Name: monitoring breast cancer growth and metastatic colony formation in mice using bioluminescence
Uploaded: 2021-11-05T13:00:00
Description: Watch this Scientific Journal Video about Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence at JoVE.com

我们感谢Y.D.S.实验室的成员。我们要感谢耶路撒冷哈达萨医学中心的Wohl转化医学研究所提供小动物成像设施。这项研究得到了以色列癌症研究基金会的研究 职业发展奖 的支持。

所有小鼠实验均在希伯来大学机构动物护理和使用委员会批准的协议MD-21-16429-5下进行。此外，希伯来大学还获得了实验动物护理评估和认证协会（AAALAC）的认证。
1. 细胞系维持
注意：本方案中使用了人乳腺癌细胞系（MCF-7，MDA-MB-468和MDA-MB-231）。
<ol><li>在Dulbecco的改良Eagle培养基（DMEM）中培养所有乳腺癌细胞系，并在37°C下在加湿的二氧化碳（5%CO 2）培?...

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(56), e3175 (2011).</li><li>Rashid, O. 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=Is+tail+vein+injection+a+relevant+breast+cancer+lung+metastasis+model.">Is tail vein injection a relevant breast cancer lung metastasis model.</a> Journal of Thoracic Disease. 5 (4), 385-392 (2013).</li><li>Fantozzi, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+breast+cancer+metastasis.">Mouse models of breast cancer metastasis.</a> Breast Cancer Research. 8 (4), 212 (2006).</li><li>Kocat&#252;rk, B., Versteeg, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+injection+of+breast+cancer+cells+into+the+mammary+fat+pad+of+mice+to+study+tumor+growth.">Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth.</a> Journal of Visualized Experiments: JoVE. (96), e51967 (2015).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+whole+picture.">The whole picture.</a> Nature. 463 (7283), 977-979 (2010).</li><li>Wang, Y., Tseng, J. -. C., Sun, Y., Beck, A. H., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+imaging+of+tumor+burden+and+molecular+pathways+in+mouse+models+of+cancer.">Noninvasive imaging of tumor burden and molecular pathways in mouse models of cancer.</a> Cold Spring Harbor Protocols. 2015 (2), 135-144 (2015).</li><li>Kim, J. E., Kalimuthu, S., Ahn, B. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+tracking+with+bioluminescence+imaging.">In vivo cell tracking with bioluminescence imaging.</a> Nuclear Medicine and Molecular Imaging. 49 (1), 3-10 (2015).</li><li>Paschall, A. 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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=Studying+triple+negative+breast+cancer+using+orthotopic+breast+cancer+model.">Studying triple negative breast cancer using orthotopic breast cancer model.</a> Journal of Visualized Experiments: JoVE. (157), e60316 (2020).</li><li>Bajikar, S. 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=Tumor-suppressor+inactivation+of+GDF11+occurs+by+precursor+sequestration+in+triple-negative+breast+cancer.">Tumor-suppressor inactivation of GDF11 occurs by precursor sequestration in triple-negative breast cancer.</a> Developmental Cell. 43 (4), 418-435 (2017).</li><li>Khatib, 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=The+glutathione+peroxidase+8+(GPX8)/IL-6/STAT3+axis+is+essential+in+maintaining+an+aggressive+breast+cancer+phenotype.">The glutathione peroxidase 8 (GPX8)/IL-6/STAT3 axis is essential in maintaining an aggressive breast cancer phenotype.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (35), 21420-21431 (2020).</li></ol>

基于动物的实验是癌症研究的必要条件7，8，9，实际上已经开发了许多方案3，6，10，11，12，13，14。然而，这些研究中的大多数仅在实验结束时确定了...

