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Ultrasound-Stimulated Microbubbles Enhance Radiation-Induced Cell Killing

Open AccessPublished:September 11, 2022DOI:https://doi.org/10.1016/j.ultrasmedbio.2022.07.001

      Abstract

      Recent in vivo studies using ultrasound-stimulated microbubbles as a localized radiosensitizer have had impressive results. While in vitro studies have also obtained similar results using human umbilical vein endothelial cells (HUVEC), studies using other cell lines have had varying results. This study was aimed at investigating any increases in radiation-induced cell killing in vitro using two carcinoma lines not previously investigated before (metastatic follicular thyroid carcinoma cells [FTC-238] and non-small cell lung carcinoma cells [NCI-H727]), in addition to HUVEC. Cells were treated using a combination of 1.6% (v/v) microbubbles, ∼90 s of 2-MHz ultrasound (mechanical index = 0.8) and 0–6 Gy of kilovolt or MV X-rays. Cell viability assays obtained 72 h post-treatment were normalized to untreated controls, and analysis of variance was used to determine statistical significance. All cells treated with combined ultrasound-stimulated microbubbles and radiation exhibited decreased normalized survival, with statistically significant effects observed for the NCI-H727 cells. No statistically significant differences in effects were observed using kV compared with MV radiation. Further studies using increased microbubble concentrations may be required to achieve statistically significant results for the FTC-238 and HUVEC lines.

      Key Words

      Introduction

      Microbubbles (MB) have long been used as contrast agents to improve image quality in ultrasound (US) cardiac imaging (
      • Kaul S.
      Myocardial contrast echocardiography: A 25-year retrospective.
      ). Their size is similar to that of the average erythrocyte, meaning they remain within the endovascular borders during their travel from the site of injection to the site of interest. This feature has resulted in MB also being investigated for a variety of focused therapeutic purposes, as they can be “burst” using higher US intensities once at the site of interest. This “bursting” creates localized biophysical disruptions in the cell's vicinity (
      • Ebben HP
      • Nederhoed JH
      • Lely RJ
      • Wisselink W
      • Yeung K.
      Microbubbles and ultrasound-accelerated thrombolysis (MUST) for peripheral arterial occlusions: Protocol for a phase II single-arm trial.
      ;
      • Zhu Q
      • Dong G
      • Wang Z
      • Sun L
      • Gao S
      • Liu Z.
      Intra-clot microbubble-enhanced ultrasound accelerates catheter-directed thrombolysis for deep vein thrombosis: A clinical study.
      ) and, if loaded with a treatment agent, can also deliver a localized therapeutic payload (
      • Liu Y
      • Miyoshi H
      • Nakamura M.
      Encapsulated ultrasound microbubbles: Therapeutic application in drug/gene delivery.
      ).

      In vivo studies

      As a result, there has been recent interest in using ultrasound-stimulated microbubbles (USMB) as localized radiosensitizers of radiation treatment (RT), with in vivo studies reporting some promising results.
      • Czarnota GJ
      • Karshafian R
      • Burns PN
      • Wong S
      • Al Mahrouki A
      • Lee JW
      • Caissie A
      • Tran W
      • Kim C
      • Furukawa M
      Tumor radiation response enhancement by acoustical stimulation of the vasculature.
      reported a statistically significant increase in mean animal survival from 19d with RT alone to 28 d using USMB and RT together (USMB + RT). PC3 human prostate cancer xenografts in mice were treated twice weekly with USMB in conjunction with 24 Gy in 12 fractions of 160-kVp X-rays delivered four times a week for 3 wk.
      • Daecher A
      • Stanczak M
      • Liu JB
      • Zhang J
      • Du S
      • Forsberg F
      • Leeper DB
      • Eisenbrey JR.
      Localized microbubble cavitation-based antivascular therapy for improving HCC treatment response to radiotherapy.
      reported a mean animal survival of 11 d with RT alone compared with 35 d using USMB + RT in treating human hepatocellular carcinoma xenografts in nude rats. In this study the xenografts were treated with USMB, followed by a single dose of 5-Gy kilovolt X-rays delivered using 4- × 1.25-Gy beams. Two animals treated with USMB + RT exhibited complete tumor control, with tumor reduction or stability observed 50 d post-treatment.
      • Eisenbrey JR
      • Shraim R
      • Liu JB
      • Li J
      • Stanczak M
      • Oeffinger B
      • Leeper DB
      • Keith SW
      • Jablonowski LJ
      • Forsberg F.
      Sensitization of hypoxic tumors to radiation therapy using ultrasound-sensitive oxygen microbubbles.
      observed a statistically significant growth delay for MDA-MB-231 human adenocarcinoma breast xenografts in mice administered USMB + RT compared with the controls administered US and oxygen MB alone (p = 0.03) or US and RT in the absence of MB (p = 0.01). Animals were treated with in-house manufactured oxygen or nitrogen USMB and a single fraction of 5 Gy using 310-kV X-rays.
      Other in vivo studies have also determined that USMB + RT increase tumor cell death 24 h after treatment.
      • Al-Mahrouki AA
      • Iradji S
      • Tran WT
      • Czarnota GJ.
      Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model.
      , using clonogenic assays from excised PC3 xenografts in mice, reported decreases in cell survival from 45.5% for 2 Gy alone to 26.8% for USMB + 2Gy, and from 38.2% for 8 Gy alone to 14.4% for USMB + 8 Gy, respectively.
      • Lai P
      • Tarapacki C
      • Tran WT
      • El Kaffas A
      • Lee J
      • Hupple C
      • Iradji S
      • Giles A
      • Al-Mahrouki A
      • Czarnota GJ.
      Breast tumor response to ultrasound mediated excitation of microbubbles and radiation therapy in vivo.
      , using in situ end labeling (ISEL) staining, reported increased rates of tumor death for MDA-MB-231 xenografts in Swiss nude mice of 3.4- and 2.3-fold respectively, for USMB + 2 Gy or USMB + 8 Gy treatments compared with 2- or 8-Gy RT alone.
      It has been proposed that USMB + RT behaves as a biophysical vascular disruptor in vivo, acting primarily on endothelial cells to disturb tumoral blood supply (
      • El Kaffas A
      • Czarnota GJ.
      Biomechanical effects of microbubbles: From radiosensitization to cell death.
      ). As a result of downstream vascular disruption, overall tumor control can be achieved at much lower radiation doses as microvascular collapse and rapid vascular shutdown lead to secondary tumor cell death once upstream cells become starved of nutrients and oxygen.

