma-Aldrich, catalogue number CLS430639). Due to supply issues, NCI-H727 cells were plated into T12.5cm2 flasks (Bio-Strategy, catalogue number BDAA353107, Tullamarine, Australia). Cells at the designated seeding densities were allowed to adhere overnight in their respective flasks for ~14-16 hours before treatment. Eight flasks were prepared for each experiment – three controls (no treatment), one USMB-alone (USMB), and two flasks for each radiation dose level – one with USMB and one without (ie. 3Gy, USMB+3Gy; 6Gy, and USMB+6Gy). Each experiment was performed in triplicate, with separate experiments run for kV and MV x-ray energies using the HUVEC and FTC-238 cell lines. Concurrent experiments were undertaken for NCI-H727 cells where 12 flasks were prepared for each experiment – four for kV and four for MV in addition to four 0Gy flasks (ie. three controls and one USMB). This experiment was also performed in triplicate.
Pre-Treatment Preparation
Two hours prior to treatment, cells grown in T25cm2 flasks (volumes used for the T12.5cm2 flasks were half that described here) were washed using 5ml Phosphate-Buffered Saline (PBS) (Thermo Fisher, catalogue number 70011069), and detached from their adherent state using 2ml 0.25% Trypsin-EDTA (Thermo Fisher, catalogue number 25200056). Both flask sizes were then filled with 30ml of cell-line specific complete media. Definity® Microbubbles (Lantheus Medical Imaging, Inc, supplied by Global Medical Solutions, Keilor Park, Australia) were activated using the Vialmix® activation device (Lantheus Medical Imaging, Inc) following the manufacturer’s instructions [24], and 480µl added to the relevant treatment flasks to give a final microbubble concentration of 1.6% v/v, which was close to that described in other in vitro studies [11–16].
The culture flasks including the untreated controls were then transported offsite to the treatment facility at either the Australian Radiation Protection and Nuclear Safety Agency (ARPANSA) (Yallambie, Australia) or the GenesisCare Epping Radiation Oncology Centre (EPROC) (Epping, Australia). Travel time to and from these facilities was around 20min each way, with the cells out of the 37°C incubator for ~90min.
Ultrasound Treatment
Once onsite at the treatment facility, Ultrasound sonication was applied using the LOGIQ i Portable Ultrasound with the 4C-RS transducer (GE Healthcare, Paramatta, Australia). Here, 2MHz frequency ultrasound was applied directly to the flask using a coupling gel, with a focal point of 3.25cm Focal Point and depth of 4cm to achieve a Mechanical Index of 0.9. These settings were the closest match possible to the mechanical index of 0.8 reported in previous studies [11–13]. The transducer was moved across the flask for ~90 seconds until the opaque, milky-white microbubbles had burst and the media had returned to its normal transparency. Previous preliminary studies using colorimetry and microscopy had shown that this visual change correlated with the bursting of the microbubbles (Data not shown). The flasks were then immediately exposed to either 0Gy, 3Gy or 6Gy of kV or MV x-rays. The three control flasks were left untreated, whilst the USMB only flask received ultrasound sonication in the absence of ionising radiation.
Radiation Treatment
6MV x-rays were delivered using either a Varian iX (EPROC) or an Elekta Synergy Linear Accelerator (ARPANSA), with the same linac used for each repeat of an individual experiment. Flasks were laid flat on the treatment couch on top of 10cm of solid water, with a source-to-surface distance (SSD) of 100cm set to the top of the solid water from a gantry angle of 0o. Next 2cm of solid water was then placed on top of the flasks to provide a build-up region, and both the RT±USMB flasks irradiated simultaneously using a 20x20cm field size to cover both flasks. A schematic diagram of this setup is shown in Figure 1.
100kVp x-rays (average energy 42.5kV) were delivered using a Model X80 X-Ray Beam irradiator by Hopewell Designs (ARPANSA). A 100cm SSD was set to the baseplate of the unit before the RT±USMB flasks were taped vertically to the baseplate (see Figure 2). A 10cm circle field size was used to expose both flasks concurrently. As the dose rate from this unit was 6mGy/s, the 3Gy and 6Gy doses took 491.8 and 983.6 seconds to deliver respectively, which was the determining factor in the selection of x-ray doses used in this study. The 3Gy dose was chosen as an approximate clinical fractional dose consistent with that used in an earlier study [15], while the 6Gy dose selected as the highest dose deliverable in a reasonable timeframe within the limitations of the 6mGy/s dose rate. Radiation field uniformity and coverage was validated in the x- and y-planes for all radiation setups using Gafchromic film (data not shown).
Post-Treatment
Following treatments, the flasks were transported back to the laboratory where they were returned into the incubator for a few minutes whilst the laminar flow hood was prepared. Once flasks were examined under the microscope to confirm cells were still detached, flask contents were transferred to a 50ml tube and centrifuged (200g for 5min) to remove any residual treatment compounds. Cells were then resuspended in a T25cm2 flask containing 10ml complete media and returned to the incubator. After 24 hours, the tissue culture media was replenished, and 48 hours after which cell viability was measured using MTS assay.
Cell viability measurements
At ~72 hours after treatment, the media was removed from each flask, and replaced with 1ml supplement free (ie. Incomplete) tissue culture media and 200µl MTS at a ratio of 5:1 per the manufacturer’s instructions [21]. Four technical replicates of 100µl incomplete media plus 20µl MTS were plated in a 96 well plate to act as media blanks. Flasks and plates were then returned to the incubator for the development times stated earlier. Four technical replicates of 120µl from each flask were then transferred to the 96 well plate and the plate read at 490nm using a CLARIOstar Plus Plate reader (BMG Labtech, Mornington, Australia). The average of the four technical replicates was calculated for each condition, and the average of the four blank wells subtracted to give the final Raw Absorbance reading for each condition. Values for the three control flasks were averaged within each experiment, and the standard deviation (S.D.) calculated. Normalised survival was then calculated by dividing the Raw Absorbance values by the averaged control for each experiment.
Statistical Analysis
Overall normalised survival was averaged across the three repeats of the same experiment, and the S.D. calculated using Excel. The key assumptions of the ANOVA test were initially validated in SPSS V26 (International Business Machines Corporation (IBM), USA), using the Shapiro-Wilk (SW) test to confirm normality of distributions; Levine’s test to confirm homogeneity of Error Variances (LHEV); and outliers identified visually on the box-and-whisker plot as flagged by the software [25,26]. Statistical significance between treatment groups was then determined via a Three-Way ANOVA using a 2x3x2 design to report on the two levels of USMB (ie. presence or absence), three radiation dose levels of 0, 3 and 6Gy, and two radiation energies of kV and MV x-rays. Post-Hoc analysis included pair-wise t-tests, with p values <0.05 reported as statistically significant [27]. Where LHEV revealed unequal variances, data was re-analysed using a One-Way Welch’s ANOVA with Games-Howell Post-Hoc testing after re-expressing the 2x3x2 design as a one-way subprogram [26,28].
A coefficient of variation was calculated using the standard error of the Raw Absorbance values from each experiment and dividing this by the overall average. This value is used to give an indication of any variation arising from differences in plating between experiments [27].