代表性数据由美国国家癌症研究所（K22CA218549至S.T.S）资助。除了协助开发本文报道的综合分析方法外，我们还感谢俄亥俄州立大学综合癌症中心比较病理学和小鼠表型共享资源（主任 - Krista La Perle，DVM，PhD）的组织学和免疫组织化学服务以及病理学成像核心的算法开发和分析。

动物使用遵循俄勒冈州立大学机构动物护理和使用委员会（IACUC）批准的协议2007A0120-R4（PI：Gina Sizemore博士）下的大学实验动物资源（ULAR）法规。
1. 乳腺癌细胞尾静脉注射
<ol><li>制备注射用细胞和注射器<ol>
<li>根据要使用的小鼠数量和细胞浓度，接种适当数量的细胞。 注意：注射的细胞数量和转移发生的时间将取决于所使用的细胞系，需要优化。在该演示中，将1 x 106 MDA-MB-231细胞静脉注射到 NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ（NSG）小鼠中，并且在注射后24天内观察到宏观肺病变。对于MVT1小鼠乳腺肿瘤细胞系17，将3 x 10 6个细胞注射到免疫感受态FVB / N小鼠中，这些小鼠在14天内观察到许多肺转移22，23。</li>
		<li>吸出培养基并用1x PBS冲洗细胞板。胰蛋白酶消化以最小体积对细胞进行胰蛋白酶消化，加入适当体积的培养基，并使用血细胞计数器或其他优选方法对细胞进行计数。台盼蓝（0.4%）或其他活/死细胞染料可用于确定活细胞计数。</li>
		<li>通过以180× g 旋转5分钟来沉淀细胞。</li>
		<li>在无菌的1x PBS中重悬适当数量的细胞，使得每只小鼠注射100μL的体积。将细胞悬浮液保持在冰上以保持活力。</li>
		<li>在注射前，用200μL或1mL移液器彻底重悬细胞以避免结块。在28G胰岛素注射器中吸取100μL（ 见材料表）。</li>
		<li>通过保持注射器垂直，点击注射器并缓慢调整柱塞来消除任何气泡。将气泡注入静脉可能会导致致命的空气/气体栓塞。</li>
	</ol></li>
	<li>尾静脉外侧静脉注射 注意：对于实验性乳腺癌转移测定，在>6周龄的雌性小鼠上进行注射。<ol>
<li>用尾巴操作鼠标，将动物滑入适当尺寸的开槽管/约束装置中（请参阅所用约束装置 的材料表 ）。</li>
		<li>插入约束装置的塞子部分，并将鼠标放置在其侧面，以便其横向尾静脉易于查看。小鼠有一条与生殖器对齐的腹侧动脉，一条背静脉和两条外侧尾静脉。</li>
		<li>用无菌擦拭物清洁小鼠尾巴的表面。用非惯用手抓住食指和拇指之间的尾巴，并施加轻微的张力。</li>
		<li>从尾巴的远端部分开始，将针头平行于静脉，斜面向上。</li>
		<li>如果允许， 请仔细 地将针头重新包裹起来，并弯曲成20-30°的角度。强烈建议使用单手方法或针头重盖装置。 注意：没有必要抽吸，因为这可能会导致静脉塌陷。然而，首次放置时可能会看到少量血迹。针头将平稳地进入静脉，并正确放置。</li>
		<li>慢慢地将整个体积分配到静脉中。当柱塞被推动时，不应该有阻力。</li>
		<li>如果感觉到任何阻力，请立即取出注射器针头。如果需要，重新插入针头（理想情况下不超过3次尝试），向尾部近端或对侧静脉移动。</li>
		<li>注射后可能会有少量血液移位。用无菌纱布轻轻按压，然后用无菌擦拭。</li>
		<li>及时将注射器丢弃在适当的尖锐容器中。</li>
		<li>将鼠标放回干净、通风的笼子中，并监测是否有遇险迹象。</li>
		<li>监测小鼠2-3次/周转移的迹象（呼吸困难，驼背姿势，体重减轻）和一般痛苦。转移的发展时间将取决于细胞系和小鼠品系。</li>
		<li>如果使用体内活体动物成像装置，则在尾静脉注射后立即对小鼠进行成像，以确认细胞注射成功并获得时间"零"数据（本文未包括体内生物发光成像的具体细节，但由Yang等人描述24）。</li>
	</ol></li>
</ol>2. 肺组织固定及转移性肺肿瘤负荷分析
<ol><li>肺组织充气以保持肺的结构形式，用于组织病理学<ol>