      In vitro studies

      Consequently, three in vitro studies were centered around human umbilical vein endothelial cells (HUVEC) using 3.3% (v/v) MB, 30 s of US with a mechanical index of 0.8 and between 0 and 8 Gy of 160-kVp X-rays. Clonogenic assays (normalized to control) revealed an increase in cell death from 67% for 8 Gy alone (p < 0.014) to 95% for USMB + 8 Gy (p < 0.002) (
      • Al-Mahrouki AA
      • Karshafian R
      • Giles A
      • Czarnota GJ.
      Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: Gene expression changes associated with radiation enhancement in vitro.
      ), and a decrease in cell survival from 8% using 2 Gy alone to 1% using USMB + 2 Gy (
      • Nofiele JIT
      • Karshafian R
      • Furukawa M
      • Al Mahrouki A
      • Giles A
      • Wong S
      • Czarnota GJ
      Ultrasound-activated microbubble cancer therapy: Ceramide production leading to enhanced radiation effect in vitro.
      ). The third study using HUVEC (
      • Al-Mahrouki AA
      • Wong E
      • Czarnota GJ.
      Ultrasound-stimulated microbubble enhancement of radiation treatments: Endothelial cell function and mechanism.
      ), found that treatment with USMB alone or USMB + RT caused cell membrane distortion, which was not evident in cells treated with RT alone, suggesting multiple mechanisms of cell damage may be achieved through the combined USMB + RT treatment.
      Other in vitro studies investigating direct tumor cell radiosensitization using USMB have also been conducted.
      • Karshafian R
      • Giles A
      • Burns PN
      • Czarnota GJ.
      Ultrasound-activated microbubbles as novel enhancers of radiotherapy in leukemia cells in vitro.
      exposed acute myeloid leukemia (AML-5) cells to varying concentrations of MB, differing US pressures and 2–8 Gy of 160-kVp X-rays, and observed that cell viability decreased with increased MB concentration until maximal death was achieved at ∼1.6% (v/v). Higher USMB levels did not increase cell killing but rather conferred a slightly protective effect. When AML-5 cells were exposed to 570-kPa US peak negative pressure (PNP) and 3.3% (v/v) USMB, cell viability was 71 ± 7% which was higher than that seen for cells exposed only to 4-Gy radiation (55 ± 5%). The order of treatment (i.e., USMB followed by RT compared with RT followed by USMB) was found to have minimal impact on cell viability. Clonogenic assays were 11% for 2 Gy alone versus 2.5% for USMB + 2 Gy normalized to control (no treatment). Clonogenic survival also fell in another study (
      • Karshafian R
      • Tchouala JIN
      • Al-Mahrouki A
      • Giles A
      • Czarnota GJ.
      Enhancement of radiation therapy by ultrasonically-stimulated microbubbles in vitro: Effects of treatment scheduling on cell viability and production of ceramide.
      ), when PC3 and KHT-C murine fibrosarcoma cells were treated with USMB + 3 Gy (PC3 17% and KHT-C 34%, respectively) compared with RT alone (PC3 50% and KHT-C 75%, respectively). In this study, treatment order and time between treatments affected clonogenic survival, with greatest decreases in clonogenic viability seen when PC3 cells were exposed to USMB followed by immediate 3-Gy treatment (17%), compared with that of KHT-C cells treated with USMB followed by a 3-h gap before being exposed to 3 Gy (30%).
      • Deng H
      • Cai Y
      • Feng Q
      • Wang X
      • Tian W
      • Qiu S
      • Wang Y
      • Li Z
      • Wu J.
      Ultrasound-stimulated microbubbles enhance radiosensitization of nasopharyngeal carcinoma.
      treated NCE-2 nasopharyngeal carcinoma cells with 3% (v/v) perfluropropane-based USMB, administered 24 h prior to exposure to 2- or 8-Gy RT. Cell viability was measured using the 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay 24, 48 and 72 hours post-treatment. Significant (p < 0.01) decreases in viability for NCE-2 cells treated with USMB + 2 Gy compared with 2 Gy were observed at 48 h, and at 72 h, USMB + RT significantly increased cell death for both radiation doses.
      At a cellular level, USMB has previously been used to increase cell membrane permeability via the induction of sonoporation effects (
      • Fan Z
      • Kumon RE
      • Deng CX.
      Mechanisms of microbubble-facilitated sonoporation for drug and gene delivery.
      ;
      • Lentacker I
      • De Cock I
      • Deckers R
      • De Smedt SC
      • Moonen CTW.
      Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms.
      ;
      • Osei E
      • Al-Asady A.
      A review of ultrasound-mediated microbubbles technology for cancer therapy: A vehicle for chemotherapeutic drug delivery.
      ), and several early phase I clinical trials have subsequently confirmed the translational possibilities of this as a novel treatment modality (
      • Dimcevski G
      • Kotopoulis S
      • Bjånes T
      • Hoem D
      • Schjøtt J
      • Gjertsen BT
      • Biermann M
      • Molven A
      • Sorbye H
      • McCormack E.
      A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer.
      ;
      • Wang Y
      • Li Y
      • Yan K
      • Shen L
      • Yang W
      • Gong J
      • Ding K.
      Clinical study of ultrasound and microbubbles for enhancing chemotherapeutic sensitivity of malignant tumors in digestive system.
      ). It is thought that the combination of these sonoporation effects on cell membranes works synergistically with DNA damage arising from ionizing radiation to produce the enhanced tumor cell death seen through the combination of USMB and RT (
      • Al-Mahrouki AA
      • Karshafian R
      • Giles A
      • Czarnota GJ.
      Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: Gene expression changes associated with radiation enhancement in vitro.
      ).
      However, in a study of human pharyngeal squamous carcinoma (FaDU) cells, no differences between cells treated with USMB + RT and those treated with RT alone were observed in clonogenic assays (
      • Lammertink BHA
      • Bos C
      • van der Wurff-Jacobs KM
      • Storm G
      • Moonen CT
      • Deckers R.
      Increase of intracellular cisplatin levels and radiosensitization by ultrasound in combination with microbubbles.
      ). In this study the cells were exposed to 1 µg/mL cisplatin and USMB (700 µL of SonoVue) 24 h before being treated with varying doses of MV γ-rays (2, 4, 6, 8 or 10 Gy). The addition of USMB did not enhance any further damage than that elicited by RT alone in these cells.
      As this study stands in stark contrast to all other research to date, the purpose of our study was to investigate the role of USMB in directly enhancing radiation-induced tumor cell killing in vitro. Two tumor cell lines not previously investigated before were used to determine whether direct USMB radiosensitization could be achieved for both a primary and metastatic cancer cell line. NCI-H727 cells are from a primary non-small cell lung carcinoma (NSCLC), while FTC-238 cells are from a metastatic follicular thyroid carcinoma deposit in lung tissue. Lung cancer is the most common cause of cancer death worldwide (

      World Health Organization. Cancer. Available at: https://www.who.int/news-room/fact-sheets/detail/cancer. Accessed February 26, 2022.

      ), and radiotherapy treatment of lung lesions is challenging (
      • Piperdi H
      • Portal D
      • Neibart SS
      • Yue NJ
      • Jabbour SK
      • Reyhan M.
      Adaptive radiation therapy in the treatment of lung cancer: An overview of the current state of the field.
      ), hence the need to identify any means of radiotherapy dose enhancement available for these types of tumors. HUVEC were also investigated to allow for comparisons with existing published data (
      • Al-Mahrouki AA
      • Karshafian R
      • Giles A
      • Czarnota GJ.
      Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: Gene expression changes associated with radiation enhancement in vitro.
      ,
      • Al-Mahrouki AA
      • Wong E
      • Czarnota GJ.
      Ultrasound-stimulated microbubble enhancement of radiation treatments: Endothelial cell function and mechanism.
      ). Definity MB were selected as they are the only commercially available MB approved for clinical use in Australia, and clinical hardware was used to provide US simulation. Both kilovolt (kV) and megavolt (MV) X-rays were used to investigate if different radiation energies enhance the cytotoxic effect of USMB, as previously it was reported that MV energies may not (
      • Lammertink BHA
      • Bos C
      • van der Wurff-Jacobs KM
      • Storm G
      • Moonen CT
      • Deckers R.
      Increase of intracellular cisplatin levels and radiosensitization by ultrasound in combination with microbubbles.
      ).

