We would like to acknowledge our sources of funding for this project and the personnel involved including: C17 Research Grant, Canada Graduate Scholarship, Ontario Student Opportunity Trust Fund, and James J. Hammond Fund.

<ol>
	<li>Skapek, S. X., 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=Rhabdomyosarcoma.">Rhabdomyosarcoma.</a> Nature Reviews Disease Primers. 5 (1), (2019).</li><li>Ferrari, A., 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=Impact+of+rhabdomyosarcoma+treatment+modalities+by+age+in+a+population-based+setting.">Impact of rhabdomyosarcoma treatment modalities by age in a population-based setting.</a> Journal of Adolescent and Young Adult Oncology. 10 (3), 309-315 (2021).</li><li>. Pediatric rhabdomyosarcoma surgery: Background, anatomy, pathophysiology Available from: <a target="_blank" href="https://emedicine.medscape.com/article/939156-overview#a2">https://emedicine.medscape.com/article/939156-overview#a2</a> (2019)</li><li>Ognjanovic, S., Linabery, A. M., Charbonneau, B., Ross, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+childhood+rhabdomyosarcoma+incidence+and+survival+in+the+United+States,+1975-2005.">Trends in childhood rhabdomyosarcoma incidence and survival in the United States, 1975-2005.</a> Cancer. 115 (18), 4218-4226 (2009).</li><li>Mulrooney, D. A., 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=Cardiac+outcomes+in+a+cohort+of+adult+survivors+of+childhood+and+adolescent+cancer:+retrospective+analysis+of+the+Childhood+Cancer+Survivor+Study+cohort.">Cardiac outcomes in a cohort of adult survivors of childhood and adolescent cancer: retrospective analysis of the Childhood Cancer Survivor Study cohort.</a> BMJ. 339, (2009).</li><li>Lipshultz, S. E., Cochran, T. R., Franco, V. I., Miller, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment-related+cardiotoxicity+in+survivors+of+childhood+cancer.">Treatment-related cardiotoxicity in survivors of childhood cancer.</a> Nature Reviews Clinical Oncology. 10 (12), 697-710 (2013).</li><li>Winter, S., Fasola, S., Brisse, H., Mosseri, V., Orbach, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapse+after+localized+rhabdomyosarcoma:+Evaluation+of+the+efficacy+of+second-line+chemotherapy.">Relapse after localized rhabdomyosarcoma: Evaluation of the efficacy of second-line chemotherapy.</a> Pediatric Blood &amp; Cancer. 62 (11), 1935-1941 (2015).</li><li>Wood, B. J., 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=Phase+I+study+of+heat-deployed+liposomal+doxorubicin+during+radiofrequency+ablation+for+hepatic+malignancies.">Phase I study of heat-deployed liposomal doxorubicin during radiofrequency ablation for hepatic malignancies.</a> Journal of Vascular and Interventional Radiology. 23 (2), 248-255 (2012).</li><li>Bulbake, U., Doppalapudi, S., Kommineni, N., Khan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+formulations+in+clinical+use:+an+updated+review.">Liposomal formulations in clinical use: an updated review.</a> Pharmaceutics. 9 (2), 12 (2017).</li><li>Zagar, T. 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=Two+phase+I+dose-escalation/pharmacokinetics+studies+of+low+temperature+liposomal+doxorubicin+(LTLD)+and+mild+local+hyperthermia+in+heavily+pretreated+patients+with+local+regionally+recurrent+breast+cancer.">Two phase I dose-escalation/pharmacokinetics studies of low temperature liposomal doxorubicin (LTLD) and mild local hyperthermia in heavily pretreated patients with local regionally recurrent breast cancer.</a> International Journal of Hyperthermia. 30 (5), 285-294 (2014).</li><li>. A phase I study of lyso-thermosensitive liposomal doxorubicin and MR-HIFU for pediatric refractory solid tumors Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT02536183">https://clinicaltrials.gov/ct2/show/NCT02536183</a> (2019)</li><li>PanDox: targeted doxorubicin in pancreatic tumours (PanDox). University of Oxford Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT04852367">https://clinicaltrials.gov/ct2/show/NCT04852367</a> (2021)</li><li>. Image-guided targeted doxorubicin delivery with hyperthermia to optimize loco-regional control in breast cancer (i-GO) Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT03749850">https://clinicaltrials.gov/ct2/show/NCT03749850</a> (2018)</li><li>De Vita, A., 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=Lysyl+oxidase+engineered+lipid+nanovesicles+for+the+treatment+of+triple+negative+breast+cancer.">Lysyl oxidase engineered lipid nanovesicles for the treatment of triple negative breast cancer.</a> Scientific Reports. 11 (1), 5107 (2021).</li><li>Sapareto, S. A., Dewey, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+dose+determination+in+cancer+therapy.">Thermal dose determination in cancer therapy.</a> International Journal of Radiation Oncology, Biology, Physics. 