Name: high-resolution ultrasonography for the analysis of orthotopic atc tumors in a genetically engineered mouse model
Uploaded: 2022-10-11T15:00:00
Description: Watch this Scientific Journal Video about High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model at JoVE.com

这项研究没有获得公共、商业或非营利资助机构的具体资助。

本研究经四川大学动物护理与利用委员会批准进行。 TPO-cre/ERT2;布拉夫CA/wt;本研究使用Trp53 Δex2-10/ Δex2-10 转基因小鼠10 （见 材料表）。如有必要，可以针对不同的动物物种修改协议步骤。这里使用了12只小鼠，包括6只雌性和6只雄性，平均年龄为93天。
1. 实验准备
<ol><li>打开超声检查系统（参见 材料?...

<ol>
	<li>Maniakas, 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=Evaluation+of+overall+survival+in+patients+with+anaplastic+thyroid+carcinoma,+2000-2019.">Evaluation of overall survival in patients with anaplastic thyroid carcinoma, 2000-2019.</a> JAMA Oncology. 6 (9), 1397-1404 (2020).</li><li>Molinaro, E., 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=Anaplastic+thyroid+carcinoma:+From+clinicopathology+to+genetics+and+advanced+therapies.">Anaplastic thyroid carcinoma: From clinicopathology to genetics and advanced therapies.</a> Nature Reviews Endocrinology. 13 (11), 644-660 (2017).</li><li>Champa, D., Di Cristofano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+anaplastic+thyroid+carcinoma+in+the+mouse.">Modeling anaplastic thyroid carcinoma in the mouse.</a> Hormones and Cancer. 6 (1), 37-44 (2015).</li><li>Vitiello, M., Kusmic, C., Faita, F., Poliseno, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+lymph+node+volume+by+ultra-high-frequency+ultrasound+imaging+in+the+Braf/Pten+genetically+engineered+mouse+model+of+melanoma.">Analysis of lymph node volume by ultra-high-frequency ultrasound imaging in the Braf/Pten genetically engineered mouse model of melanoma.</a> Journal of Visualized Experiments. (175), e62527 (2021).</li><li>Wang, Y., 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=Low+intensity+focused+ultrasound+(LIFU)+triggered+drug+release+from+cetuximab-conjugated+phase-changeable+nanoparticles+for+precision+theranostics+against+anaplastic+thyroid+carcinoma.">Low intensity focused ultrasound (LIFU) triggered drug release from cetuximab-conjugated phase-changeable nanoparticles for precision theranostics against anaplastic thyroid carcinoma.</a> Biomaterials Science. 27 (1), 196-210 (2018).</li><li>Mohammed, 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=Early+detection+and+prevention+of+pancreatic+cancer:+Use+of+genetically+engineered+mouse+models+and+advanced+imaging+technologies.">Early detection and prevention of pancreatic cancer: Use of genetically engineered mouse models and advanced imaging technologies.</a> Current Medicinal Chemistry. 19 (22), 3701-3713 (2012).</li><li>Wege, A. K., 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+resolution+ultrasound+including+elastography+and+contrast-enhanced+ultrasound+(CEUS)+for+early+detection+and+characterization+of+liver+lesions+in+the+humanized+tumor+mouse+model.">High resolution ultrasound including elastography and contrast-enhanced ultrasound (CEUS) for early detection and characterization of liver lesions in the humanized tumor mouse model.</a> Clinical Hemorheology and Microcirculation. 52 (2-4), 93-106 (2012).</li><li>Greco, 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=Preclinical+imaging+for+the+study+of+mouse+models+of+thyroid+cancer.">Preclinical imaging for the study of mouse models of thyroid cancer.</a> International Journal of Molecular Sciences. 18 (12), 2731 (2017).</li><li>Renault, 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=High-resolution+ultrasound+imaging+of+the+mouse.">High-resolution ultrasound imaging of the mouse.</a> Journal of Radiologie. 87, 1937-1945 (2006).</li><li>McFadden, D. 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=p53+constrains+progression+to+anaplastic+thyroid+carcinoma+in+a+Braf-mutant+mouse+model+of+papillary+thyroid+cancer.">p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (16), 1600-1609 (2014).</li><li>Garassini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+principles+of+ultrasonic+diagnosis.">Basic principles of ultrasonic diagnosis.</a> GEN. 39 (4), 283-289 (1985).</li><li>Aldrich, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+physics+of+ultrasound+imaging.">Basic physics of ultrasound imaging.</a> Critical Care Medicine. 35, 131-137 (2007).</li><li>Mancini, 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=Morphological+ultrasound+microimaging+of+thyroid+in+living+mice.">Morphological ultrasound microimaging of thyroid in living mice.</a> Endocrinology. 150 (10), 4810-4815 (2009).</li><li>Ying, M., Yung, D. M., Ho, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+ultrasound+measurement+of+thyroid+gland+volume:+a+new+equation+with+higher+correlation+with+3-D+ultrasound+measurement.">Two-dimensional ultrasound measurement of thyroid gland volume: a new equation with higher correlation with 3-D ultrasound measurement.</a> Ultrasound in Medicine &amp; Biology. 34 (1), 56-63 (2008).</li></ol>