      Methods

      Cell lines and culture conditions

      Experiments were performed using HUVEC, FTC-238 (lung metastasis of follicular thyroid carcinoma) and NCI-H727 (non-small cell lung carcinoma). HUVEC (Catalog No. C2519A, pooled donor) were purchased from Lonza (Walkersville, MD, USA). FTC-238 and NCI-H727 cell lines were supplied by the European Collection of Cell Cultures (ECACC; Salisbury, UK) as Catalog Nos. 94060902 and 94060303, respectively, and were purchased from CellBank Australia (Westmead, Australia). All cell lines have been previously described (
      • Takahashi T
      • Nau MM
      • Chiba I
      • Birrer MJ
      • Rosenberg RK
      • Vinocour M
      • Levitt M
      • Pass H
      • Gazdar AF
      • Minna JD.
      p53: A frequent target for genetic abnormalities in lung cancer.
      ;
      • Goretzki P
      • Frilling A
      • Simon D
      • Roeher HD.
      Growth regulation of normal thyroids and thyroid tumors in man. In: Hormone-related malignant tumors.
      ;
      • Dos Santos M
      • Paget V
      • Ben Kacem M
      • Trompier F
      • Benadjaoud MA
      • François A
      • Guipaud O
      • Benderitter M
      • Milliat F
      Importance of dosimetry protocol for cell irradiation on a low X-rays facility and consequences for the biological response.
      ).
      Cells were maintained according to the manufacturer's instructions and incubated at 37°C, 5% CO2 and 95% humidity. HUVEC were cultured using the EGM-2 Endothelial Cell Growth Medium-2 BulletKit from Lonza (Catalog No. CC-13162). FTC-238 cells were cultured using DMEM:F12 (Catalog No. D8437, Sigma-Aldrich, Sydney, Australia), supplemented with 5% fetal bovine serum (FBS, Corning, Catalog No. 35-076-CV supplied by Fisher Biotec, Wembley, Australia) and 1% penicillin–streptomycin (Pen-Strep, Catalog No. 15070063, Sigma-Aldrich). NCI-H727 cells were cultured in RPMI-1640 (Catalog No. 11875093, Thermo Fisher, Scorsby Australia), supplemented with 10% FBS and 1% Pen-Strep.

      Plating and MTS assay optimization

      Cell proliferation assays were initially optimized in the absence of treatments using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Catalog No. 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 (
      • Deng H
      • Cai Y
      • Feng Q
      • Wang X
      • Tian W
      • Qiu S
      • Wang Y
      • Li Z
      • Wu J.
      Ultrasound-stimulated microbubbles enhance radiosensitization of nasopharyngeal carcinoma.
      ), and is bio-reduced by viable cells into a colored formazan product that can be analyzed via colorimetry. Cells are incubated with the reagent for between 1 and 4 h, and then absorbance readings were taken at 490 nm 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 ().
      Cell seeding densities and MTS development times were optimized to achieve 80% confluence at 72 h post-treatment for each cell line, as summarized in Table 1. This level of confluence was chosen to avoid any potential effects of contact-dependent cell signaling or contact inhibition, and 72 h is consistent with the manufacturer's instructions for cell proliferation studies and other experiments using this assay (
      • Smith DJ
      • Jaggi M
      • Zhang W
      • Galich A
      • Du C
      • Sterrett SP
      • Smith LM
      • Balaji K.
      Metallothioneins and resistance to cisplatin and radiation in prostate cancer.
      ; ;
      • Youkhana EQ
      • Feltis B
      • Blencowe A
      • Geso M.
      Titanium dioxide nanoparticles as radiosensitisers: An in vitro and phantom-based study.
      ) and the MTT assay (
      • Deng H
      • Cai Y
      • Feng Q
      • Wang X
      • Tian W
      • Qiu S
      • Wang Y
      • Li Z
      • Wu J.
      Ultrasound-stimulated microbubbles enhance radiosensitization of nasopharyngeal carcinoma.
      ). Because of the number of treatment conditions investigated in this study, the MTS assay was selected as it allows for high-volume throughput and is similar to the MTT assay that has demonstrated equivalence to the clonogenic assay (
      • Buch K
      • Peters T
      • Nawroth T
      • Sänger M
      • Schmidberger H
      • Langguth P.
      Determination of cell survival after irradiation via clonogenic assay versus multiple MTT assay—A comparative study.
      ).
      Table 1Cell seeding densities and MTS development times
      Cell lineFlask size (cm)Seeding density (cells/flask)MTS development time (min)
      HUVECT258,00025
      FTC-238T2512,00025
      NCI-H727T12.550,00015
      HUVEC = human umbilical vein endothelial cells; MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.
      HUVEC and FTC-238 cells from stock cultures were plated into T25 tissue culture flasks (Sigma-Aldrich, Catalog No. CLS430639). Because of supply issues, NCI-H727 cells were plated into T12.5 flasks (Bio-Strategy, Catalog No. BDAA353107, Tullamarine, Australia) which were as high as the T25 flasks (2.62 cm), but were 3.6 cm longer and 0.79 cm wider. Cells at the designated seeding densities were allowed to adhere overnight in their respective flasks for ∼14–16 h before treatment. Eight flasks were prepared for each experiment—three technical replicate control flasks (no treatment), one USMB-alone flask (USMB), and two flasks for each radiation dose level, one with USMB and one without (i.e., 3 Gy, USMB + 3 Gy, 6 Gy and USMB + 6 Gy). 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 in which 12 flasks were prepared for each experiment: four for kV flasks and four for MV flasks in addition to four 0-Gy flasks (i.e., three controls and one USMB). This experiment was also performed in triplicate.

      Pre-treatment preparation

      Two hours prior to treatment, cells grown in T25 flasks (volumes used for the T12.5 flasks were half that described here) were washed using 5 mL of phosphate-buffered saline (PBS, Thermo Fisher, Catalog No. 70011069), and detached from their adherent state using 2 mL of 0.25% trypsin–EDTA (Thermo Fisher, Catalog No. 25200056). Flasks of both sizes were then filled with 30 mL of cell-line specific complete medium and were periodically gently shaken throughout treatments to ensure cells did not re-attach. Definity Microbubbles (Lantheus Medical Imaging, Inc, supplied by Global Medical Solutions, Keilor Park, Australia) were activated using the Vialmix activation device (

      DEFINITY®. Package insert. Lantheus Medical Imaging, Inc.; 2013.

      ) according to the manufacturer's instructions (

      DEFINITY®. Package insert. Lantheus Medical Imaging, Inc.; 2013.

      ), and 480 µL was added to the relevant treatment flasks to give a final microbubble concentration of 1.6% (v/v) (∼2.8 × 109 MB/flask), which was close to that described in other in vitro studies (
      • Karshafian R
      • Giles A
      • Burns PN
      • Czarnota GJ.
      Ultrasound-activated microbubbles as novel enhancers of radiotherapy in leukemia cells in vitro.
      ,
      • Karshafian R
      • Tchouala JIN
      • Al-Mahrouki A
      • Giles A
      • Czarnota GJ.
      Enhancement of radiation therapy by ultrasonically-stimulated microbubbles in vitro: Effects of treatment scheduling on cell viability and production of ceramide.
      ;
      • Al-Mahrouki AA
      • Karshafian R
      • Giles A
      • Czarnota GJ.
      Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: Gene expression changes associated with radiation enhancement in vitro.
      ,
      • Al-Mahrouki AA
      • Wong E
      • Czarnota GJ.
      Ultrasound-stimulated microbubble enhancement of radiation treatments: Endothelial cell function and mechanism.
      ;
      • Nofiele JIT
      • Karshafian R
      • Furukawa M
      • Al Mahrouki A
      • Giles A
      • Wong S
      • Czarnota GJ
      Ultrasound-activated microbubble cancer therapy: Ceramide production leading to enhanced radiation effect in vitro.
      ;
      • Deng H
      • Cai Y
      • Feng Q
      • Wang X
      • Tian W
      • Qiu S
      • Wang Y
      • Li Z
      • Wu J.
      Ultrasound-stimulated microbubbles enhance radiosensitization of nasopharyngeal carcinoma.
      ).
      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 20 min each way, with the cells out of the 37°C incubator for ∼90 min.

