The authors would like to thank the staff of the Syracuse University Department of Physics workshop for their innovative assistance in matters relating to our system design.

1. Cleaning Cell Sonifier System
<ol>
	<li>Clean the union between the horn and converter using chloroform and a swab of cotton. Apply a light oil or mineral oil to the threads of the horn and converter to further remove buildup of metal shavings, rust, or oxidation.</li>
	<li>Use chloroform to remove the oil and any other contaminants from the union after wiping the threads with oil.</li>
</ol>
2. Reassembling the Cell Sonifier and Setting up the System&#160;
<ol>
	<li>Reattach the horn to the converter. To properly torque the system, remove the assembled converter and horn from the cup assembly by pulling the horn out of the bottom of the cup (Figure 2A). Place a slot wrench inside one of the three small holes near the top of the converter (Figure 2B). Then, position a torque-wrench fitted with a &#189; inch crow&#8217;s foot on the slotted part of the horn (Figure 2B). Tighten in standard clockwise direction until the torque meter reads 54.23 N.m (Figure 2C), the torque recommended by the manufacturer16. Fit the tightened converter and horn back into the cup, and mount the horn and converter to a ring stand in an upright position. Reattach the converter to the power supply before setting the system for experimental pulse dosing.</li>
	<li>Set the system for pulse dosing using 1 sec of sonication with 1 sec between pulses. Different dosing models may be used for other experimental conditions. Many variables, such as water volume and horn diameter would potentially require changes to this dosing. Using the aforementioned cell sample types, volume, and concentration, empirical data and observation have led to the formulation of this dosing regimen.</li>
	<li>Adjust the system to the desired amplitude. In this protocol, use 33% and 50% amplitude to prevent complete cell destruction, which would render the efficacy of sonosensitizers difficult or impossible to gauge. 
	NOTE: In this study the amplitudes are compared.</li>
</ol>
3. Assessing System Intensity and Function&#160;
<ol>
	<li>Hold the cavitation meter at 1.5 cm, which is the level of the sample sonication. Note the readings to assess that the system is running at an expected intensity. Using the current system and water levels, expect between 100-110 W/cm2 for 33% amplitude and 115-125 W/cm2 for 50% amplitude. Note the cavitation meter readings from the meter display which represents cavitation energy in W/cm2 directly. Take these readings at the surface of the water, and ensure no bubbles are present at the face of the meters probe. 
	NOTE: It is important to note that these procedures are done without the cap for access above the horn. They need not be repeated after every sonication. Rather, they should be performed only after cleaning, reassembling, and setting up the system.</li>
	<li>Place the hydrophone in a sample vial containing 12.5 ml of water in order to fully immerse the hydrophone sensor. Suspend the hydrophone using the ring stand.</li>
	<li>Attach the hydrophone to the input of the oscilloscope in order to assess waveform characteristics. Examine the oscilloscope for a clear sine wave, as aberrant waves can alter the frequency of the system from the specifications of the manufacturer. Both the cavitation meter and oscilloscope give a frequency reading, but only the oscilloscope will show aberrant waves that are indicative of a faulty system or improper assembly.</li>
</ol>
4. Preparation of Cells and Medium
<ol>
	<li>Prepare the medium by using 240 ml Iscove&#8217;s Modified Dubecco&#8217;s Medium (IMDM) and 50 ml fetal bovine serum. Do not thaw fetal bovine serum in a hot water bath, as rapid increases in temperature can lead to a compromise in serum stability. Combine 240 &#181;l gentamicin 50 mg/ml and 5 ml penicillin/ streptomycin 100x in a standard culture flask, and add to the medium. Store the medium at 4 &#176;C.</li>
	<li>Seed U937 cells at ~4 x 104 cells/ml in a 25 cm2 flask using the prepared medium. Assess cells for concentration and viability using a TC20 cell counter and trypan blue exclusion by placing 15-20 &#181;l of cells and 15-20 &#181;l trypan blue into a micro-centrifuge tube and mixing the contents. Pipette15-20 &#181;l of this mixture into a TC20 sampling slide, and initiate counts by placing the slide into the TC20 cell counter.</li>
	<li>Incubate cells at 37 &#176;C and 5% CO2, and check for cell viability using trypan blue exclusion and the TC20 cell counter. When the cells are at an appropriate concentration and/or have been treated with sonosensitizers for an appropriate time frame, transfer 3 ml of cells from the culture flask to a 20 ml glass scintillation vial. In this protocol, grow U937 cells for 48 hr, and then treat&#160;with 0.5, 1, or 5 mM Me&#946;CD for 30 min to sufficiently deplete the cholesterol of cells prior to sonication.</li>
</ol>
5. Cell Sonication
<ol>
	<li>Degas deionized, distilled water using a vacuum and Buchner flask. Transfer the water to the cup horn, and fill to a level of 15 mm above the top of the horn. The sonication process causes further degassing of the water for a period of a few minutes, and can lead to inconsistencies over the course of the experiment if the system is not run prior to sample sonication. Therefore, run the system for ~7 min prior to sample sonication.</li>
	<li>Attach the scintillation vial containing the cells to the holding device. Then, attach the holding device to the top of the cup horn and set to an elevation of 15 mm from the top of the horn (at the water surface using the sliding mechanism).</li>