10 (6), 787-800 (1984).</li><li>Kok, H. P., 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=Heating+technology+for+malignant+tumors:+a+review.">Heating technology for malignant tumors: a review.</a> International Journal of Hyperthermia. 37 (1), 711-741 (2020).</li><li>Kokuryo, D., Kumamoto, E., Kuroda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+technological+advancements+in+thermometry.">Recent technological advancements in thermometry.</a> Advanced Drug Delivery Reviews. 163, 19-39 (2020).</li><li>Bongiovanni, A., 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=3-T+magnetic+resonance-guided+high-intensity+focused+ultrasound+(3+T-MR-HIFU)+for+the+treatment+of+pain+from+bone+metastases+of+solid+tumors.">3-T magnetic resonance-guided high-intensity focused ultrasound (3 T-MR-HIFU) for the treatment of pain from bone metastases of solid tumors.</a> Support Care Cancer. 30 (7), 5737-5745 (2022).</li><li>Seifert, G., 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=Regional+hyperthermia+combined+with+chemotherapy+in+paediatric,+adolescent+and+young+adult+patients:+current+and+future+perspectives.">Regional hyperthermia combined with chemotherapy in paediatric, adolescent and young adult patients: current and future perspectives.</a> Radiation Oncology. 11, 65 (2016).</li><li>Dewhirst, M. W., Lee, C. -. T., Ashcraft, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+biology+in+driving+the+field+of+hyperthermia.">The future of biology in driving the field of hyperthermia.</a> International Journal of Hyperthermia. 32 (1), 4-13 (2016).</li><li>Dewhirst, M. W., Vujaskovic, Z., Jones, E., Thrall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-setting+the+biologic+rationale+for+thermal+therapy.">Re-setting the biologic rationale for thermal therapy.</a> International Journal of Hyperthermia. 21 (8), 779-790 (2005).</li><li>Repasky, E. A., Evans, S. S., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+matters!+And+why+it+should+matter+to+tumor+immunologists.">Temperature matters! And why it should matter to tumor immunologists.</a> Cancer Immunology Research. 1 (4), 210-216 (2013).</li><li>Hijnen, N., 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=Thermal+combination+therapies+for+local+drug+delivery+by+magnetic+resonance-guided+high-intensity+focused+ultrasound.">Thermal combination therapies for local drug delivery by magnetic resonance-guided high-intensity focused ultrasound.</a> Proceedings of the National Academy of Sciences. 114 (24), E4802-E4811 (2017).</li><li>Shultz, L. D., 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=Human+cancer+growth+and+therapy+in+immunodeficient+mouse+models.">Human cancer growth and therapy in immunodeficient mouse models.</a> Cold Spring Harbor Protocols. 2014 (7), 694-708 (2014).</li><li>De Vita, A., 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=Deciphering+the+genomic+landscape+and+pharmacological+profile+of+uncommon+entities+of+adult+rhabdomyosarcomas.">Deciphering the genomic landscape and pharmacological profile of uncommon entities of adult rhabdomyosarcomas.</a> International Journal of Molecular Sciences. 22 (21), 11564 (2021).</li><li>McKinnon, T., 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=Functional+screening+of+FGFR4-driven+tumorigenesis+identifies+PI3K/mTOR+inhibition+as+a+therapeutic+strategy+in+rhabdomyosarcoma.">Functional screening of FGFR4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma.</a> Oncogene. 37 (20), 2630-2644 (2018).</li><li>Zaporzan, B., 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=MatMRI+and+MatHIFU:+software+toolboxes+for+real-time+monitoring+and+control+of+MR-guided+HIFU.">MatMRI and MatHIFU: software toolboxes for real-time monitoring and control of MR-guided HIFU.</a> Journal of Therapeutic Ultrasound. 1, (2013).</li><li>Dunne, 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=Heat-activated+drug+delivery+increases+tumor+accumulation+of+synergistic+chemotherapies.">Heat-activated drug delivery increases tumor accumulation of synergistic chemotherapies.</a> Journal of Controlled Release. 308, 197-208 (2019).</li><li>Zhao, Y. X., Hu, X. Y., Zhong, X., Shen, H., Yuan, Y. <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+as+an+alternative+regimen+for+myxofibrosarcoma.">High-intensity focused ultrasound treatment as an alternative regimen for myxofibrosarcoma.</a> Dermatologic Therapy. 34 (2), 14816 (2021).</li><li>Vanni, S., 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=Myxofibrosarcoma+landscape:+diagnostic+pitfalls,+clinical+management+and+future+perspectives.">Myxofibrosarcoma landscape: diagnostic pitfalls, clinical management and future perspectives.</a> Therapeutic Advances in Medical Oncology. 14, 17588359221093973 (2022).</li></ol>