      Ultrasound treatment

      Once onsite at the treatment facility, ultrasound sonication was applied using the LOGIQi Portable Ultrasound with the 4C-RS transducer in simple 2-D B-mode (GE Healthcare, Paramatta, Australia). Here, 2-MHz frequency ultrasound was applied directly to the flask using a coupling gel, with a focal point of 3.25 cm and depth of 4 cm, resulting in a frame rate of 61 Hz. Two megahertz was selected to provide a wide beam that exposed the greatest surface area and minimized attenuation from the polystyrene walls of the flask. The depth of 4 cm was selected to ensure the entire height of the flask was exposed within the near zone. The transducer was moved across the flask for ∼90 s until the opaque, milky-white microbubbles had burst and the medium had returned to its normal transparency. Previous preliminary studies using colorimetry and microscopy had revealed that this visual change correlated with the bursting of the microbubbles and this visual change, along with the ultrasound setup, can be seen in Figure 1. The flasks were then immediately exposed to either 0, 3 or 6 Gy of kV or MV X-rays. The three control flasks were left untreated, and the USMB-only flask received ultrasound sonication in the absence of ionizing radiation.
      Fig 1
      Fig. 1Images of the ultrasound exposure revealing how the transducer was moved across the surface of the flask for ∼90 s until the milky white microbubbles as seen in (A) had burst and the medium returned to its normal transparency (B).

      Radiation treatment

      Six-megavolt X-rays were delivered using either a Varian iX (EPROC) or an Elekta Synergy Linear Accelerator (ARPANSA), with the same linear accelerator used for each repeat of an individual experiment. Flasks were laid flat on the treatment couch on top of 10 cm of solid water, with a source-to-surface distance (SSD) of 100 cm set to the top of the solid water from a gantry angle of 0o. Next, 2 cm of solid water was then placed on top of the flasks to provide a buildup region, and both RT ± USMB flasks were irradiated simultaneously using a 20- × 20-cm field size to cover both flasks. Figure 2 is a schematic of this setup.
      Fig 2
      Fig. 2Schematic illustrating the experimental setup for megavolt X-ray exposures. Radiation therapy ± ultrasound-stimulated microbubble flasks were placed on top of 10 cm of solid water, with a 100-cm source-to-surface distance set to the top of the solid water. Then 2 cm of solid water was placed on top of the flasks to provide a buildup region. The flasks were exposed to a 6-MV X-ray beam at a gantry angle of 0o and field size of 20 × 20 cm.
      The 100-kVp X-rays (average energy: 42.5 kV) were delivered using a Model X80 X-Ray Beam irradiator by Hopewell Designs (ARPANSA). A 100-cm SSD was set to the baseplate of the unit before the RT ± USMB flasks were taped vertically to the baseplate (see Fig. 3). A 10-cm circle field size was used to expose both flasks concurrently. As the dose rate from this unit was 6 mGy/s, the 3- and 6-Gy doses took 491.8 and 983.6 s to deliver, respectively, which was the determining factor in the selection of X-ray doses used in this study. The 3-Gy dose was chosen as an approximate clinical fractional dose consistent with that used in an earlier study (
      • Karshafian R
      • Tchouala JIN
      • Al-Mahrouki A
      • Giles A
      • Czarnota GJ.
      Enhancement of radiation therapy by ultrasonically-stimulated microbubbles in vitro: Effects of treatment scheduling on cell viability and production of ceramide.
      ), while the 6-Gy dose was selected as the highest dose deliverable in a reasonable time frame within the limitations of the 6 mGy/s dose rate. Radiation field uniformity and coverage were validated in the x- and y-planes for all radiation setups using Gafchromic film (data not shown).
      Fig 3
      Fig. 3Image of experimental setup for kilovolt X-ray exposures. Treatment flasks taped vertically to the baseplate (A) of the Model X80 X-Ray Beam irradiator (B). The source-to-surface distance was set at 100 cm to the front of the baseplate.

      Post-treatment

      After treatments, the flasks were transported back to the laboratory where they were returned to the incubator for a few minutes while 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 50-mL tube and centrifuged (200g for 5 min) to remove any residual treatment compounds. Cells were then resuspended in a T25 flask containing 10 mL of complete medium and returned to the incubator. After 24 h, the tissue culture medium was replenished, and 48 h later cell viability was measured using the MTS assay.

      Cell viability measurements

      At ∼72 h after treatment, the medium was removed from each flask, and replaced with 1 mL of supplement-free (i.e., Incomplete) tissue culture medium and 200 µL of MTS at a ratio of 5:1 per the manufacturer's instructions (). Four technical replicates of 100 µL of incomplete medium plus 20 µL of MTS were plated in a 96-well plate to act as medium blanks. Flasks and plates were then returned to the incubator for the development times stated earlier. Four technical replicates of 120 µL from each flask were then transferred to the 96-well plate, and the plate was read at 490 nm 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 (SD) calculated. Normalized survival was then calculated by dividing the raw absorbance values for each treatment condition by the averaged raw absorbance control value for each experiment.

      Statistical analysis

      Overall normalized survival was averaged across the three repeats of the same experiment, and the SD was calculated using Excel. The key assumptions of the analysis of variance (ANOVA) test were initially validated in SPSS version 27 software (IBM, Armonk, NY, USA), using the Shapiro–Wilk (SW) test to confirm the normality of distributions; Levine's test to confirm the homogeneity of error variance (LHEV); and outliers identified visually on the box-and-whisker plot as flagged by the software (;

      Laerd Statistics. Testing for Normality using SPSS Statistics when you have only one independent variable. Available at: https://statistics.laerd.com/spss-tutorials/testing-for-normality-using-spss-statistics.php. Accessed October 9, 2020.

      ). Statistical significance between treatment groups was then determined via a three-way ANOVA using a 2 × 3 × 2 design to report on the two levels of USMB (i.e., presence or absence); three radiation dose levels of 0, 3 and 6 Gy; and two radiation energies of kilovolt and MV X-rays. Post hoc analysis included pairwise t-tests, with p values <0.05 reported as statistically significant (

      Fay DS, Gerow K. WormBook: The online review of C. elegans biology [Internet]. Available at: https://www.ncbi.nlm.nih.gov/books/NBK153593/. Accessed March 20, 2020.

      ). Where the LHEV revealed unequal variances, data were re-analyzed using a one-way Welch's ANOVA with Games–Howell post hoc testing after re-expressing the 2 × 3 × 2 design as a one-way subprogram (

      Fay DS, Gerow K. WormBook: The online review of C. elegans biology [Internet]. Available at: https://www.ncbi.nlm.nih.gov/books/NBK153593/. Accessed March 20, 2020.

      ; ).

      Results

      NCI-H727 cells

      Three-way ANOVA revealed statistically significant decreases in irradiated NCI-H727 cell survival across all RT dose levels and energies through the addition of USMB (Fig. 4). USMB alone in the absence of radiation resulted in significant cell death, producing a large, extremely significant overall difference in normalized survival compared with untreated controls (ηp2 = 0.776, p < 0.0001).
      Fig 4
      Fig. 4Effect of USMB + RT on the viability of NCI-H727 cells. Cells were treated with 1.6% MB, ∼90 s of US and either 0, 3 or 6 Gy of kV (red) and MV (blue) X-rays. Results are expressed as the mean + standard deviation for three biological replicates. The value for the untreated controls was assigned as unity (1), and the values for the treated cells were expressed as a fraction of this number. Both kV and MV experiments were carried out concurrently for this cell line. Significance values were calculated using a three-way analysis of variance. kV = kilovolt; MV = megavolt; RT = radiation therapy; USMB = ultrasound-stimulated microbubbles.
      No overall difference in normalized cell survival was seen between cells treated with kV X-rays and those treated with MV X-rays (ηp2 = 0.025, p = 0.420), with comparisons between equivalent cohorts treated using the same radiation dose levels and USMB conditions revealing no significant difference in survival between the two radiation energies.
      Radiation dose had the largest overall effect (ηp2 = 0.796, p < 0.0001), with normalized survival decreasing as radiation dose increased. However, dose–response analysis revealed only one significant decrease in normalized survival (NS) in response to increases in radiation dose, and this was for cells treated with 3 Gy compared with 6 Gy of kV radiation in the absence of USMB (NS = 0.62 for 3 Gy kV alone vs. NS = 0.47 for 6 Gy kV alone, p = 0.031). Almost no dose response was detected when radiation dose was increased from 3 to 6 Gy of MV radiation in the presence of USMB (NS = 0.36 for USMB + 3 Gy MV vs. NS = 0.35 for USMB + 6 Gy MV, p = 0.868).