<li>遵循批准的安乐死程序（例如，室容积的30-70%位移/分钟的二氧化碳）后，将小鼠尸体固定在带有销钉的解剖板上。喷洒或施用70%乙醇，以防止在解剖过程中阻挡小鼠的皮毛。 注意：二氧化碳窒息可引起肺出血，作为预期的背景病变，特别是在流速较慢时。</li>
		<li>用中线切口打开胸部，将切口从颅骨/尾部延伸穿过腹膜，并通过抓住椎骨过程切除隔膜。</li>
		<li>使用一套单独的剪刀，以免刀片变钝，沿着胸骨两侧切开肋骨，并小心地取出肋骨笼，为肺部扩张留出空间。</li>
		<li>通过切除下颌下唾液腺和舌骨下肌肉组织来隔离气管。在气管的两侧放置销钉可以防止在针插入过程中不必要的移动。</li>
		<li>在 26 G 注射器中加入 2-3 mL 10% 中性缓冲福尔马林，然后插入气管。</li>
		<li>缓慢注射福尔马林，观察肺部扩张（通常需要约1.5毫升福尔马林）。</li>
		<li>一旦福尔马林开始从肺部泄漏（避免过度充气），用一对镊子捏住气管，取出注射器针头并取下整个呼吸装置。将肺、心脏等直接放入福尔马林中，因为固定后可以对组织进行额外的修剪。</li>
		<li>使用标准方法完成组织加工，包埋，切片以及苏木精和曙红（H&E）染色。</li>
	</ol></li>
	<li>转移性肺肿瘤负荷分析<ol>
<li>在高分辨率载玻片扫描仪上以40倍放大倍率扫描H&E染色的肺部切片（图3）。</li>
		<li>将图像导入图像分析软件（例如，Visiopharm 图像分析）以定量肺转移。 注意：我们建议新用户获得现场或在线培训以使用图像分析软件。许多网络研讨会也可以通过商业网页获得。</li>
		<li>从软件的应用程序库中选择Visiopharm 10118 H&E Lung Metastasis App。 注意：此应用程序的目的是标记和量化H&E染色载玻片上的肺转移。作为10118 H&E肺转移应用程序的一部分，第一个图像处理步骤使用组织检测应用程序分割肺组织。第二个图像处理步骤使用转移检测应用程序来识别肺组织内的转移。转移通过形状以及形状太畸形，太红或太稀疏的区域来识别，无法识别为转移。</li>
		<li>调整定义形状和稀疏度的参数，以最适合代表性图像。肿瘤转移和正常肺组织的分割区域可以使用每种组织类型的不同颜色标签显示。 注意：如果应用程序无法准确地将转移从正常肺组织中分离出来，则可能需要编写使用Visiopharm决策森林程序的自定义应用程序，就像实验一样（参见 图2 和 图3）。下面详细介绍了编写自定义算法。否则，请继续执行步骤 2.2.9。</li>
		<li>打开决策森林程序，该程序通过在所需图像上训练多个 类 [即肺组织（非肿瘤）、转移、红细胞、上皮和/或空白]来工作。在 图2中，肿瘤转移是蓝色的，正常组织是绿色的，支气管上皮是黄色的。此外，红细胞是红色的，空气空间是粉红色的。</li>
		<li>按照提示的一系列是或否问题进行相应的训练，以便为映像适当地训练每个 类 。算法的准确性将决定是/否问题的数量。对于分析，编写自定义算法/应用时，精度设置为 50（范围 0-100）。</li>
		<li>通过应用滤镜来调整每个类的功能，以锐化，模糊，按形状排序等，以提高算法/应用程序的准确性。功能会更改类查看图像的方式，以显示某些颜色或强度。 注意：对于自定义算法，测量为8500μm2及以上的转移被标记为转移并测量。这解释了尺寸差异和转移太小而无法检测到的原因。将8500 μm2以下的小畸形区域和小转移区域纳入正常组织定量。</li>
		<li>保存来自应用程序或自定义算法的修改设置，然后将算法/应用程序应用于整套或一系列H&E染色组织。</li>
		<li>最后，导出所有输出变量，其中包括 表 1 中列出的变量。可以针对每种组织类型量化以微米平方（μm2）为单位的面积，并且百分比来自标本总净组织面积（即总组织减去空气空间）。</li>
		<li>创建自定义算法时，请咨询由美国兽医病理学家学会认证的兽医病理学家委员会，以检查组织标记，以确保准确的测量并区分组织类型。</li>
	</ol></li>
</ol>