The authors have no financial interests or conflicts of interest to disclose.

The protocol developed herein was used to target hind limb tumors using MRgHIFU for mild HT treatment and release encapsulated drugs from liposomes in vivo. Several critical steps were encountered in this protocol during the pilot study, and optimizing these critical steps accounted for the improved treatment success over the pilot study. First is the complete removal of the hair on the area to be sonicated. Any gas trapping within the fur prevents the ultrasound beam from passing and blocks ultrasound passage i...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64544">Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model </a>. The Authors section was updated from:
Claire Wunker1,2 
Karolina Piorkowska3 
Ben Keunen3 
Yael Babichev2 
Suzanne M. Wong3,4 
Maximilian Regenold5 
Michael Dunne5 
Julia Nomikos1,2 
Maryam Siddiqui6 
Samuel Pichardo6 
Warren Foltz7 
Adam C. Waspe3,8 
Justin T. Gerstle3,9 
Rebecca A. Gladdy1,2,10 
1 Institute of Medical Science, University of Toronto 
2 2Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital 
3 The Wilfred and Joyce Posluns Centre for Image-Guided Innovation and Therapeutic Intervention, The Hospital for Sick Children 
4 Institute of Biomedical Engineering, University of Toronto 
5 Leslie Dan Faculty of Pharmacy, University of Toronto 
6 Departments of Radiology and Clinical Neurosciences, University of Calgary 
7 Department of Radiation Oncology, University of Toronto 
8 Department of Medical Imaging, University of Toronto 
9 Department of Pediatric Surgery, University of Toronto 
10 Department of Surgery, University of Toronto
to:
Claire Wunker1,2 
Karolina Piorkowska3 
Ben Keunen3 
Yael Babichev2 
Suzanne M. Wong3,4 
Maximilian Regenold5 
Michael Dunne5 
Julia Nomikos1,2 
Maryam Siddiqui6 
Samuel Pichardo6 
Warren Foltz7 
Adam C. Waspe3,8 
Justin T. Gerstle3,9 
James M. Drake1,3,4,10 
Rebecca A. Gladdy1,2,10 
1 Institute of Medical Science, University of Toronto 
2 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital 
3 The Wilfred and Joyce Posluns Centre for Image-Guided Innovation and Therapeutic Intervention, The Hospital for Sick Children 
4 Institute of Biomedical Engineering, University of Toronto 
5 Leslie Dan Faculty of Pharmacy, University of Toronto 
6 Departments of Radiology and Clinical Neurosciences, University of Calgary 
7 Department of Radiation Oncology, University of Toronto 
8 Department of Medical Imaging, University of Toronto 
9 Department of Pediatric Surgery, University of Toronto 
10 Department of Surgery, University of Toronto

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64544">Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model
</a>. The Authors section was updated.

Erratum: Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Rhabdomyosarcoma (RMS) is a skeletal muscle tumor that most commonly occurs in children and young adults1. Localized disease is often treated with multimodal treatment, including chemotherapy, ionizing radiation, and surgery. The use of multi-drug chemotherapy regimens is more prevalent in pediatric patients, with improved outcomes compared to their adult counterparts2; however, despite ongoing research efforts, the 5-year survival rate remains at around 30% in the most aggressive form of the disease3,4. The chemotherapy standard of care is a multidrug regimen that includes vincristine, cyclophosphamide, and actinomycin D. In cases of relapsed or recurrent disease, alternate chemotherapies are used, including standard (free) doxorubicin (FD) and ifosfamide1. While all these chemotherapies have systemic toxicities, the cardiotoxicity of doxorubicin imposes a life-long dose limitation5-7. To increase the amount of the drug delivered to the tumor and to minimize systemic toxicity, alternative formulations have been developed, including liposomal encapsulation. These can be non-thermosensitive doxorubicin, which has been approved for the treatment of breast cancer and hepatocellular carcinoma, or thermosensitive doxorubicin, for which clinical trials are ongoing8,9,10,11,12,13. Alternative methods for delivering liposomal encapsulated drugs such as multi-vesicular liposomes and ligand-targeted liposomes have been evaluated and show promise for the treatment of tumors9. In this study, the addition of heat has multifactorial impacts, including drug release14. The combination of hyperthermia (HT) generated with magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) and thermosensitive liposomal doxorubicin (TLD) is a novel multimodal therapeutic approach for using this toxic yet effective drug to treat RMS, while minimizing dose-limiting toxicity and potentially increasing the immune response to the tumor.
Doxorubicin releases rapidly from TLD at temperatures &#62;39 &#176;C, well above the average human body temperature of 37 &#176;C but not high enough to cause tissue damage or ablation; this starts to occur at 43 &#176;C, but occurs more rapidly as temperatures approach 60 &#176;C15. Various methods have been used to generate HT in vivo, including lasers, microwaves, radiofrequency ablation, and focused ultrasound, many of which are invasive heating methods16. MRgHIFU is a noninvasive, nonionizing heating method that facilitates precise temperature settings within the target tissue in situ. Magnetic resonance (MR) imaging crucially provides real-time imaging, where computer software can be used, to calculate a thermometry measurement of the tissue throughout treatment; subsequently, this data can be used to control the ultrasound therapy in real time to reach and maintain a desired temperature set point17. MRgHIFU has been tested in various tissue types and can be used for a wide range of temperature treatments, from mild HT to ablation, as well as clinically to successfully treat painful bone metastases18. Additionally, HT has been shown to cause tumor cytotoxicity, modulate protein expression, and alter the immune response in the tumor microenvironment19,20,21,22. One study combined mild HT with TLD, followed by ablation with MRgHIFU, in a synergetic R1 rat model23, resulting in necrosis in the tumor core and drug delivery to the periphery. Traditionally, radiotherapy has been used as an adjunct therapy to damage tumor cells and decrease local disease recurrence. However, its use is limited by lifetime dosing and off-target damage1. Thus, HT is unique in that it can cause some of the same effects without the same toxicities or limitations.
Preclinical animal models for RMS include syngeneic immunocompetent models and patient derived xenografts (PDX) in immunocompromised hosts. While the immunocompromised models allow for growth of the human tumors, they lack the appropriate tumor microenvironment and are limited in their ability to study immune response24. FGFR4-activating mutation is a promising marker for poor prognosis and a potential therapeutic target in adult and pediatric RMS1,25. In the syngeneic RMS models developed in the Gladdy lab, the tumors are able to grow in an immunocompetent host, which develops innate and adaptive immune responses to the tumor26. As HT influences the immune response, observation of the change in the murine immune response is a valuable advantage of this tumor model. To test both the tumor response to TLD in comparison to FD, as well as the change in the immune response of the tumor to both chemotherapy and HT, a protocol was developed and employed to treat syngeneic murine RMS tumors in vivo using MRgHIFU and TLD, which is the focus of this study.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5mL Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>1kb plus DNA Ladder</td>