      FTC-238 cells

      Decreases in normalized cell survival were observed through the addition of USMB; however, these was not statistically significant for FTC-238 cells as determined with Welch's ANOVA (Fig. 5). Unlike the NCI-H727 cells, treatment with USMB alone had little effect on FTC-238 cell survival compared with untreated controls, with only a small decrease in survival observed for the kV cohort (NS = 0.93 for kV, NS = 1.0 for MV).
      Fig 5
      Fig. 5Effect of USMB + RT on the viability of FTC-238 cells. Cells were treated with 1.6% MB, ∼90 s of US and either 0, 3 or 6 Gy of (A) kV (red) and (B) MV(blue) X-rays. Results are expressed as the mean + standard deviation for three biological replicates. The value for the untreated controls was assigned as unity (1), and the values for the treated cells were expressed as a fraction of this number. Significance values were calculated using Welch's analysis of variance as the data did not demonstrate homogeneity of variances. kV = kilovolt; MV = megavolt; RT = radiation therapy; USMB = ultrasound-stimulated microbubbles.
      A slight increase in overall cell kill was observed for cells treated with kV compared with MV radiation; however, pairwise comparisons between equivalent treatment conditions were statistically significant only for the USMB + 6 Gy cohorts (NS = 0.46 for USMB + 6 Gy kV vs. NS = 0.60 for USMB + 6 Gy MV, p = 0.038).
      For MV radiation, consistent decreases in survival with increases in dose were seen for both MV-alone and USMB + MV cohorts; however, these were not significant at any dose level. Normalized survival decreased by 0.17 when radiation dose was increased from 0 to 3 Gy in both the absence and presence of USMB, respectively (i.e., 0–3Gy MV-alone vs. USMB + 0–3 Gy MV). This trend continued as dose was increased from 3 to 6 Gy, with further decreases in survival of 0.21 and 0.23. For kV radiation, however, much larger decreases in survival were observed at lower doses, with normalized survival decreasing by 0.30 and 0.33 from 0 to 3 Gy for kV alone compared with USMB + kV. However, further dose increases from 3–6 Gy yielded a more diminished response in the USMB + kV cohorts, where a decrease of 0.14 was seen for USMB +3 Gy kV and USMB + 6 Gy kV compared with 0.21 between 3 Gy kV alone and 6 Gy kV alone.

      Human umbilical vein endothelial cells

      Much like the FTC-238 cells, decreases in normalized cell survival were observed through the addition of USMB; however, these was not statistically significant for HUVEC as determined with Welch's ANOVA (Fig. 6). Non-significant variations in cell survival were observed when HUVEC were treated with USMB alone. For the MV cohort, a decrease in cell survival compared with untreated controls was seen, while an increase was noted for the kV group (0.92 for MV compared with 1.06 for kV).
      Fig 6
      Fig. 6Effect of USMB + RT on the viability of human umbilical vein endothelial cells. Cells were treated with 1.6% MB, ∼90 s of US and either 0, 3 or 6 Gy of (A) kV and (B) MV x-rays. Results expressed as the mean + standard deviation for three biological replicates. The value for the untreated controls was assigned as unity (1), and the values for the treated cells were expressed as a fraction of this number. Significance values were calculated using a Welch's analysis of variance (ANOVA) as the data did not demonstrate homogeneity of variances. kV = kilovolt; MV = megavolt; RT = radiation therapy; USMB = ultrasound-stimulated microbubbles.
      No statistically significant differences were observed in cells treated using kV versus MV radiation, with the largest mean difference between equivalent conditions observed in the USMB + 3 Gy cohorts (NS = 0.45 for USMB + 3 Gy kV vs. NS = 0.61 for USMB + 3 Gy MV, p = 0.95).
      A statistically significant dose–response relationship was seen across all radiation doses for cells treated using kV radiation in the absence of USMB. Normalized survival decreased by 0.46 when radiation dose was increased from 0 to 3 Gy (p < 0.0001) and by 0.19 when increased from 3 to 6 Gy (p = 0.037). A similar trend was observed with the addition of USMB; however, this was statistically significant only for the 0- to 3-Gy dose level increase (NS decreased by 0.61, p = 0.024, for 0–3 Gy, compared with 0.11, p = 0.167, for 3–6 Gy). For cells treated with MV radiation, non-significant decreases in survival were consistent across the two dose levels, with the greatest difference observed when radiation dose was increased from 0 to 3 Gy MV in the absence of USMB (survival decreased by 0.35 between the 0- and 3-Gy MV-alone cohorts, and 0.22 between 3 and 6 Gy, compared with 0.27 between 0- and 3-Gy USMB + MV and 0.27 between 3- and 6-Gy USMB + MV).

      Discussion

      This study was aimed at investigating the role of USMB in directly enhancing radiation-induced tumor cell killing in vitro, given the conflicting results previously reported elsewhere. As there was strong evidence to support the effect as being cell type dependent, a variety of cell types were used, with a focus on lung tumors given the potential benefit radiosensitization may have in overcoming challenges of radiotherapy treatment delivery to the chest (
      • Piperdi H
      • Portal D
      • Neibart SS
      • Yue NJ
      • Jabbour SK
      • Reyhan M.
      Adaptive radiation therapy in the treatment of lung cancer: An overview of the current state of the field.
      ). Both kV and MV energies were also used to investigate radiation energy as another potential source of the reported variation, as no direct comparison between the two has been investigated to date, despite both being used in previous studies.

      USMB directly enhance radiation-induced tumor cell killing in vitro in a cell type- dependent manner