	<li>The cells are typically sonicated using three 1 sec pulses of ultrasound, with 1 sec spacing in between each of these pulses. For this protocol, sonicate cells at 33% and 50% amplitude using the abovementioned pulse dosing.</li>
</ol>
6. Assessing Cells for Damage Post-sonication
<ol>
	<li>Assess the suspended cells for damage and viability using trypan blue and the TC20 cell counter. In addition, analyze the sample using a Z2 counter. For Z2 counter analysis, pipette a 100 &#181;l cell suspension into 20 ml isotonic saline (200:1 ratio). Prior to analysis, flush the aperture of the Z2 particle analyzer at least twice using isotonic saline. Place the Z2 sample in the counter holder, and raise the platform to the aperture before initiating counts.</li>
	<li>Acquire the data from the TC20 and Z2 counters using software provided by the manufacturer. Analyze these data for cell damage, viability, and the creation of cellular debris from sonication.</li>
</ol>
7. XTT Assay to Determine Cell Viability and Mitochondrial Activity of Treated U937 and THP1 Cells
<ol>
	<li>Prior to the XTT assays, grow U937 and THP1 cells under the same conditions. Pretreat cells with the same concentration range of Me&#946;CD for 30 min prior to sonication. Use the same ultrasound parameters as before except that cells are now sonicated using a range of 1-3 one sec pulses spaced one sec apart to develop a range of damage for the XTT kit to assess. 
	Note: Many of these steps are taken directly from the XTT kit manual and are only repeated for the convenience of interested laboratories.</li>
	<li>Collect cells by centrifugation at 200 x g for 10 min. Resuspend cell pellet in the growth medium previously described. Resuspend cells at ~1 x 105 cells/ml. Seed U937 cells at 100 &#181;l per well into a flat-bottom 96-well microtiter plate in triplicate, and then repeat for THP1 cells using a separate 96-well plate. Include 3 control wells containing 100 &#181;l of complete growth medium alone as blank absorbance readings. Then, incubate the inoculated plates for 24 hr.</li>
	<li>Defrost two aliquots of the XTT reagent and the activation reagent at 37 &#176;C prior to use. Swirl aliquots gently until clear solutions are obtained. Add 0.1 ml of the activation reagent to 5.0 ml of the XTT reagent, which forms enough activated XTT solution for one 96-well microtiter plate assay. Repeat the process for the other plate.</li>
	<li>Add 50 &#181;l of the activated-XTT solution to each well. Return the plate to the cell culture CO2 incubator for 2 hr. Shake the plate gently following the incubation period to evenly distribute the orange color in the positive wells. Measure the absorbance of the assay wells containing the cells and the blank background control wells at a wavelength between 450-500 nm wavelength using a microtiter plate reader.</li>
	<li>Either zero the microtiter plate reader using the blank control wells or subtract their average value from the specific results. Measure the absorbance of all the assay wells again at a wavelength between 630-690 nm and subtract the values from the 450-500 nm values obtained. Note: This second absorbance determination helps eliminate non-specific readings from the assay results. The XTT assay was repeated four times for both cell lines.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+shock+wave+lithotripsy:+a+comprehensive+review.">Optimizing shock wave lithotripsy: a comprehensive review.</a> Rev Urol. 15 (2), 49-60 (2013).</li><li>Mitragotri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healing+sound:+the+use+of+ultrasound+in+drug+delivery+and+other+therapeutic+applications.">Healing sound: the use of ultrasound in drug delivery and other therapeutic applications.</a> Nat Rev Drug Discov. 4 (3), 255-260 (2005).</li><li>Cordeiro, E. R., Cathelineau, X., Th&#252;roff, S., Marberger, M., Crouzet, S., de la Rosette, J. 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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+focused+ultrasound+treatment+for+patients+with+unresectable+pancreatic+cancer.">High-intensity focused ultrasound treatment for patients with unresectable pancreatic cancer.</a> Hepatobiliary Pancreat Dis Int. 11 (6), 655-660 (2012).</li><li>Xiaoping, L., Leizhen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+of+high+intensity+focused+ultrasound+(HIFU)+for+pancreatic+cancer.">Advances of high intensity focused ultrasound (HIFU) for pancreatic cancer.</a> Int J Hyperthermia. 29 (7), 678-682 (2013).</li><li>Lukka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-intensity+focused+ultrasound+for+prostate+cancer:+a+systematic+review.">High-intensity focused ultrasound for prostate cancer: a systematic review.</a> Clin Oncol (R Coll Radiol). 23 (2), 117-127 (2011).</li><li>So, A. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-site+sonoporation+disrupts+actin+cytoskeleton+organization.">Single-site sonoporation disrupts actin cytoskeleton organization.</a> J R Soc Interface. 11 (95), 20140071 (2014).</li><li>Trendowski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+enlargement+of+leukemia+cells+using+cytoskeletal-directed+agents+and+cell+cycle+growth+control+parameters+to+induce+sensitivity+to+low+frequency+ultrasound.">Preferential enlargement of leukemia cells using cytoskeletal-directed agents and cell cycle growth control parameters to induce sensitivity to low frequency ultrasound.</a> Cancer Lett. In press, (2015).</li><li>Dumontet, C., Sikic, B. 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The authors declare that they have no competing financial interests.