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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=Syngeneic+mouse+mammary+carcinoma+cell+lines:+two+closely+related+cell+lines+with+divergent+metastatic+behavior.">Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior.</a> Clinical and Experimental Metastasis. 22 (1), 47-59 (2005).</li><li>Yang, 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=Immunocompetent+mouse+allograft+models+for+development+of+therapies+to+target+breast+cancer+metastasis.">Immunocompetent mouse allograft models for development of therapies to target breast cancer metastasis.</a> Oncotarget. 8 (19), 30621-30643 (2017).</li><li>Resch, M., Neels, T., Tichy, A., Palme, R., Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+assessment+of+tail-vein+injection+in+mice+using+a+modified+anaesthesia+induction+chamber+versus+a+common+restrainer+without+anaesthesia.">Impact assessment of tail-vein injection in mice using a modified anaesthesia induction chamber versus a common restrainer without anaesthesia.</a> Laboratory Animals. 53 (2), 190-201 (2019).</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>Goodale, D., Phay, C., Postenka, C. O., Keeney, M., Allan, 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=Characterization+of+tumor+cell+dissemination+patterns+in+preclinical+models+of+cancer+metastasis+using+flow+cytometry+and+laser+scanning+cytometry.">Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry.</a> Cytometry Part A. 75 (4), 344-355 (2009).</li><li>Goddard, E. T., Fischer, J., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Portal+Vein+Injection+Model+to+Study+Liver+Metastasis+of+Breast+Cancer.">A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.</a> Journal of Visualized Experiments. (118), (2016).</li><li>Wright, L. 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=Murine+models+of+breast+cancer+bone+metastasis.">Murine models of breast cancer bone metastasis.</a> BoneKEy Reports. 5, 804 (2016).</li><li>Simmons, J. 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=Animal+Models+of+Bone+Metastasis.">Animal Models of Bone Metastasis.</a> Veterinary Pathology. 52 (5), 827-841 (2015).</li><li>Liu, 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=Improving+orthotopic+mouse+models+of+patient-derived+breast+cancer+brain+metastases+by+a+modified+intracarotid+injection+method.">Improving orthotopic mouse models of patient-derived breast cancer brain metastases by a modified intracarotid injection method.</a> Scientific Reports. 9 (1), 622 (2019).</li><li>Kodack, D. P., Askoxylakis, V., Ferraro, G. B., Fukumura, D., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+strategies+for+treating+brain+metastases+from+breast+cancer.">Emerging strategies for treating brain metastases from breast cancer.</a> Cancer Cell. 27 (2), 163-175 (2015).</li><li>Brown, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Stereology+Applications+for+the+Pathologist.">Practical Stereology Applications for the Pathologist.</a> Veterinary Pathology. 54 (3), 358-368 (2017).</li><li>Aeffner, F., 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=Digital+Microscopy,+Image+Analysis,+and+Virtual+Slide+Repository.">Digital Microscopy, Image Analysis, and Virtual Slide Repository.</a> Institute for Laboratory Animal Research Journal. 59 (1), 66-79 (2018).</li></ol>

作者没有什么可透露的。

随着研究人员继续使用静脉注射肿瘤细胞作为转移的实验模型，缺乏分析由此产生的转移性肿瘤负担的标准实践。在某些情况下，可以在宏观上观察到操作特定细胞系和/或使用化合物时转移性肿瘤负担的显着差异。然而，在其他情况下，如果没有彻底的病理分析，转移性播种和生长的细微差异可能会被忽视或误解。本研究通过包括转移性肺肿瘤负荷分析的综合方法，推进了先前发表的尾静脉注射...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61270/pathological-analysis-lung-metastasis-following-lateral-tail-vein">Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells</a>. The author list was updated.
The author list was updated from:
Katie A. Thies1,&#160;Sue E. Knoblaugh2, and Steven T. Sizemore1 
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 
2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University
to:
Katie A. Thies1, Sarah Steck1, Sue E. Knoblaugh2, and Steven T. Sizemore1 
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 
2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61270/pathological-analysis-lung-metastasis-following-lateral-tail-vein">Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells</a>. The author list was updated.