      Our results indicated that cells treated with combined USMB and radiation have decreased normalized survival compared with cells treated with radiation alone, with statistically significant effects observed for the NCI-H727 cells. USMB had a significant effect on the survival of irradiated NCI-H727 cells. This effect could be cell type dependent, which had been suggested earlier (
      • El Kaffas A
      • Czarnota GJ.
      Biomechanical effects of microbubbles: From radiosensitization to cell death.
      ). These cell type-specific responses to USMB + RT have previously been linked to ASMase gene expression and ceramide signaling associated with apoptosis. This was determined by
      • Nofiele JIT
      • Karshafian R
      • Furukawa M
      • Al Mahrouki A
      • Giles A
      • Wong S
      • Czarnota GJ
      Ultrasound-activated microbubble cancer therapy: Ceramide production leading to enhanced radiation effect in vitro.
      using ASMase deficient–/– astrocytes and ASMase+/+ astrocytes treated with sphingosine-1-phosphate (S1P) to counteract the ceramide-mediated apoptosis pathway. ASMase+/+ astrocytes were more sensitive to USMB + RT compared with ASMase–/– or S1P-ASMase+/+ astrocytes where ASMase and ceramide production had been genetically or chemically inhibited. As NCI-H727 cells elicited a strong response to USMB + RT, changes in ASMase gene expression and ceramide levels produced in response to this treatment should be investigated to determine whether this could be used as an indicator of cell sensitivity to such treatments. In a study of pre-operative blood serum samples extracted from 61 patients diagnosed with NSCLC, significantly increased levels of ASMase activity were detected compared with those in healthy controls (
      • Kachler K
      • Bailer M
      • Heim L
      • Schumacher F
      • Reichel M
      • Holzinger CD
      • Trump S
      • Mittler S
      • Monti J
      • Trufa DI
      • Rieker RJ
      • Hartmann A
      • Sirbu H
      • Kleuser B
      • Kornhuber J
      • Finotto S.
      Enhanced acid sphingomyelinase activity drives immune evasion and tumor growth in non–small cell lung carcinoma.
      ), which may explain why the NSCLC cell line investigated here exhibited such a strong response compared with the other two cell lines. Immunostaining cells for ASMase activity and/or ceramide production may be of benefit in future studies—both as a screening tool to identify candidate cells lines for further USMB + RT research and to better understand responses to USMB + RT treatment.
      Given the increased risks associated with delivering radiotherapy to centrally lying lung tumors (
      • Wu AJ
      • Williams E
      • Modh A
      • Foster A
      • Yorke E
      • Rimner A
      • Jackson A.
      Dosimetric predictors of esophageal toxicity after stereotactic body radiotherapy for central lung tumors.
      ;
      • Haseltine JM
      • Rimner A
      • Gelblum DY
      • Modh A
      • Rosenzweig KE
      • Jackson A
      • Yorke ED
      • Wu AJ.
      Fatal complications after stereotactic body radiation therapy for central lung tumors abutting the proximal bronchial tree.
      ), methods of localized radiotherapy sensitization as offered by USMB are of particular interest. The identification of specific cell types demonstrating radiosensitization effects through the addition of USMB provides exciting avenues for further research with direct clinical relevance. Although the logistics of microbubble delivery and ultrasound administration to the chest may require further optimization, solutions may be found in other areas of research, such as nanoparticle radiosensitization research, in which direct tumoral injection and inhalation administration methods have been investigated (
      • Boateng F
      • Ngwa W.
      Delivery of nanoparticle-based radiosensitizers for radiotherapy applications.
      ). Given microbubbles have long been used as a contrast agent to improve cardiac ultrasound imaging, microbubble stimulation of centrally lying lung tumors proximal to the mediastinum is not unfeasible and could be investigated further.
      HUVEC, however, have also been reported to have ∼20-fold higher levels of ASMase compared with other cells (
      • Marathe S
      • Schissel SL
      • Yellin MJ
      • Beatini N
      • Mintzer R
      • Williams KJ
      • Tabas I.
      Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase: Implications for early atherogenesis and ceramide-mediated cell signaling.
      ), and so should have also exhibited decreased viability when treated with USMB + RT in this study if ASMase gene expression is the primary mechanism involved. Further investigations of other factors that may be responsible for the dramatic effect USMB have on the viability of irradiated NCI-727 cells are underway. These factors include the use of T12.5 flasks, as the acoustic field applied to the NCI-H727 cells may have differed from that applied to the HUVEC and FTC-238 cells treated in T25 flasks, potentially resulting in differences in the level of sonoporation effects on these cells.
      • Kinoshita M
      • Hynynen K.
      Key factors that affect sonoporation efficiency in in vitro settings: The importance of standing wave in sonoporation.
      reported that cell viability was substantially decreased in the presence of standing waves, and the interaction of the US field with the T12.5 flask may have created such a phenomenon, which was absent in the T25 flasks. It is interesting to note that this study also reported differences in cell viability when cells were exposed to USMB in a suspended state compared with an adherent state.
      • Lammertink BHA
      • Bos C
      • van der Wurff-Jacobs KM
      • Storm G
      • Moonen CT
      • Deckers R.
      Increase of intracellular cisplatin levels and radiosensitization by ultrasound in combination with microbubbles.
      also exposed the cells in an adherent state, further introducing yet another point of difference from all other research using USMB + RT in which cells were treated in suspension. This may also explain why their results were so different and is another factor under investigation.
      Although slight dose enhancement was noted for HUVEC treated with USMB + RT compared with those treated with RT alone, the difference was not significant and less than that seen in previous studies (
      • Al-Mahrouki AA
      • Karshafian R
      • Giles A
      • Czarnota GJ.
      Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: Gene expression changes associated with radiation enhancement in vitro.
      ,
      • Al-Mahrouki AA
      • Wong E
      • Czarnota GJ.
      Ultrasound-stimulated microbubble enhancement of radiation treatments: Endothelial cell function and mechanism.
      ;
      • Nofiele JIT
      • Karshafian R
      • Furukawa M
      • Al Mahrouki A
      • Giles A
      • Wong S
      • Czarnota GJ
      Ultrasound-activated microbubble cancer therapy: Ceramide production leading to enhanced radiation effect in vitro.
      ). This difference could be related to the experimental setup, in which different cell numbers and culture vessels, MB concentrations, US applications, and x-ray fields were used. The 1.6% (v/v) MB concentration used in the current study was less than the 3.3% (v/v) used in those studies on HUVEC. MB concentrations were reported to be optimal at 1.6% (v/v) in enhancing the effect of RT on acute myeloid leukemia cells (
      • Karshafian R
      • Giles A
      • Burns PN
      • Czarnota GJ.
      Ultrasound-activated microbubbles as novel enhancers of radiotherapy in leukemia cells in vitro.
      ); however,
      • Lammertink BHA
      • Bos C
      • van der Wurff-Jacobs KM
      • Storm G
      • Moonen CT
      • Deckers R.
      Increase of intracellular cisplatin levels and radiosensitization by ultrasound in combination with microbubbles.
      observed no significant difference in the survival of FaDU cells when MB at 7% (v/v) were added to cells exposed to RT. Unlike HUVEC, the effect of MB on the cytotoxic effect of RT has not been examined for either FTC-238 and NCI-H727 cells, and as such, 1.6% v/v was chosen as the MB concentration to use in these experiments. As the effects elicited with 1.6% (v/v) MB on the cytotoxic effect of RT on HUVEC reported here were less than those seen in earlier studies (
      • Al-Mahrouki AA
      • Karshafian R
      • Giles A
      • Czarnota GJ.
      Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: Gene expression changes associated with radiation enhancement in vitro.
      ,
      • Al-Mahrouki AA
      • Wong E
      • Czarnota GJ.
      Ultrasound-stimulated microbubble enhancement of radiation treatments: Endothelial cell function and mechanism.
      ;
      • Nofiele JIT
      • Karshafian R
      • Furukawa M
      • Al Mahrouki A
      • Giles A
      • Wong S
      • Czarnota GJ
      Ultrasound-activated microbubble cancer therapy: Ceramide production leading to enhanced radiation effect in vitro.
      ), repeat experiments using 3.3% (v/v) are now warranted. Similarly, further quantification of the exact ultrasound pressures applied to the microbubbles may be of benefit, given the differences in the ultrasound equipment used in this study compared with previous studies (i.e., the use of a diagnostic US probe here compared with those used specifically for US research).