To attain optimal results, special care should be taken to carefully position the sample and clean the converter-horn union. The placement of the sample in the horn is important for obtaining consistent cell destruction, as changing the distance from the horn will alter the acoustic foci, and therefore alter the energy the sample is exposed to. The acoustic energy within the cup horn can be mapped using the cavitation meter to find the position of maximum cavitation. In addition, the cavitation meter, along with the oscilloscope are vital for determining the sound intensity the cells are being exposed to, as well as the homogeneity of the waveform. Therefore, these instruments should be used to detect problems with the system, and help determine what troubleshooting may be needed to correct system instability.
As previously mentioned, the low frequency system may act to further degas the water throughout the experiment if not run for several minutes prior to sample sonication. This initial run must be performed to yield a relatively degassed sonication medium and thus consistent results during experiments. In addition, cells should not be sonicated at or near the maximum amplitude when assessing the efficacy of sonosensitizers, as the true extent of sensitization would be difficult to assess. Using 33% amplitude on the 40 kHz system is an ideal setting, as it produces notable damage, but provides sonosensitizers ample room to demonstrate their efficacy, as demonstrated with Me&#946;CD against U937 and THP1 cells (Figure 7). These data also confirm that Me&#946;CD sensitizes multiple leukemia lines to low frequency ultrasound in a dose dependent manner.
There have been a number of experiments done with higher frequency in the range of 0.75 MHz to 8 MHz showing evidence of intramembrane cavitation bubbles being generated through sonication17-19. However, questions still remain in regards to the exact mechanism of ultrasound-induced cell lysis18. We have shown a link between fluidizing the cytoskeleton and increased sonic sensitivity using low frequency ultrasound15, a phenomenon demonstrated by other laboratories20, 21. In addition, we have found that microfilament-disrupting agents such as cytochalasin B potentiate ultrasonic sensitivity in multiple leukemia lines, but not hHSCs or leukocytes22, suggesting that inhibition of actin polymerization may be a sonosensitizing mechanism of particular interest. We have also observed that vincristine, a microtubule-disrupting agent that inhibits tubulin polymerization23, 24, markedly increases the ultrasonic sensitivity of different leukemia types in vitro including acute myeloid leukemia, chronic myeloid leukemia, and acute lymphoid leukemia. By contrast, cytoskeletal-directed agents that stabilize cytoskeletal components (paclitaxel and jasplakinolide) appear to make cells resistant to sonication, reflected by lower rates of cell lysis22. Taken together, these data support the hypothesis that fluidizing the cytoskeletal components of neoplastic cells is indeed an important factor in increasing the efficacy of SDT25. The present study also demonstrates that cholesterol depletion may be another method by which to further potentiate the ultrasonic sensitivity of neoplastic cells, as Me&#946;CD-treated U937 cells are markedly sensitized to 40 kHz ultrasound.
While our sonication protocols have demonstrated marked antineoplastic activity in vitro, the current methodology is limited to work in culture and small vertebrate models that are able to fit in the vials used for sonication. We have shown that zebrafish can be safely sonicated using pulsed low frequency ultrasound (20 kHz), and that their tolerance to chemotherapeutic agents is quantitatively comparable to doses tolerated by murine models26, suggesting that tumor-bearing zebrafish may be used in preliminary investigations to assess the in vivo antineoplastic activity of these protocols. Nevertheless, administering chemotherapeutic agents prior to sonication of mammalian models has been reported in the MHz range1, and such protocols can likely be extended to incorporate low frequency ultrasound, as well as cholesterol-depleting and cytoskeletal-directed agents.
Potential clinical applications of this form of SDT may involve extracorporeal blood sonication in which antineoplastic agents are administered intravenously (i.v.) prior to the blood being removed for sonication25. This method removes potential sound barriers posed by human anatomy, and may be an effective way to damage leukemic blasts, as well as metastases from solid tumors. It is also possible that cholesterol-depleting and cytoskeletal-directed agents could be used in HIFU protocols that are already being examined in the clinic in an attempt to improve the efficacy of this treatment modality.
The methods described in the present study are capable of assessing the value of potential sonosensitizers, and further system refinement may enhance this utility. However, there are many variables to be considered when using such ultrasonic devises, including power supply quality, acoustic foci, and individual variation among converters. Therefore, future research will focus on visualizing the sonic waves and understanding their influence on results. SDT has shown to enhance cell lysis in vitro and may prove to be clinically viable if more in vivo data in mammalian models are acquired. Experiments examining other potentially exploitable characteristics of malignant cells, as well as various combined modalities involving multiple agents and ultrasound continue in our laboratory.