Erratum: Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

尽管转移是一个高度复杂且低效的过程1，但转移是癌症患者发病率和死亡率的重要因素2。事实上，大多数癌症相关死亡归因于疾病的转移性传播3，4。为了使肿瘤细胞成功转移，它们必须从原发部位脱离，通过相邻的基质侵入，侵入血液循环或淋巴管，行进到次级部位的毛细血管床，外渗到次级组织，并增殖或生长形成转移性病变5。小鼠模型的使用对于进一步了解负责转移性播种和生长的分子机制至关重要6，7。在本文中，我们专注于乳腺癌转移，为此经常使用转基因小鼠模型和移植方法 - 每种方法都有自己的一套优点和局限性。
基因工程乳腺肿瘤模型利用乳腺特异性启动子，包括 MMTV-LTR （小鼠乳腺肿瘤病毒长末端重复）和 WAP （乳清酸性蛋白），以驱动乳腺上皮中转基因的表达8。包括中位T抗原（PyMT）、 ErbB2/Neu、 c-Myc、 Wnt-1和猿猴病毒40（SV40）在内的癌基因已经以这种方式表达9，10，11，12，13，虽然这些遗传模型有助于研究原发性肿瘤的发生和进展，但很少有易转移至远处器官的遗传基因。此外，这些遗传小鼠模型通常比自发或实验转移模型花费更多的时间和成本。鉴于大多数基因工程乳腺肿瘤模型在研究转移方面的局限性，移植技术已成为研究这一复杂过程的有吸引力的方法。这包括原位、尾静脉、心内和颅内注射合适的细胞系。
虽然几种乳腺癌细胞系在将原位注射到乳腺脂肪垫14，15后容易转移，但转移性肿瘤负荷的一致性和可重复性可能是一个挑战，并且此类研究的持续时间可能长达数月。特别是对于评估肺转移，静脉注射到尾静脉通常是一种更具可重复性和时间有效的方法，转移性扩散通常发生在几周内。然而，由于静脉注射模型绕过了转移性级联反应的初始步骤，因此在解释这些研究的结果时必须小心。在这个演示中，我们展示了乳房肿瘤细胞的尾静脉注射以及准确而全面的分析方法。
尽管研究界在了解乳腺癌转移的复杂过程方面取得了重大进展，但据估计，目前有超过150，000名女性患有转移性乳腺癌16。在IV期乳腺癌患者中，>36%的患者有肺转移17;然而，位点特异性模式和转移发生率可能因分子亚型而异18，19，20，21。乳腺癌相关肺转移患者的中位生存期仅为 21 个月，这凸显了确定该疾病的有效治疗方法和新型生物标志物的必要性17。实验转移模型的使用，包括静脉注射肿瘤细胞，将继续推进我们对这一重要临床挑战的了解。当与本方案中描述的数字成像病理学和转移性肺肿瘤负荷分析方法相结合时，尾静脉注射是乳腺癌转移研究的宝贵工具。

<table>
	<tbody>
		<tr>
			<td>alcohol prep pads</td>
			<td>Fisher Scientific</td>
			<td>22-363-750</td>
			<td>for cleaning tail prior to injection</td>
		</tr>
		<tr>
			<td>dissection scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-5</td>
			<td>for mouse dissection and lung tissue inflation</td>
		</tr>
		<tr>
			<td>DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>MT10013CV</td>
			<td>cell culture media base for MDA-MB-231 and MVT1 cell lines</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Salt Solution 1x</td>
			<td>Fisher Scientific</td>
			<td>MT21030CV</td>
			<td>used for resuspending tumor cells for injection</td>
		</tr>
		<tr>
			<td>ethanol (70 % solution)</td>
			<td>OSU</td>
			<td></td>
			<td>used to minimize mouse&#39;s fur during dissection; use caution - flammable</td>
		</tr>
		<tr>
			<td>Evan&#39;s blue dye</td>
			<td>Millipore Sigma</td>
			<td>E2129</td>
			<td>used at 1 % in sterile PBS for practice with tail-vein injection method; use caution - dangerous reagent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Millipore Sigma</td>
			<td>F4135</td>
			<td>cell culture media additive; used at 10% in DMEM</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fisher Scientific</td>
			<td>10-270</td>
			<td>for dissection and lung tissue inflation</td>
		</tr>
		<tr>
			<td>FVB/NJ mice</td>
			<td>The Jackson Laboratory</td>
			<td>001800</td>
			<td>syngeneic mouse strain for MVT1 cells</td>
		</tr>
		<tr>
			<td>hemacytometer (Bright-Line)</td>
			<td>Millipore Sigma</td>
			<td>Z359629</td>
			<td>for use in cell culture to obtain cell counts</td>
		</tr>
		<tr>
			<td>insulin syringe (28 G)</td>
			<td>Fisher Scientific</td>
			<td>14-829-1B</td>
			<td>for tail-vein injections (BD 329424)</td>
		</tr>
		<tr>
			<td>MDA-MB-231 cells</td>
			<td>ATCC</td>
			<td></td>
			<td>human breast cancer cell line</td>
		</tr>
		<tr>
			<td>MVT1 cells</td>
			<td></td>
			<td></td>
			<td>mouse mammary tumor cells</td>
		</tr>
		<tr>
			<td>needles (26 G)</td>
			<td>Fisher Scientific</td>
			<td>14-826-15</td>
			<td>used to inflate the mouse&#39;s lungs</td>
		</tr>
		<tr>
			<td>neutral buffered formalin (10%)</td>
			<td>Fisher Scientific</td>
			<td>245685</td>
			<td>used as a tissue fixative and to inflate lung tissue; use caution - dangerous reagent</td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice</td>
			<td>The Jackson Laboratory</td>
			<td>005557</td>
			<td>maintained by OSUCCC Target Validation Shared Resource</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin 100x</td>
			<td>ThermoFisher</td>
			<td>15140163</td>
			<td>cell culture media additive</td>
		</tr>
		<tr>
			<td>sterile gauze</td>
			<td>Fisher Scientific</td>
			<td>NC9379092</td>
			<td>for applying pressue to mouse&#39;s tail if bleeding occurs</td>
		</tr>
		<tr>
			<td>syringe (5 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-955-458</td>
			<td>used to inflate mouse lung tissue</td>
		</tr>
		<tr>
			<td>tail-vein restrainer</td>
			<td>Braintree Scientific, Inc.</td>
			<td>TV-150 STD</td>
			<td>used to restrain mouse for tail-vein injections</td>
		</tr>
		<tr>
			<td>Trypan blue (0.4 %)</td>
			<td>ThermoFisher</td>
			<td>15250061</td>
			<td>used in cell culture to assess viability</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25 %</td>
			<td>ThermoFisher</td>
			<td>25200-114</td>
			<td>used in cell culture to detach tumor cells from plate</td>
		</tr>
	</tbody>
</table>