      Radiation energy is not a likely influence on results

      The FTC-238 cells are derived from a metastatic tumor deposit of a follicular thyroid carcinoma. The addition of MB at 1.6% (v/v) did not have a significant effect on the viability of these secondary tumor cells when exposed to either kV or MV radiation. Of interest was that at the same dose (3 or 6 Gy), kV radiation was more cytotoxic than MV radiation, with a statistically significant result observed between USMB + 6 Gy kV and USMB + 6 Gy MV. This difference between the cytotoxic effect elicited by kV and MV radiation was seen to a much lesser extent in the HUVEC, where a similar trend was observed; however, no statistically significant differences were seen between cohorts treated using the same radiation dose levels and USMB conditions using either kV or MV radiation. For the NCI-H727 cells, three-way ANOVA revealed no overall difference in normalized cell survival for cells treated using kV compared with MV X-rays, suggesting that for this cell line in particular, radiation energy has little influence on results.
      It is unlikely that such responses to differences in radiation energy are cell type dependent and are more likely due to the differences in raw absorbance readings recorded for the control flasks, which varied by ∼26% between the two X-ray treatment types for the FTC-238 cell line. This could have arisen from differences in travel times to the various treatment facilities that resulted in these cells being out of the incubator for different lengths of time, or the difference in medium-to-cell ratio between the T12.5 and T25 flasks during the ∼90-min treatment time. Subsequent research is required to investigate the impacts of these differences further.
      In the study conducted by
      • Lammertink BHA
      • Bos C
      • van der Wurff-Jacobs KM
      • Storm G
      • Moonen CT
      • Deckers R.
      Increase of intracellular cisplatin levels and radiosensitization by ultrasound in combination with microbubbles.
      , FaDU cells were exposed to MV X-rays, and the survival curves for cells treated with USMB + RT and those treated with RT were similar. This differs from other studies in which cells exposed to kV X-rays were treated with USMB and there was a reduction in cell survival rates (
      • Karshafian R
      • Giles A
      • Burns PN
      • Czarnota GJ.
      Ultrasound-activated microbubbles as novel enhancers of radiotherapy in leukemia cells in vitro.
      ,
      • Karshafian R
      • Tchouala JIN
      • Al-Mahrouki A
      • Giles A
      • Czarnota GJ.
      Enhancement of radiation therapy by ultrasonically-stimulated microbubbles in vitro: Effects of treatment scheduling on cell viability and production of ceramide.
      ;
      • Deng H
      • Cai Y
      • Feng Q
      • Wang X
      • Tian W
      • Qiu S
      • Wang Y
      • Li Z
      • Wu J.
      Ultrasound-stimulated microbubbles enhance radiosensitization of nasopharyngeal carcinoma.
      ). In our study, flasks of FTC-238 cells and HUVEC were exposed to kV and MV X-rays at different times, and it is unknown if a similar result would be obtained if the flasks were irradiated by both X-ray energies concurrently, as done for the NCI-H727 cells. We observed that in the NCI-727 cells, there was minimal difference in survival at similar dose levels for kV and MV X-rays, compared with the differences seen when the different cohorts were treated separately as for FTC-238 cells and HUVEC.

      Responses to USMB alone may be a predictor of dose enhancement

      The most likely explanation for the NCI-H727 results seen here is that these cells are inherently more sensitive to the effects of USMB, which is supported by the very highly significant difference (p < 0.001) in normalized survival between the control and USMB-only cohorts. Previous sonoporation studies have also determined different responses across cell lines of similar origin treated with USMB alone (
      • Bjånes T
      • Kotopoulis S
      • Murvold ET
      • Kamčeva T
      • Gjertsen BT
      • Gilja OH
      • Schjøtt J
      • Riedel B
      • McCormack E.
      Ultrasound- and microbubble-assisted gemcitabine delivery to pancreatic cancer cells.
      ). Where the HUVEC and FTC-238 cells may benefit from increases in MB concentration, further experiments at lower concentrations for the NCI-H727 cells may be required to establishing toxicity profiles for US, MB and USMB alone in the absence of RT.

      Conclusions

      Although USMB did enhance radiation-induced cell killing in vitro in the cell lines examined, the difference was statistically significant only for NCI-H727 non-small cell lung carcinoma cells. This suggests that enhancement is most likely cell type dependent, which is consistent with previous findings in the literature. However, there may also be other factors influencing these results, such as variations in applied acoustic fields, differences in the cytotoxicity profiles for USMB alone and cell culture state (i.e., suspension vs. adherent). Given the overall trends observed here, this study illustrates that this type of research is feasible using clinical hardware and that further opportunities exist to understand the specific mechanisms involved in the direct radiosensitization of tumor cells using USMB.

      Declaration of Competing Interest

      The authors declare no conflict of interest.

      Acknowledgments

      The authors thank the Australian Radiation Protection and Nuclear Safety Agency and the staff at GenesisCare Epping Radiation Oncology Centre for their support and use of their equipment. They also acknowledge the Australian Government's Department of Education and Training for providing financial support via the Joint Research Engagement Engineering Cadetship.

      References

        • Al-Mahrouki AA
        • Karshafian R
        • Giles A
        • Czarnota GJ.
        Bioeffects of ultrasound-stimulated microbubbles on endothelial cells: Gene expression changes associated with radiation enhancement in vitro.
        Ultrasound Med Biol. 2012; 38: 1958-1969
        • Al-Mahrouki AA
        • Iradji S
        • Tran WT
        • Czarnota GJ.
        Cellular characterization of ultrasound-stimulated microbubble radiation enhancement in a prostate cancer xenograft model.
        Dis Model Mech. 2014; 7: 363
        • Al-Mahrouki AA
        • Wong E
        • Czarnota GJ.
        Ultrasound-stimulated microbubble enhancement of radiation treatments: Endothelial cell function and mechanism.
        Oncoscience. 2015; 2: 944-957
        • Bjånes T
        • Kotopoulis S
        • Murvold ET
        • Kamčeva T
        • Gjertsen BT
        • Gilja OH
        • Schjøtt J
        • Riedel B
        • McCormack E.
        Ultrasound- and microbubble-assisted gemcitabine delivery to pancreatic cancer cells.
        Pharmaceutics. 2020; 12: 141
        • Boateng F
        • Ngwa W.
        Delivery of nanoparticle-based radiosensitizers for radiotherapy applications.
        Int J Mol Sci. 2019; 21: 273
        • Buch K
        • Peters T
        • Nawroth T
        • Sänger M
        • Schmidberger H
        • Langguth P.
        Determination of cell survival after irradiation via clonogenic assay versus multiple MTT assay—A comparative study.
        Radiat Oncol. 2012; 7: 1-6
        • Czarnota GJ
        • Karshafian R
        • Burns PN
        • Wong S
        • Al Mahrouki A
        • Lee JW
        • Caissie A
        • Tran W
        • Kim C
        • Furukawa M
        Tumor radiation response enhancement by acoustical stimulation of the vasculature.
        Proc Natl Acad Sci USA. 2012; 109: E2033-E2041
        • Daecher A
        • Stanczak M
        • Liu JB
        • Zhang J
        • Du S
        • Forsberg F
        • Leeper DB
        • Eisenbrey JR.
        Localized microbubble cavitation-based antivascular therapy for improving HCC treatment response to radiotherapy.
        Cancer Lett. 2017; 411: 100-105
        • Deng H
        • Cai Y
        • Feng Q
        • Wang X
        • Tian W
        • Qiu S
        • Wang Y
        • Li Z
        • Wu J.
        Ultrasound-stimulated microbubbles enhance radiosensitization of nasopharyngeal carcinoma.
        Cell Physiol Biochem. 2018; 48: 1530-1542
        • Dimcevski G
        • Kotopoulis S
        • Bjånes T
        • Hoem D
        • Schjøtt J
        • Gjertsen BT
        • Biermann M
        • Molven A
        • Sorbye H
        • McCormack E.
        A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer.
        J Control Release. 2016; 243: 172-181
        • Dos Santos M
        • Paget V
        • Ben Kacem M
        • Trompier F
        • Benadjaoud MA
        • François A
        • Guipaud O
        • Benderitter M
        • Milliat F
        Importance of dosimetry protocol for cell irradiation on a low X-rays facility and consequences for the biological response.
        Int J Radiat Biol. 2018; 94: 597-606
        • Ebben HP
        • Nederhoed JH
        • Lely RJ
        • Wisselink W
        • Yeung K.
        Microbubbles and ultrasound-accelerated thrombolysis (MUST) for peripheral arterial occlusions: Protocol for a phase II single-arm trial.
        BMJ Open. 2017; 7e014365
        • Eisenbrey JR
        • Shraim R
        • Liu JB
        • Li J
        • Stanczak M
        • Oeffinger B
        • Leeper DB
        • Keith SW
        • Jablonowski LJ
        • Forsberg F.
        Sensitization of hypoxic tumors to radiation therapy using ultrasound-sensitive oxygen microbubbles.
        Int J Radiat Oncol Biol Phys. 2018; 101: 88-96
        • El Kaffas A
        • Czarnota GJ.
        Biomechanical effects of microbubbles: From radiosensitization to cell death.
        Future Oncol. 2015; 11: 1093-1108
        • Fan Z
        • Kumon RE
        • Deng CX.
        Mechanisms of microbubble-facilitated sonoporation for drug and gene delivery.
        Ther Deliv. 2014; 5: 467-486
      1. Fay DS, Gerow K. WormBook: The online review of C. elegans biology [Internet]. Available at: https://www.ncbi.nlm.nih.gov/books/NBK153593/. Accessed March 20, 2020.