Ultrasound refers to any oscillating sound pressure wave with a frequency greater than the upper limit of human auditory capacities (~ 20 kHz)1. Low frequency ultrasound in the 20-60 kHz range has been utilized in the laboratory as a means of generating emulsions, preparing cellular samples for nucleic acid extraction, for tissue disruption, and for a variety of other tests. The utility of low frequency ultrasound has also been extended to the industrial setting for welding, cleaning various materials, and in materials processing. Commercially available ultrasound generators come in frequencies ranging from 18-60 kHz, and full-scale wattages from 100-1,200 W.
Although ultrasound has long been used in the clinical setting for diagnostic imaging, it has been applied as a therapeutic modality only recently. Ultrasound &#8805;1 MHz is capable of safely disrupting urinary calculi (kidney stones) and biliary calculi (stones in the gallbladder or in the liver) in patients to reduce symptoms2,3. This approach known as extracorporeal shockwave lithotripsy (ESWL) is now widely applied in the clinic (more than one million patients are treated annually with ESWL in the United States alone4), and offers a modality by which to non-invasively break up calculi with minimal collateral damage through the use of externally applied, focused, high intensity acoustic pulses2-4.
Due to the unique direct shearing forces, as well as cavitation bubbles generated by high intensity ultrasound, these methodologies have been examined in cancer therapy for the treatment of castration-resistant prostate carcinoma and pancreatic adenocarcinoma in an approach known as high intensity focused ultrasound (HIFU)5-8. In a manner very similar to ESWL, HIFU uses multiple ultrasound beams and focuses them on a selected focal area to generate temperatures of 60 &#176;C or higher through the use of acoustic energy, inducing coagulative necrosis in the targeted tissue5. Although other modalities of thermal ablation currently exist (radiofrequency ablation and microwave ablation), HIFU offers a distinct advantage over these methods in that it is the only non-invasive hyperthermic modality5. HIFU has attained mixed results in the clinic and is currently only available in clinical trials8-11. Nevertheless, the limited success it has achieved, and the very promising in vivo data acquired from preclinical mammalian models have demonstrated the potential of ultrasound in cancer therapy.
In an effort to improve HIFU, researchers have attempted to combine ultrasound with appropriate antineoplastic agents to generate a form of sonochemotherapy. Sonodynamic therapy (SDT) is a promising novel treatment modality that has demonstrated impressive antineoplastic activity in both in vitro and in vivo studies1. It has been shown that ultrasound preferentially damages malignant cells based on the size differential between such cells and those of normal histology1,5. SDT incorporates specialized agents known as sonosensitizers to increase the extent of preferential damage exerted by ultrasound against neoplastic cells. While therapeutic applications of SDT have been previously examined, ultrasonic systems used typically employ higher frequency ultrasound (&#8805;1 MHz), and the effects of low kHz frequency ultrasound has yet to be fully explored. Lower frequencies of ultrasound are often more proficient at producing inertial cavitation, a phenomenon that results in the destruction of cells due to the rapid collapsing of microbubbles, inducing physicochemical damage12-14. This difference in the generation of inertial cavitation between MHz and low kHz ultrasound has been attributed to the fact that lower wave frequencies enable microbubbles more time to grow by rectified diffusion in the expansion half cycle, consequently producing more violent collapses during the following compression half cycle12.
We have previously shown that U937 human monocytic leukemia cells are sensitive to low frequency ultrasound (23.5 kHz), and that this sensitivity can be markedly increased through the application of antineoplastic agents that perturb the cytoskeleton15. Further, we have demonstrated that cells are preferentially damaged based on size, with larger cells exhibiting higher ultrasonic sensitivity. In addition, normal human hematopoietic stem cells (hHSCs) and leukocytes at comparable cell sizes are much more resistant to sonication than their neoplastic counterparts15, tentatively suggesting that low frequency ultrasound may be used to preferentially damage malignant cells in the presence of normal tissue.
To further examine the unique properties of low frequency ultrasound for potential therapeutic use, we have developed cleaning and stabilization procedures to increase the efficacy and reliability of one of our current sonication systems, the Branson Model SLPe 150 W, 40 kHz Cell Disrupter, equipped with a 20 mm horn fitted into a 7.62 cm cup. In addition, we have been able to determine accurate sample cavitation energies, as well as consistent waveforms and amplitude within the 40 kHz range using a cavitation meter and oscilloscope with hydrophone. By refining and systematizing our protocols, we have been able to establish consistency in our experimental sonications, allowing us to quantitatively compare the sonic sensitivities of neoplastic and normal cells of different histogenetic lineages. Our protocol for the 40 kHz system is presented in extensive detail in order for interested laboratories to be capable of performing comparable experiments, and to evaluate our findings of the antineoplastic effects elicited by low frequency ultrasound. In addition, we examine the dose dependent effects of methyl-&#946;-cyclodextrin (Me&#946;CD; Figure 1), a cholesterol-depleting agent, on increasing the ultrasonic sensitivity of U937 and THP1 human monocytic leukemia cells.