乳腺癌是全世界常见的癌症类型，美国每年诊断出约250，000例新病例1。尽管发病率很高，但一套新的抗癌药物显着改善了乳腺癌患者的预后2。然而，这些治疗仍然不足，因为许多患者经历疾病复发和转移扩散到重要器官2，这是患者发病和死亡的主要原因。因此，乳腺癌研究的主要挑战之一是确定调节远端转移形成的分子机制，以开发抑制其发展的新方法。
癌症转移是一个动态过程，其中细胞从原发肿瘤中分离并通过血液循环侵入邻近组织。因此，其中细胞经历类似转移级联反应的动物模型可以促进识别控制该过程的机制3，4。此外，这些 体内 模型对于开发乳腺癌治疗剂5，6是必不可少的。然而，这些原位模型不能指示实际的肿瘤生长动力学，因为效果仅在终止时确定。因此，我们建立了一种基于荧光素酶的工具，以实时检测肿瘤发展和转移定植。此外，这些细胞表达GFP以检测转移集落。这种方法相对简单，不涉及任何侵入性操作3。因此，结合荧光素酶和荧光检测是推进乳腺癌治疗和疾病管理的临床前研究的有用策略。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.7 mL eppendorf tubes</td>
			<td>Lifegene</td>
			<td>LMCT1.7B-500</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L tips</td>
			<td>Lifegene</td>
			<td>LRT10</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L tips</td>
			<td>Lifegene</td>
			<td>LRT1000</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Lifegene</td>
			<td>LTB15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L tips</td>
			<td>Lifegene</td>
			<td>LRT200</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well cell culture plate</td>
			<td>COSTAR</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Plates BLACK flat bottom</td>
			<td>Bar Naor</td>
			<td>BN30496</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counters</td>
			<td>Thermofisher</td>
			<td>A50298</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III sorter</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Microlance 3 Needles 27 G (3/4&#39;&#39;)</td>
			<td>BD</td>
			<td>302200</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Plastipak Syringes 1 mL x 120</td>
			<td>BD</td>
			<td>303172</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 100 mm x 20 mm Style Dish</td>
			<td>CORNING</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 150 mm x 20 mm Style Dish</td>
			<td>CORNING</td>
			<td>430599</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess cell counting chamber slides</td>
			<td>Thermofisher</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), high glucose, no glutamine</td>
			<td>Biological Industries</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse 80i microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>eppendorf Centrifuge 5810 R</td>
			<td>Sigma Aldrich</td>
			<td>EP5820740000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biological Industries</td>
			<td>04-127-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>FUW GFP</td>
			<td></td>
			<td></td>
			<td>Gifted from Dr. Yossi Buganim&#39;s lab (Hebrew University of Jerusalem)</td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td></td>
			<td></td>
			<td>Gifted from Dr. Lior Nissim&#39;s lab (Hebrew University of Jerusalem)</td>
		</tr>
		<tr>
			<td>Isoflurane, USP Terrell</td>
			<td>Piramal</td>
			<td>NDC 66794-01-25</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine Solution</td>
			<td>Biological industries</td>
			<td>03-020-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>bioluminescence measurement</td>
		</tr>
		<tr>
			<td>MCF-7</td>
			<td>ATCC</td>
			<td>ATCC HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>ATCC HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-468</td>
			<td>ATCC</td>
			<td>ATCC HTB-132</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>NORMAX</td>
			<td>2430-475</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hylabs</td>
			<td>BP655/500D</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-dR8.2-dvpr</td>
			<td>Addgene</td>
			<td>#8455</td>
			<td>Provided by David M. Sabatini&#8217;s lab (Whitehead institute, Boston, USA)</td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Addgene</td>
			<td>#8454</td>
			<td>Provided by David M. Sabatini&#8217;s lab (Whitehead institute, Boston, USA)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Biological Industries</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 90 mm (90x15)</td>
			<td>MINI PLAST</td>
			<td>820-090-01-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 10ml</td>
			<td>Lifegene</td>
			<td>LG-GSP010010S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 25ml</td>
			<td>Lifegene</td>
			<td>LG-GSP010050S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 5ml</td>
			<td>Lifegene</td>
			<td>LG-GSP010005S</td>
			<td></td>
		</tr>
		<tr>
			<td>pLX304 Luciferase-V5 blast plasmid</td>
			<td>Addgene</td>
			<td>#98580</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma Aldrich</td>
			<td>#107689</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 9</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Reservoirs</td>
			<td>Bar Naor</td>
			<td>BN20621STR200TC</td>
			<td></td>
		</tr>
		<tr>
			<td>SMZ18 Stereo microscopes</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Bio-Lab</td>
			<td>190359400</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Lifegene</td>
			<td>LG-FPV403030S</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.5% solution</td>
			<td>Biological industries</td>
			<td>03-102-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA Solution B (0.25%), EDTA (0.05%)</td>
			<td>Biological Industries</td>
			<td>03-052-1a</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum driven Filters</td>
			<td>SOFRA LIFE SCIENCE</td>
			<td>SPE-22-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Virusolve</td>
			<td></td>
			<td>disinfectant</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoGlo Luciferin, In Vivo Grade</td>
			<td>Promega</td>
			<td>P1043</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE HP DNA Transfection Reagent</td>
			<td>Sigma Aldrich</td>
			<td>#6366236001</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring breast cancer growth and metastatic colony formation in mice using bioluminescence

乳腺癌是一种常见的异质性恶性肿瘤，也是女性死亡的第二大原因，主要是由于远处的器官转移。已经产生了几种动物模型，包括广泛使用的原位小鼠模型，其中癌细胞被注射到乳腺脂肪垫中。然而，这些模型不能帮助监测肿瘤生长动力学和转移定植。在小鼠中实时监测癌细胞的尖端工具将显着促进对肿瘤生物学的理解。 
在这里，建立了稳定表达荧光素酶和绿色荧光蛋白（GFP）的乳腺癌细胞系。具体而言，该技术包含两个连续的步骤，通过在 体外 测量荧光素酶活性而启动，然后将癌细胞植入非肥胖严重联合免疫缺陷（NOD-SCID）小鼠的乳腺脂肪垫中。注射后，通过无创生物发光成像系统实时监测肿瘤生长和转移定植。然后，通过荧光显微镜检查肺部表达GFP转移的定量，以验证观察到的生物发光结果。这种复杂的系统结合了荧光素酶和基于荧光的检测工具，可评估体内的癌症转移 ， 这在乳腺癌治疗和疾病管理中具有巨大的应用潜力。

我们生成了表达GFP和荧光素酶载体的乳腺癌细胞系（MDA-MB-231，MCF-7和MDA-MB-468）。具体来说，这是通过顺序感染实现的。首先，乳腺癌细胞系感染了表达荧光GFP的慢病毒载体。GFP阳性细胞（GFP+）在感染后2天进行分类（图1A，B）并感染pLX304荧光素酶-V5载体。然后，使用杀菌素选择荧光素酶以产生指示的（GFP +，Luc +）细胞。为了验证 体?...

Watch this Scientific Journal Video about Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence at JoVE.com

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

在这里，我们描述了一种涉及荧光素酶和绿色荧光蛋白在各种乳腺癌细胞系中表达的非侵入性监测方法。该协议提供了一种在小鼠中实时监测肿瘤形成和转移定植的技术。

使用生物发光监测小鼠乳腺癌生长和转移集落形成

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

使用这种复杂的非侵入性工具，我们可以实时监测小鼠的肿瘤生长动力学和转移定植，这在乳腺癌治疗和疾病管理方面具有巨大的潜力。结合荧光素酶和荧光检测是推进乳腺癌进展和疾病管理的临床前研究的有用策略。这可以应用于抗癌药物研究。该方案不仅限于乳腺癌，可应用于其他癌症，如肺癌和胰腺癌。首先，在16厘米的平板中分别生长MCF-7，MDA-MB-468和MDA-MB-231 GFP和荧光素酶阳性细胞，以达到80%汇合度。通过胰蛋白酶消化收获细胞后，将每个孔中越来越多的细胞接种到黑色96孔板中。然后，用100微升DMEM填充所有孔，并在37摄氏度，5%CO 2培养箱中孵育16至24小时。以每毫升1.5毫克的浓度在PBS中制备荧光素溶液，并将溶液储存在零下20摄氏度。孵育后，用PBS轻轻洗涤细胞一次，然后将100微升荧光素溶液加入每个孔中。等待两分钟，然后使用生物发光测量所有乳腺癌细胞中的荧光素酶活性。在注射小鼠之前，将500万MCF-7和MDA-MB-468细胞转移到200微升PBS和200万MDA-MB-231 GFP和荧光素酶阳性细胞中，进入100微升PBS。通过将锥体以仰卧姿势放在头部上来准备麻醉小鼠注射。接下来，使用棉签用乙醇擦拭乳腺上方的小鼠腹部区域，并用镊子抬起第四乳腺，然后在脂肪垫下插入27号针头，缓慢注射100微升制备的细胞悬浮液。注射后，将小鼠从罩中取出，并在观察下将其转移到新的笼子中，直到它恢复意识。在生物发光检测之前，通过用左手握住其颈部并将手向左倾斜来抑制有意识的小鼠，导致小鼠的脸向上，下半身处于仰卧位置。使用一毫升注射器腹膜内注射100微升每毫升30毫克荧光素到小鼠左下腹象限。在没有麻醉的情况下将小鼠保持七分钟，然后在麻醉室内保持三分钟，然后测量肿瘤动力学。当鼠标处于孵育状态时，打开软件并通过单击“初始化”按钮初始化成像系统。保持设置自动曝光，自动曝光时间为60秒，将介质分档为f/stop 1，激发滤光片被阻挡，发射滤光片打开。初始化结束后，选择“成像向导”和“生物发光”，然后单击“下一步，打开滤镜”以在图像主体中选择鼠标。在景视图中，选择 10 厘米处的舞台 C 和 1.50 厘米处的被摄体高度。然后，单击图像设置阶段到 C.确保将鼠标放置在舞台上正确的仰卧位置，然后再关闭门并单击“获取”按钮。等待图像出现在屏幕上。对另一只鼠标重复该过程。为了检测肺转移，请使用厚纸覆盖原发性肿瘤的强信号，并仅将肺部的腹侧暴露在相机上。使用前面所述的相同参数捕获映像。乳腺癌细胞系感染表达荧光GFP的慢病毒载体，并进行FAC分选。GFP阳性细胞在感染后2天进行分类，用荧光显微镜进行接种和观察。体外荧光素酶活性随着荧光素酶活性水平的细胞数依赖性增加而得到证实。此外，发现荧光素酶活性与细胞数量之间存在线性相关性。注射乳腺癌细胞系后，每两周对小鼠进行一次生物发光读数，以确定肿瘤生长动力学。MDA-MB-231细胞系中的肿瘤生长动力学比侵袭性较低的细胞系MCF-7和MDA-MB-468更快。对三个细胞系的发光活性进行定量。注射后六周，发现收获的肿瘤维持GFP表达。此外，从整个小鼠的肺部实时获得阳性生物发光读数。收获肺以验证阳性转移集落，并观察转移集落的GFP和生物发光。更重要的是在执行注射程序时要小心。注射不当可能导致肿瘤生长速率偏差或肿瘤缺失。我们可以通过检测GFP表达来检查体内和离体的肺转移。此外，我们还可以通过基于免疫组化学的染色来研究肺部的组织病理学。这种技术可用于探索新问题，例如药物疗效研究的效果。

Research

JoVE Journal

Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

乳腺癌相关的淋巴水肿的超声评价

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

球体、菌落形成测定和斑马鱼移植对抗癌香豆素 OT48 生物活性的临床前评价

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Performing Data Mining And Integrative Analysis Of Biomarker in Breast Cancer Using Multiple Publicly Accessible Databases

利用多个可公开访问的数据库对乳腺癌生物标志物进行数据挖掘和综合分析

In recent years, emerging databases were designed to lower the barriers for approaching the intricate cancer genomic datasets, thereby, facilitating ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...