      2. Gignac GE. How2statsbook. Available at: https://sites.google.com/site/how2statsbook1/Table%20of%20Contents%20-%202019.pdf?attredirects=0&d=1. Accessed July 8, 2020.

        • Goretzki P
        • Frilling A
        • Simon D
        • Roeher HD.
        Growth regulation of normal thyroids and thyroid tumors in man. In: Hormone-related malignant tumors.
        Springer, Berlin/Heidelberg1990: 48-63
        • Haseltine JM
        • Rimner A
        • Gelblum DY
        • Modh A
        • Rosenzweig KE
        • Jackson A
        • Yorke ED
        • Wu AJ.
        Fatal complications after stereotactic body radiation therapy for central lung tumors abutting the proximal bronchial tree.
        Pract Radiat Oncol. 2016; 6: e27-e33
        • Kachler K
        • Bailer M
        • Heim L
        • Schumacher F
        • Reichel M
        • Holzinger CD
        • Trump S
        • Mittler S
        • Monti J
        • Trufa DI
        • Rieker RJ
        • Hartmann A
        • Sirbu H
        • Kleuser B
        • Kornhuber J
        • Finotto S.
        Enhanced acid sphingomyelinase activity drives immune evasion and tumor growth in non–small cell lung carcinoma.
        Cancer Res. 2017; 77: 5963-5976
        • Karshafian R
        • Giles A
        • Burns PN
        • Czarnota GJ.
        Ultrasound-activated microbubbles as novel enhancers of radiotherapy in leukemia cells in vitro.
        Proc IEEE Int Ultrason Symp. 2009; : 1792-1794
        • Karshafian R
        • Tchouala JIN
        • Al-Mahrouki A
        • Giles A
        • Czarnota GJ.
        Enhancement of radiation therapy by ultrasonically-stimulated microbubbles in vitro: Effects of treatment scheduling on cell viability and production of ceramide.
        Proc IEEE Int Ultrason Symp. 2010; : 2115-2118
        • Kaul S.
        Myocardial contrast echocardiography: A 25-year retrospective.
        Circulation. 2008; 118: 291-308
        • Kinoshita M
        • Hynynen K.
        Key factors that affect sonoporation efficiency in in vitro settings: The importance of standing wave in sonoporation.
        Biochem Biophys Res Commun. 2007; 359: 860-865
      3. Laerd Statistics. Testing for Normality using SPSS Statistics when you have only one independent variable. Available at: https://statistics.laerd.com/spss-tutorials/testing-for-normality-using-spss-statistics.php. Accessed October 9, 2020.

        • Lai P
        • Tarapacki C
        • Tran WT
        • El Kaffas A
        • Lee J
        • Hupple C
        • Iradji S
        • Giles A
        • Al-Mahrouki A
        • Czarnota GJ.
        Breast tumor response to ultrasound mediated excitation of microbubbles and radiation therapy in vivo.
        Oncoscience. 2016; 3: 98-108
        • Lammertink BHA
        • Bos C
        • van der Wurff-Jacobs KM
        • Storm G
        • Moonen CT
        • Deckers R.
        Increase of intracellular cisplatin levels and radiosensitization by ultrasound in combination with microbubbles.
        J Control Release. 2016; 238: 157-165
      4. DEFINITY®. Package insert. Lantheus Medical Imaging, Inc.; 2013.

        • Lentacker I
        • De Cock I
        • Deckers R
        • De Smedt SC
        • Moonen CTW.
        Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms.
        Adv Drug Deliv Rev. 2014; 72: 49-64
        • Liu Y
        • Miyoshi H
        • Nakamura M.
        Encapsulated ultrasound microbubbles: Therapeutic application in drug/gene delivery.
        J Control Release. 2006; 114: 89-99
        • Marathe S
        • Schissel SL
        • Yellin MJ
        • Beatini N
        • Mintzer R
        • Williams KJ
        • Tabas I.
        Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase: Implications for early atherogenesis and ceramide-mediated cell signaling.
        J Biol Chem. 1998; 273: 4081-4088
        • Nofiele JIT
        • Karshafian R
        • Furukawa M
        • Al Mahrouki A
        • Giles A
        • Wong S
        • Czarnota GJ
        Ultrasound-activated microbubble cancer therapy: Ceramide production leading to enhanced radiation effect in vitro.
        Technol Cancer Res Treat. 2013; 12: 53-60
        • Osei E
        • Al-Asady A.
        A review of ultrasound-mediated microbubbles technology for cancer therapy: A vehicle for chemotherapeutic drug delivery.
        J Radiother Pract. 2020; 19: 291-298
        • Piperdi H
        • Portal D
        • Neibart SS
        • Yue NJ
        • Jabbour SK
        • Reyhan M.
        Adaptive radiation therapy in the treatment of lung cancer: An overview of the current state of the field.
        Front Oncol. 2021; 11770382
      5. Promega Corp. CellTiter 96® AQueous One Solution Cell Proliferation Assay. Available at: https://www.promega.com.au/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/celltiter-96-aqueous-one-solution-cell-proliferation-assay-_mts_/?catNum=G3582. Accessed August 30, 2017.

        • Smith DJ
        • Jaggi M
        • Zhang W
        • Galich A
        • Du C
        • Sterrett SP
        • Smith LM
        • Balaji K.
        Metallothioneins and resistance to cisplatin and radiation in prostate cancer.
        Urology. 2006; 67: 1341-1347
        • Takahashi T
        • Nau MM
        • Chiba I
        • Birrer MJ
        • Rosenberg RK
        • Vinocour M
        • Levitt M
        • Pass H
        • Gazdar AF
        • Minna JD.
        p53: A frequent target for genetic abnormalities in lung cancer.
        Science. 1989; 246: 491-494
        • Wang Y
        • Li Y
        • Yan K
        • Shen L
        • Yang W
        • Gong J
        • Ding K.
        Clinical study of ultrasound and microbubbles for enhancing chemotherapeutic sensitivity of malignant tumors in digestive system.
        Chin J Cancer Res. 2018; 30: 553-563
      6. World Health Organization. Cancer. Available at: https://www.who.int/news-room/fact-sheets/detail/cancer. Accessed February 26, 2022.

        • Wu AJ
        • Williams E
        • Modh A
        • Foster A
        • Yorke E
        • Rimner A
        • Jackson A.
        Dosimetric predictors of esophageal toxicity after stereotactic body radiotherapy for central lung tumors.
        Radiother Oncol. 2014; 112: 267-271
        • Youkhana EQ
        • Feltis B
        • Blencowe A
        • Geso M.
        Titanium dioxide nanoparticles as radiosensitisers: An in vitro and phantom-based study.
        Int J Med Sci. 2017; 14: 602
        • Zhu Q
        • Dong G
        • Wang Z
        • Sun L
        • Gao S
        • Liu Z.
        Intra-clot microbubble-enhanced ultrasound accelerates catheter-directed thrombolysis for deep vein thrombosis: A clinical study.
        Ultrasound Med Biol. 2019; 45: 2427-2433