<table><tbody><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium w/ NaHCO3 &amp;amp; 25mM Hepes&amp;nbsp;</td><td>Life Technologies</td><td>12440079</td><td></td></tr><tr><td>Amphotericin B Solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2942&amp;nbsp;</td><td></td></tr><tr><td>Penicillin/Streptomycin 100x Solution&amp;nbsp;</td><td>Life Technologies</td><td>10378-016</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>12105</td><td></td></tr><tr><td>Branson SLPe 40kHz Cell Disruptor with 3&quot; (25mm) Cuphorn</td><td>Branson Ultrasonics</td><td>101-063-726</td><td>sonication device</td></tr><tr><td>Brisk Heat SDC Benchtop Digital temperature Controler w/ 1000 ml Beaker Heater</td><td>Brisk Heat</td><td>SDCJF1A-GBH1000-1</td><td>heater used for temperature control</td></tr><tr><td>Beckman-Coulter Z2 Cell Sizer with AccuComp&amp;reg; Software</td><td>Beckman-Coulter</td><td>6605700</td><td></td></tr><tr><td>Bio-Rad TC20 Automated Cell Counter</td><td>Bio-Rad</td><td>145-0102</td><td></td></tr><tr><td>Gentamicin 50 mg/ml</td><td>Sigma-Aldrich</td><td>G1397&amp;nbsp;</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>Falcon 50 ml &amp;amp; 25 ml Vented Culture Flasks</td><td>Fisher Scientific</td><td>353082</td><td></td></tr><tr><td>Lonza L-Glutamine 200 mM 0.85% NaCl</td><td>Lonza</td><td>17-605C&amp;nbsp;</td><td></td></tr><tr><td>Seal-Rite 1.5 ml Microcentrifuge Tubes</td><td>USA Scientific</td><td>1615-5510</td><td></td></tr><tr><td>Beckman-Coulter Accuvette ST 25 ml Vials and caps</td><td>Beckman-Coulter</td><td>&amp;nbsp;A35473</td><td></td></tr><tr><td>AccuJet Pro Auto Pipet&amp;nbsp;</td><td>BrandTech Scientific</td><td>26330</td><td></td></tr><tr><td>USA Scientific 10 ml Disposable Serological Pipets</td><td>USA Scientific</td><td>1071-0810</td><td></td></tr><tr><td>Tip One 100 &amp;#956;l and 1,000 &amp;#956;l Filter Tips</td><td>USA Scientific</td><td>1120-1840, 1126-7810</td><td></td></tr><tr><td>100 &amp;#956;l Micropipette&amp;nbsp;</td><td>Wheaton</td><td>851164</td><td></td></tr><tr><td>1,000 &amp;#956;l Micropipette</td><td>Wheaton</td><td>851168</td><td></td></tr><tr><td>BioRad Dual Chamber Counting Slides</td><td>Bio-Rad</td><td>145-0015</td><td></td></tr><tr><td>Forma Scientific Dual chamber water jacketed Incubator</td><td>Forma Scientific</td><td>3131</td><td></td></tr><tr><td>Tektronix&amp;nbsp; DPO 2002B Digital Phosphor Oscilloscope</td><td>Tektronix</td><td>DPO2002B</td><td>used to measure the ultrasonic waveform</td></tr><tr><td>PPB MegaSonics Model PB-500 Ultrasonic Energy Meter</td><td>PPB Megasonics</td><td>PB-500&amp;nbsp;</td><td>used to assess the sound intensity in W/cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Teledyne RESON TC4013-1 Hydrophone</td><td>Teledyne</td><td>TC4013-1&amp;nbsp;</td><td>connects to the oscilloscope&amp;nbsp;</td></tr><tr><td>Wheaton 250 ml Flasks</td><td>Sigma-Aldrich</td><td>Z364827</td><td></td></tr><tr><td>20 ml Glass Scintillation Vials&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Z190527</td><td></td></tr><tr><td>Beckman-Coulter Isotonic Saline Solution&amp;nbsp;</td><td>Beckman-Coulter</td><td>N/A</td><td>diluent for Z2 counter</td></tr><tr><td>Chloroform 99%</td><td>Sigma-Aldrich</td><td>C2432</td><td></td></tr><tr><td>Ethanol 200 Proof Anhydrous&amp;nbsp;</td><td>Sigma-Aldrich</td><td>459836</td><td></td></tr><tr><td>Mineral Oil</td><td>N/A</td><td></td><td></td></tr><tr><td>XTT Cell Proliferation Assay Kit</td><td>ATCC</td><td>30-1011K</td><td></td></tr><tr><td>96-Well Microplate Reader</td><td>Cole-Palmer</td><td>&amp;nbsp;EW-13055-54</td><td></td></tr><tr><td>U937 Human Monocytic Leukemia Cells</td><td>ATCC</td><td>CRL&amp;shy;1593.2</td><td></td></tr><tr><td>THP1 Human Monocytic Leukemia Cells</td><td>ATCC</td><td>TIB-202</td><td></td></tr></tbody></table>