该协议使用高分辨率超声检查来分析基因工程小鼠模型中的原位ATC肿瘤。转基因模型，基因型为 TPO-cre/ERT2; 布拉夫CA/wt;Trp53 Δex2-10/Δex2-10，由我们的实验室开发。动物过度表达BrafV600E 而缺乏Trp53;腹膜内注射他莫昔芬导致肿瘤生长约1个月10。肿瘤生长迅速，并在50天内达到可测量的大小。该方案用于监测肿瘤生长4个月。
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虽然间变性甲状腺癌（ATC）占甲状腺癌的不到2%，但它每年导致超过50%的甲状腺癌相关死亡。诊断为 ATC 后的中位生存时间仅为约 6 个月，并且没有可显著提高生存率的治疗方法1，2。
ATC的罕见性阻碍了研究疾病如何开始和积极进展的研究。最近出现了模仿该疾病的基因工程小鼠模型，这些模型提供了对该疾病及其对可能治疗方法的反应的见解3，4，5。此类研究需要精确的肿瘤成像进行测量和监测，这通常使用磁共振成像、计算机断层扫描或高分辨率超声检查进行6，7。超声检查已广泛用于小鼠器官。它比磁共振成像和计算机断层扫描具有优势，因为它可以实时进行并且不会使受试者暴露于辐射中，并且必要的设备足够小，可以携带8，9。然而，使用超声连续监测ATC生长的研究很少;因此，这项工作探讨了超声在这种情况下的效用。
在这里，提出了一种使用高分辨率超声检查在ATC小鼠模型中准确扫描，监测和测量肿瘤的方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesive tape</td>
			<td>Winner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia system</td>
			<td>RWDlifescience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brafflox/wt mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber for anesthesia induction</td>
			<td>RWDlifescience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Winner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electric heating blanket</td>
			<td>Petbee</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>RWDlifescience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical gloves</td>
			<td>Winner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>Breeze</td>
			<td>B914JY</td>
			<td></td>
		</tr>
		<tr>
			<td>TPO-cre/ERT2 mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trp53flox/wt mice</td>
			<td>Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Keppler</td>
			<td>KL-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound machine</td>
			<td>VisualSonics</td>
			<td>Vevo 3100</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution ultrasonography for the analysis of orthotopic atc tumors in a genetically engineered mouse model

间变性甲状腺癌（ATC）与预后不良和中位生存时间短有关，但没有有效的治疗方法可显著改善结局。模仿ATC进展的基因工程小鼠模型可能有助于研究人员研究这种疾病的治疗方法。杂交三种不同基因型的小鼠， TPO-cre / ERT2;布拉夫CA/wt;建立了Trp53 Δex2-10/Δex2-10转基因ATC模型。腹腔注射他莫昔芬，过表达BrafV600E ，Trp53缺失诱导ATC小鼠模型，约1个月内产生肿瘤。应用高分辨率超声研究肿瘤的发生和发展，通过测量肿瘤大小获得动态生长曲线。与磁共振成像（MRI）和计算机断层扫描相比，超声在观察ATC鼠模型方面具有无创，便携，实时和无辐射暴露的优势。高分辨率超声适用于动态和多重测量。然而，小鼠甲状腺的超声检查需要相关的解剖学知识和经验。本文提供了利用高分辨率超声扫描转基因ATC模型中肿瘤的详细程序。同时，列出了超声参数调整、超声扫描技巧、动物的麻醉和恢复以及其他过程中需要注意的要素。

研究开始时的平均右侧ATC尺寸为4.867 mm 2，平均左侧ATC尺寸为5.189 mm2。在第四次测量中，平均右侧ATC大小已增长到11.844mm 2，而左叶的肿瘤大小已增长到9.280mm2。总 ATC 尺寸从 10.057 mm 2 增加到 15.843 mm2。在研究的后期，ATC迅速增长。就标记为“P92”的小鼠（表1）而言，第四次测量的肿瘤大小几乎是初始测量的四倍。四只小鼠的代表性测量?...

Watch this Scientific Journal Video about High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model at JoVE.com

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

本协议描述了用于可视化整个小鼠甲状腺和监测间变性甲状腺癌生长的高频超声检查。

用于分析基因工程小鼠模型中原位ATC肿瘤的高分辨率超声检查

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

该协议可帮助用户利用高分辨率超声检查来扫描，监测和测量间变性甲状腺癌小鼠模型中的肿瘤。超声检查已被证明在小鼠中是可靠的，用于成像与人体组织占据相似身体位置的组织，包括肝脏、甲状腺和胎儿。首先，打开超声检查系统，并创建一个用于捕获图像和收集数据的新文件夹。然后，选择40兆赫的线探针，单击浅表组织模式以激活浅表组织换能器，并使用所得B模式进行甲状腺成像。清洁动物成像平台，并打开加热按钮，使平台温度达到38至40摄氏度，然后再将动物放在上面。麻醉鼠标，并将其放在加热的平台上。用连接到麻醉出口的鼻锥盖住鼻子。然后，在鼠标仰卧位的情况下，使用胶带将其四肢固定在加热的平台上。对于肿瘤成像，调整成像系统以优化分辨率。将二维增益设置为 25 到 30 分贝，将图像深度设置为 10 毫米，将中心设置为 3 到 6 毫米。设置参数后，将超声波凝胶大量涂抹在通过使用脱毛膏实现的裸露皮肤区域。握住探头，将其与胸部的超声凝胶接触。然后，从胸部向颈部扫描以定位甲状腺。根据解剖位置和超声超声从气管和绑带肌肉中识别间变性甲状腺癌肿瘤。根据总肿瘤的印象，确认肿瘤从左到右直径最大的图像切片。然后，按下冻结按钮，并使用超声卡尺测量前后和从左到右的肿瘤直径。完成扫描后，擦拭超声波凝胶，并从动物的四肢上取下约束胶带。在四个时间间隔内测量左右叶的ATC肿瘤大小。在第四次测量中，平均右侧ATC大小增长到11.844平方毫米，而左叶的肿瘤大小增长到9.280平方毫米，使总大小增加到15.843平方毫米。有趣的是，在标记为P92的小鼠中，肿瘤大小几乎是初始测量大小的四倍。该技术的主要优点是超声波是灵活的，可以在没有任何辐射的情况下实时进行。

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