			<td>Froggabio</td>
			<td>DM015-R500</td>
			<td></td>
		</tr>
		<tr>
			<td>2x HS-Red Taq (PCR mix)</td>
			<td>Wisent</td>
			<td>801-200-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>7 Tesla MRI BioSpec</td>
			<td>Bruker</td>
			<td>T184931</td>
			<td>70/30 BioSpec, Bruker, Ettlingen, Germany</td>
		</tr>
		<tr>
			<td>C1000 Thermal cycler</td>
			<td>Biorad</td>
			<td>1851148</td>
			<td></td>
		</tr>
		<tr>
			<td>Clippers</td>
			<td>Whal Peanut</td>
			<td>8655</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed ultrasound gel</td>
			<td>Aquaflex</td>
			<td>HF54-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Convection heating device</td>
			<td>3M Bair Hugger</td>
			<td>70200791401</td>
			<td></td>
		</tr>
		<tr>
			<td>Depiliatory cream</td>
			<td>Nair</td>
			<td>61700222611</td>
			<td>Shopper&#39;s Drug Mart</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Wisent</td>
			<td>219-065-LK</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy extraction kit</td>
			<td>Qiagen&#160;</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Wisent</td>
			<td>311-420-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Drug injection system</td>
			<td>Harvard Apparatus</td>
			<td>PY2 70-2131</td>
			<td>PHD 22/2200 MRI compatible Syringe Pump</td>
		</tr>
		<tr>
			<td>Eye lubricant</td>
			<td>Optixcare</td>
			<td>50-218-8442</td>
			<td></td>
		</tr>
		<tr>
			<td>F10 Media</td>
			<td>Wisent</td>
			<td>318-050-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Wisent</td>
			<td>081-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Froggarose</td>
			<td>FroggaBio</td>
			<td>A87</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Molecular Imager</td>
			<td>BioRad</td>
			<td>GelDocXR</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Wisent</td>
			<td>609-065-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Lamp</td>
			<td>Morganville Scientific</td>
			<td>HL0100&#160;</td>
			<td>Similar to this product</td>
		</tr>
		<tr>
			<td>Intravascular Polyethylene tubing (0.015&#34; ID x 0.043&#34; OD, 20G)</td>
			<td>SAI infusion</td>
			<td>PE-20-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr>
			<td>M25FV24C Cell line</td>
			<td>Gladdy Lab</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter Syringe</td>
			<td>Hamilton</td>
			<td>01-01-7648</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Imager Gel Doc XR</td>
			<td>Biorad</td>
			<td>170-8170</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse holder</td>
			<td>The 3D printing material used was ABS-M30i, and it was printed on FDM Fortus 380mc machine&#160;</td>
			<td>N/A</td>
			<td>Dimensions: length = 43 mm, outer radius = 15 mm, inner width (where the mouse would sit) = 20.7 mm.&#160;</td>
		</tr>
		<tr>
			<td>MyRun Machine</td>
			<td>Cosmo Bio Co Ltd</td>
			<td>CBJ-IMR-001-EX</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 8000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-8000-GL</td>
			<td></td>
		</tr>
		<tr>
			<td>p53 primers</td>
			<td>Eurofins</td>
			<td>N/A</td>
			<td>Custom Primers</td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Diamed</td>
			<td>SSI3131-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Wisent</td>
			<td>450-200-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteus software&#160;</td>
			<td>Pichardo lab</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Respiratory monitoring system</td>
			<td>SAII</td>
			<td>Model 1030</td>
			<td>MR-compatible monitoring and gating system for small animals</td>
		</tr>
		<tr>
			<td>Small Bore HIFU device, LabFUS</td>
			<td>Image Guided Therapy</td>
			<td>N/A</td>
			<td>LabFUS, Image Guided Therapy, Pessac, France Number of elements 8 
			frequency 2.5 MHz 
			diameter&#160; 25 mm 
			radius of curvature 20 mm 
			Focal spot size 0.6 mm x 0.6 mm x 2.0 mm 
			 
			Motor: axes 2 
			 
			Generator: 
			Number of channels 8 
			Maximum electrical power/channel Wel 4 
			Maximum electrical power Wel 32 
			Bandwidth 0.5 - 5 MHz 
			Control per channel: Freq., Phase and. amplitude 
			Measurements per channel: Vrms, Irms, cos(theta) 
			Duty Cycle at 100% power % 100% for 1 min. 
			 
			Transducer: 
			Number of elements 8 
			frequency&#160; 2.5 MHz 
			diameter 25 mm 
			radius of curvature 20 mm 
			Focal spot size&#160; 0.6 mm x 0.6 mm x 2.0 mm</td>
		</tr>
		<tr>
			<td>SYBR Safe</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE</td>
			<td>Wisent</td>
			<td>811-540-FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tail vein catheter (27G 0.5&#34; )</td>
			<td>Terumo Medical Corp</td>
			<td>15253</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal probes</td>
			<td>Rugged Monitoring</td>
			<td>L201-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>ThermoFisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Wisent</td>
			<td>325-052-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound Gel</td>
			<td>Aquasonic</td>
			<td>PLI 01-08</td>
			<td></td>
		</tr>
	</tbody>
</table>

magnetic resonance-guided high intensity focused ultrasound generated hyperthermia: a feasible treatment method in a murine rhabdomyosarcoma model

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Using the MRgHIFU-generated hyperthermia protocol, the tumors in the hind limb were able to be consistently heated to the desired set temperature for the duration of the treatment (Figure 4 shows a representative treatment, 10 or 20 min, n = 65). To consider a treatment to be successful, the ROI had to be maintained above 39 &#176;C for the entirety of the treatment, with &#60;6 &#176;C variation throughout the treatment and without heating of off-target tissue. Additionally, the core temper...

Watch this Scientific Journal Video about Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model at JoVE.com

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time ...

magnetic-resonance-guided-high-intensity-focused-ultrasound-generated

Research

JoVE Journal

Cancer Research

2.2K Views.  University of Toronto. Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

Video: Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients do not have many treatment options and are usually offered palliative chemotherapy or best supportive care. Endobronchial ultrasound (EBUS)-guided intratumoral injection of cisplatin (ITC) is a novel approach for these patients. The procedure is performed under conscious sedation. The lesion is located with a bronchoscopy using EBUS, and a 22-gauge EBUS needle is advanced through the working channel of the scope and locked in position. Under ultrasound guidance, the wall of the tracheobronchial tree is punctured and the needle is moved into the target lesion. The needle stylet is then removed and cisplatin (40 mg/40 mL) is injected into the lesion. One to two sites are treated per session. Details of the procedure are described in the protocol section of paper. At our center, 50 sites were treated in 36 patients (19 males, 17 females). The mean age of our cohort was 61.9 &#177;8.5 years. We performed final analyses on 35 patients and 41 sites. 24/35 (69%) had complete or partial response (responders), whereas 11/35 (31%) had stable or progressive disease (non-responders). Overall, survival in our group was 8 months (95% CI of 6-11 months), with patients who responded having significantly better survival than the ones who did not.

The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field.

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% of patients. Surgery has been widely used in association with radiofrequency, cryotherapy, or microwaves to expand the number of treatments performed with a curative intent. Nevertheless, several limitations have been documented when using these techniques (i.e., a traumatic puncture of the parenchyma, a limited size of lesions, and an inability to monitor the treatment in real-time).High-intensity focused ultrasound (HIFU) technology can achieve precise ablations of biological tissues without incisions or radiation. Current HIFU devices are based on an extracorporeal approach with limited access to the liver. We have developed a HIFU device designed for intraoperative use. The use of a toroidal transducer enables an ablation rate (10 cm3&#183;min-1) higher than any other treatment and is independent of perfusion.
The feasibility, safety, and accuracy of intraoperative HIFU ablation were evaluated during an ablate-and-resect prospective study. This clinical phase I and IIa study was performed in patients undergoing hepatectomy for liver metastases. The HIFU treatment was performed on healthy tissue scheduled for resection.Liver metastases measuring less than 20 mm will be targeted during phase IIb (currently ongoing). This set-up allows the real-time evaluation of HIFU ablation while protecting participating patients from any adverse effects related to this new technique.
Fifteen patients were included in phase I&#8211;IIa and 30 HIFU ablations were safely created within 40 s and with a precision of 1&#8211;2 mm. The average dimensions of the HIFU ablations were 27.5 x 21.0 mm2, corresponding to a volume of approximately 7.5 cm3. The aim of the ongoing phase IIb is to ablate metastases of less than 20 mm in diameter with a 5 mm margin.

Here, we present an ablate-and-resect prospective study to evaluate the feasibility, safety, and accuracy of intraoperative high-intensity focused ultrasound ablation in patients undergoing hepatectomy for liver metastases.

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...