pathological analysis of lung metastasis following lateral tail-vein injection of tumor cells

转移是大多数癌症患者发病和死亡的主要原因，在小鼠中进行临床前建模可能具有挑战性。很少有自发性转移模型可用。因此，涉及尾静脉注射合适细胞系的实验转移模型是转移研究的主要内容。当癌细胞被注射到外侧尾静脉时，肺是它们首选的定植部位。该技术的一个潜在局限性是转移性肺肿瘤负担的准确定量。虽然一些研究人员对预定义大小的大转移酶进行计数和/或包括组织切片后的微转移酶，但其他研究人员则确定相对于正常组织区域的转移性病变区域。当转移负担很高时，这两种定量方法都可能非常困难。在本文中，我们展示了肺转移的静脉注射模型，然后使用图像分析软件量化转移性肿瘤负荷的先进方法。该过程允许研究多个终点参数，包括平均转移大小，转移总数和总转移面积，以提供全面的分析。此外，这种方法已经过美国兽医病理学家学会（SEK）认证的兽医病理学家委员会的审查，以确保准确性。

如果使用未标记的细胞进行尾静脉注射，则可能难以确认肺定植，直到（1）如果可以观察到大转移酶，则尸检时间或（2）如果存在微观转移，则在组织学分析之后。随着广泛的转移性肺肿瘤负担，小鼠将有劳动呼吸。与任何肿瘤研究一样，在整个研究期间应仔细监测小鼠。使用标记细胞是确认尾静脉注射成功的简单方法;因此，在演示中使用了荧光素酶标记的MDA-MB-231细胞。然而，根据实验设计和...

Watch this Scientific Journal Video about Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells at JoVE.com

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

静脉注射癌细胞常用于转移研究，但转移性肿瘤负担可能难以分析。在本文中，我们展示了转移的尾静脉注射模型，并包括一种分析所得转移性肺肿瘤负担的新方法。

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

pathological-analysis-lung-metastasis-following-lateral-tail-vein

Research

JoVE Journal

Cancer Research

8.6K 次观看.  The Ohio State University Medical Center. 静脉注射癌细胞常用于转移研究，但转移性肿瘤负担可能难以分析。在本文中，我们展示了转移的尾静脉注射模型，并包括一种分析所得转移性肺肿瘤负担的新方法。

视频: Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

肺肿瘤细胞招募分析

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

颈动脉肿瘤细胞注射，产生脑转移瘤小鼠模型

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

脱细胞和细胞肺模型研究肿瘤转移

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size 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 ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

通过炎症性乳腺癌细胞的尾静脉注射模拟脑转移

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

门静脉注射结直肠癌类器官研究肝转移基质

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...