generation and quantitative analysis of pulsed low frequency ultrasound to determine the sonic sensitivity of untreated and treated neoplastic cells

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In addition, this method can be used in combination with specialized agents known as sonosensitizers to increase the extent of preferential damage exerted by ultrasound against neoplastic cells, an approach referred to as sonodynamic therapy (SDT). The methodology for generating and applying low frequency ultrasound in a preclinical in vitro setting is presented to demonstrate that reproducible cell destruction can be attained in order to examine and compare the effects of sonication on neoplastic and normal cells. This offers a means by which to reliably sonicate neoplastic cells at a level of consistency required for preclinical therapeutic assessment. In addition, the effects of cholesterol-depleting and cytoskeletal-directed agents on potentiating ultrasonic sensitivity in neoplastic cells are discussed in order to elaborate on mechanisms of action conducive to sonochemotherapeutic approaches.

System stability is paramount in obtaining repeatable and reliable results. The proper torque specification of 54.23 N.m achieved proper coupling of the transducer and sonotrode2, with the consistency of the waveform being assessed through the use of an oscilloscope and hydrophone (Figure 3A). Figure 3B depicts measurements inside the sample vial, and Figure 3C assesses stability within the cup horn. As expected, the amplitude was slightly reduced due to some energy being absorbed by the glass. However, the waveform was not perturbed or distorted in either panel B or C, which is necessary for reliable delivery of sonic energy to the sample. A waveform that does not appear as a clear singular sine wave indicates a faulty transducer or improper setup. This is expressed in Figure 3D which shows multiple aberrant waves, a clear absence of a sine wave, and a frequency reading not expected with a properly functioning system. The cavitation meter used to measure the cavitation energy generated (Figure 4) found the sound intensity to be 100-110 W/cm2 at 33% amplitude, and 125-130 W/cm2 at 50% amplitude.
Once the 40 kHz system was appropriately calibrated and assembled in its entirety (Figure 5), reliable cell destruction was readily attained, as determined by both the TC20 and Z2 counters (Figure 6). As expected, more extensive cell damage was observed in cell populations sonicated at 50%, rather than at 33% amplitude. Nevertheless, 33% amplitude is useful as a starting point to detect potential sonic sensitizing effects of administered agents, as it produces noticeable damage, but leaves room for more damage to be detected when agents are applied to assess their potentiation of ultrasonic sensitivity. This is demonstrated in Figure 7A, where the dose dependent effects of Me&#946;CD on potentiating the ultrasonic sensitivity of U937 cells are presented. Although non-sonicated Me&#946;CD-treated cells were very similar in viability to non-sonicated, untreated cells, the viability markedly dropped after three pulses of 1 sec 40 kHz ultrasound was applied. In addition, the decrease in viability after sonication appears to have been dose dependent, as 5 mM Me&#946;CD produced the largest drop in cell count, followed by 1 mM, and then 0.5 mM Me&#946;CD.
Similar effects on cell viability and mitochondrial activity were observed in both U937 and THP1 cells that were treated with varying concentrations of Me&#946;CD prior to sonication, as assessed with the XTT kit (Figure 7B). Although THP1 cells appear to be slightly more sonic resistant than U937 cells, both human leukemia lines are damaged in a dose dependent manner. Higher concentrations of Me&#946;CD and more pulses of ultrasound produced the lowest reduction of XTT to a soluble, brightly colored orange derivative for both U937 and THP1 cells, corresponding to lower cell viability and mitochondrial activity.
<img alt="Figure 1" src="/files/ftp_upload/53060/53060fig1.jpg" /> 
Figure 1.&#160;Molecular structure of methyl-&#946;-cyclodextrin. (A) Complete structure of methyl-&#946;-cyclodextrin (Me&#946;CD). (B) Me&#946;CD is a polymer of seven repeating units with a R group of either H or CH3. Molecular Formula: C56H98O35. Molecular Weight = 1331.36.
<img alt="Figure 2" src="/files/ftp_upload/53060/53060fig2.jpg" /> 
Figure 2.&#160;Properly torqueing the 40 kHz ultrasound system. (A) The horn and converter are removed from the cup prior to torqueing. (B) Position the slot wrench and torque wrench at their respective positions. (C) Apply 54.23 N.m of torque in a clockwise direction before reattaching the horn and converter to the cup.
<img alt="Figure 3" src="/files/ftp_upload/53060/53060fig3.jpg" /> 
Figure 3.&#160;Use of an oscilloscope to characterize the waveform of the ultrasonic system. (A) An oscilloscope with hydrophone can be used to evaluate amplitude and waveform consistency. (B) A recording taken with the cup directly above the horn at 1.5 cm and set at 33% amplitude. (C) The waveform pictured here is from a reading acquired when the hydrophone is placed within the 20 ml glass scintillation vial. The vial is positioned at 1.5 cm above the horn and again set at 33% amplitude. (D) An oscilloscope screenshot of a system with aberrant frequencies as would be expected with a faulty system or improper coupling of the horn converter union. <a href="https://www.jove.com/files/ftp_upload/53060/53060fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53060/53060fig4.jpg" /> 
Figure 4. Use of a cavitation meter to characterize the sound intensity of the ultrasonic system. The cavitation meter seen here is used to represent cavitation energy in W/cm2. This is a very important instrument for assessing consistency of energy with respect to the sample.
<img alt="Figure 5" src="/files/ftp_upload/53060/53060fig5.jpg" /> 
Figure 5. Key components of the 40 kHz ultrasound system. The ultrasonic system is shown complete with cup horn (A), heating apparatus (B), the converter (C), and sample positioning mechanism (D). This design allows for easy positioning of the sample (E) above the horn (F).
<img alt="Figure 6" src="/files/ftp_upload/53060/53060fig6.jpg" /> 
Figure 6. Effects of different levels of amplitude on the ultrasonic lysis of U937 cells using the TC20 and Z2 counters. Cells were sonicated with the 40 kHz cell dismembrator at 33% amplitude (100-110 W/cm2) or 50% amplitude (125-130 W/cm2) using three 1 sec pulses of ultrasound, with 1 sec spacing in between each of these pulses. &#160;(A) A screenshot from the Z2 counter software demonstrates the effect of sonication on U937 cells.&#160;(B) Data from the TC20 counter were compiled in a graph to demonstrate the reproducibility of the results. These data include the total and live cell counts, as well as the trypan blue assessed cell viability. Bars reflect the standard error of the mean (SEM) for 4 independent cell populations. <a href="https://www.jove.com/files/ftp_upload/53060/53060fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53060/53060fig7.jpg" /> 
Figure 7. Effects of methyl-&#946;-cyclodextrin on potentiating the ultrasonic sensitivity of U937 and THP1 cells. (A) U937 cells were treated with varying concentration of Me&#946;CD for 30 min prior to being sonicated with 40 kHz ultrasound (105 W/cm2) using three 1 sec pulses spaced 1 sec apart. Cell populations were grouped into &#8805;12 &#181;m and &#8805;17 &#181;m. (B) U937 or THP1 cells were again treated with varying concentrations of Me&#946;CD for 30 min prior to being sonicated with the 40 kHz system using 1-3 pulses of 1 sec ultrasound spaced 1 sec apart. Cell viability and mitochondrial activity were assessed with the XTT kit. 100 &#181;l of cells were seeded per well into a flat-bottom 96-well microtiter plate. The plate was incubated for 24 hr prior to the addition of XTT solution. Cells were then incubated for an additional 2 hr before the wavelength was read. Bars reflect SEM for 4 independent cell populations. <a href="https://www.jove.com/files/ftp_upload/53060/53060fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells at JoVE.c

Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells

Selective damage of human leukemia cells can be achieved through a novel approach of applying low frequency ultrasound both with and without chemotherapeutic pretreatment of leukemic and normal hematopoietic cells.

Low frequency ultrasound in the 20 to 60 kHz range is a novel physical modality by which to induce selective cell lysis and death in neoplastic cells. In ...

generation-quantitative-analysis-pulsed-low-frequency-ultrasound-to

Research

JoVE Journal

Medicine

11.1K Views.  Syracuse University. Selective damage of human leukemia cells can be achieved through a novel approach of applying low frequency ultrasound both with and without chemotherapeutic pretreatment of leukemic and normal hematopoietic cells. 

Video: Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Bioengineering

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Cancer Research

Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) for the controllable nucleation of cavitation in tissue-mimicking gel phantoms. This in vitro protocol was developed to demonstrate the feasibility of this approach, for both enhancement of imaging and therapeutic applications for cancer. The same apparatus can be used for both imaging and therapeutic applications by varying the exposure duration of the HIFU system. For short duration exposures (10 &#181;s), broadband acoustic emissions were generated through the controlled nucleation of inertial cavitation around the gold nanoparticles. These emissions provide direct localization of nanoparticles. For future applications, these particles may be functionalized with molecular-targeting antibodies (e.g. anti-HER2 for breast cancer) and can provide precise localization of cancerous regions, complementing routine diagnostic ultrasound imaging. For continuous wave (CW) exposures, the cavitation activity was used to increase the localized heating from the HIFU exposures resulting in larger thermal damage in the gel phantoms. The acoustic emissions generated from inertial cavitation activity during these CW exposures was monitored using a passive cavitation detection (PCD) system to provide feedback of cavitation activity. Increased localized heating was only achieved through the unique combination of nanoparticles, laser light and HIFU. Further validation of this technique in pre-clinical models of cancer is necessary.

This protocol demonstrates the controllable nucleation of cavitation in gel phantoms, through simultaneous exposure to both near-infrared pulsed laser light and high intensity focused ultrasound (HIFU). The cavitation activity can then be used for enhancing imaging and/or therapeutic uses of HIFU.

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) ...

Engineering

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Developmental Biology

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Generation and Quantitative Analysis of Pulsed Low Frequency Ultrasound to Determine the Sonic Sensitivity of Untreated and Treated Neoplastic Cells

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Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents