Name: practical considerations in studying metastatic lung colonization in osteosarcoma using the pulmonary metastasis assay
Uploaded: 2018-03-12T13:00:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay at JoVE.com

我们要感谢 Arnulfo 门多萨博士, 他提供了美洲狮技术的训练。 此外, 我们还要承认 Drs. 卡拉姆·昌德·沃赫拉肯纳, 苏珊. 加菲尔德 (NIH) 和 Sam 加亚尔多·阿帕里西奥 (BC 癌症机构) 在本研究过程中提供他们的显微镜使用。这项研究是支持 (部分) 由国家卫生研究院的内部研究计划, 癌症研究中心, 儿科肿瘤科。M.M.L. 得到美国国立卫生研究院 (15335 奖) 的支持, 目前在转移研究中得到了琼帕克奖学金的支持。P.H.S. 得到不列颠哥伦比亚省癌症基金会的支持。

获得影像数据的所有动物协议均经国家卫生研究院国家癌症研究所动物护理和使用委员会批准。在文章视频中讨论和描述的所有动物协议都已被英属哥伦比亚大学动物保育委员会批准。
1. 注射用肿瘤细胞和美洲狮模型材料的制备
注意: 对于1鼠标, 解决方案和单元格的数量将足够。如果在研究中使用更多的小鼠, 就需要扩大。对于媒体食谱, 请参阅表 1和表 2。
<ol><li>在 37 oC 水浴中, 在15毫升锥形管中预热5毫升的 a 介质。</li>
	<li>使用实验室微波熔融1.2% 低熔点琼脂糖溶液 (在无菌水中)。</li>
	<li>将5毫升的熔融琼脂糖转化为15毫升锥形管, 并在 37 oC 水浴中保持温暖。确保融化的琼脂糖是在 37 oC 和液体之前, 肺喷洒步骤。</li>
	<li>预冷30毫升细胞培养级 PBS 补充与1X 笔/链球菌在一个冰桶。</li>
	<li>在6井板的一个井中, 预浸泡1.5 毫升 B 介质中的明胶海绵。海绵将是肺切片的支撑锚。</li>
	<li>确保操作系统单元格在过程的当天大约70-90% 汇合。不要使用过度汇合的细胞, 或者如果媒体是橙色到黄色, 因为细胞通常被强调并且在这一点上降低了生存能力。对于骨肉瘤细胞系, MNNG 和居屋, 5 x 105在一卷 100 ul 将被用来注入到尾部静脉的1鼠标。因此, 如果更多的老鼠被用于研究, 那么高档。</li>
	<li>用0.25% 胰蛋白酶-EDTA (3 毫升的 T75 烧瓶或2毫升的10厘米培养皿) 收割肿瘤细胞。当细胞开始从盘子中抬起时, 用完全的培养基中和胰蛋白酶-EDTA。旋转细胞和冲洗一次与细胞培养级 PBS。并用重悬颗粒在5毫升的 PBS。</li>
	<li>按标准方法执行单元格计数。最好是做更多的细胞悬浮比实际需要的, 因为细胞悬浮脱落往往发生在针的绘制和失败的注射尝试。</li>
	<li>对细胞悬浮的样品进行台盼蓝排斥试验, 以评估细胞的生存能力。仅当单元格显示90% 的生存能力或更高时才进行。</li>
	<li>在 100 ul 的卷中注入 5 x 105单元格。要准备超过2X 的这一量, 1 x 106细胞应该被旋转下来, 并悬浮在0.2 毫升的 HBSS 中。</li>
	<li>在准备注射的小鼠时, 将细胞悬浮在冰桶中。</li>
</ol>2. 尾静脉注射和肺喷洒
注意: 本节中的解决方案和单元格的数量将足够1鼠标。如果在研究中使用更多的小鼠, 就需要扩大。有关以下步骤中使用的设备、材料和外科工具的列表, 请参阅表 3和表 4。
<ol><li>温暖一只雌性老鼠 (年龄6-8 星期) 在热化灯之下为5分钟为了使他们的尾巴静脉更加明显地明显。对于小鼠 K7M2 或 K12 细胞, 使用 Balb/c 鼠;对于人类 MG63.3、MG63、MNNG 和居屋细胞, 使用严重的联合免疫缺陷小鼠。</li>
	<li>通过轻轻摇动管子, 确保细胞悬浮均匀。小心地将细胞悬浮在没有针头的1毫升注射器中。用27口径针把注射器盖上。确保针锥与针上的音量标记在同一侧。</li>
	<li>将鼠标放在抑制剂中, 用酒精擦拭拭尾。</li>
	<li>进行尾静脉注射, 并注射 100 ul 的细胞悬浮。注射后5分钟, 将鼠标放置在 CO2室中, 并开始安乐死标准操作程序 (如您的机构动物护理委员会的大纲)。颈椎脱位不应作为安乐死的手段, 因为手术会损害气管。</li>
	<li>一旦老鼠被安乐死, 开始准备的层流罩为喷洒的小鼠肺与琼脂糖/A 介质溶液。在层流罩内, 设置一个工作区与您的消毒垫。在这个垫子上, 你将放置你的消毒仪器, iv 导管, iv 延伸集, 和重力灌注装置 (参见补充图 1)。</li>
	<li>把鼠标放在背 recumbancy。用无菌小剪刀, 仔细解剖出胸骨, 露出胸腔。注意不要刺穿肺部, 因为琼脂糖/A 介质溶液将被用来 insufflate 肺。</li>
	<li>解剖胸骨时, 解剖过胸口两侧的气管。通过解剖周围的软组织来暴露气管。</li>
	<li>用20口径静脉注射导管 Cannulate 气管。用无菌线缝合在空心气管周围的外科结。</li>
	<li>将 IV 延伸集从插管气管连接到重力灌注装置的10毫升注射器。</li>
	<li>将预热后的 37 oC 琼脂糖 (5 毫升) 和 a 介质 (5 毫升) 组合成1:1 混合物。将液体琼脂糖/A 介质溶液倒入重力灌注装置的10毫升注射器中。在喷洒肺前用琼脂糖/a 介质溶液, 确保延长集的整个长度已被琼脂糖/a 介质, 从而否定无意喷洒肺标本与空气。</li>
	<li>填充肺琼脂糖/A 介质溶液, 直到肺完全 insufflated。</li>
	<li>一旦肺完全 insufflated, 取出套管并牢固地栓在手术结上, 防止 agarase/A 介质溶液通过气管渗漏。</li>
	<li>从胸腔切开 (气管、心脏和肺部)。注意不要刺穿或损坏肺部的表面。</li>
	<li>将采摘 (在没有特定方向) 在预冷30毫升的 PBS 补充与1X 笔/链球菌, 并允许琼脂糖/A 介质固化20分钟。</li>
	<li>使用细剪刀和镊子, 切割小块的肺 (3 毫米 x 1.5 毫米), 如图 1A所示。更小的肺切片可以很容易地成像使用2.5X 目标。每个实验条件下可以切割多个切片, 每个组通常为4-10 切片。 注: 对于每个实验组, 将肺切片放入一个单独的井 (6 井板) 中, 其中含有 2 x 2 厘米的明胶海绵, 预浸泡在 B 介质中。每个井的介质数量应该是1.5 毫升。每2-3 天更换一次媒体。对于药物研究, 改变媒介/药物的频率是用户确定的。</li>
</ol>3. 肺切片的 Widefield 荧光成像及分析
注: 对于 widefield 荧光成像, 更小的切片被切割 (3 毫米 x 1.5 毫米 x 1 毫米), 以适合肺部分成1图像使用2.5X 的目标。
widefield 荧光显微镜上的图像采集:
<ol><li>肺部切片通常在注射后0、3、7和14天进行成像。在生物橱柜的无菌环境中, 小心地将肺切片从明胶海绵转移到无菌的35毫米玻璃底圆碟。注意从肺部切片中抹去多余的液体, 因为多余的液体会作为镜子, 反射来自肿瘤细胞的荧光光。</li>
	<li>按图 1A中描述的类似方式排列肺切片。每一列将代表不同的实验条件。小心不要用镊子把肺部弄脏。在处理另一实验组的肺切片前, 用70% 乙醇冲洗并干燥。</li>
	<li>优化成像参数 (即增益, 偏移, 曝光时间, binning), 提供最佳对比的荧光肿瘤细胞和背景肺组织在控制 (车辆未经治疗) 组。使用控制组中的相同参数对实验组进行图像处理。另存为 tiff 图像格式。</li>
	<li>对于刻度引用, 请在同一目标上以千分尺的数字图像。</li>
	<li>随着时间的推移, 肺部自动荧光的变化, 成像参数必须调整到控制肺部每一个成像会话。一个会话的成像参数不一定是后续映像会话的最佳选择。</li>
</ol>图像分析: 注意: 下面的图像处理步骤是使用 ImageJ 1.51h 软件包17完成的。
<ol><li>在 ImageJ 中打开图像文件 (图 3A)。</li>
	<li>减去背景: 过程 > 减去背景 > 滚动球半径 (从50像素开始), 取消选中 "浅色背景" (图 3B)。</li>
	<li>将图像转换为8位格式: 图像 > 类型 > 8 位 (图 3C)。</li>
	<li>将单位设置为像素: 图像 > 在长度单位中输入 "像素", 在 "像素宽度"、"像素高度"、"体素深度" 中输入1。选中 "全局" 框可应用于后续图像。</li>
	<li>使用多边形选择工具, 勾勒出整个肺切片的形状, 并确定总肺切片的面积 (像素2)。这个值将用于计算肺的肿瘤负担百分比。</li>
	<li>阈值图像: 图像 > 调整 > 阈值 > 突出显示 "默认" 和 "B & W" > 使用滑块阈值的图像, 使大多数肿瘤细胞被准确地强调。按下 "应用" 将导致黑色和白色的图像, 其中肺全是黑色的, 荧光损伤是白色的 (图 3D)。第二次按下 "应用" 将反转所有荧光结构现在为黑色形状的图像 (图 3E)。</li>
	<li>病变数量和形状的量化: 分析 > 设置测量 > 检查 "区域"。取消选中所有其他框。 分析粒子 > 大小 (像素2): 0-无穷大表示 ImageJ 将枚举的形状范围。经验主义地确定最小的病变, 被认为是一个单一的肿瘤细胞在0天的车辆图像。使用此单个肿瘤细胞的面积作为数据集中剩余图像的下限。对于本文所介绍的工作, 11 像素2设置为下限。对于视频示例中显示的工作, 24 像素2设置为下限。将 "循环" 范围保留为0-1。"显示" 可以设置为 "无"。或者, 如果选择了 "显示" 和 "轮廓", 则会生成所有轮廓形状的绘图。检查 "显示结果" 以单独的测量窗口弹出。如果选中 "添加到管理器" 复选框, 则会将所有量化的形状保存到 ROI 管理器中, 这可以保存以供将来参考。按 OK 后, 将弹出一个 "结果" 窗口, 其中包含所有枚举的形状和区域度量 (图 3F)。</li>
	<li>将数据从 "结果" 窗口复制并粘贴到电子表格中。使用 Excel 中的 sum 数学函数对该特定肺切片的转移病灶的所有区域求和。目的: 评价肺切片肺肿瘤负荷的百分比, 将转移性病变面积与肺切片总面积的总和分开。此参数也称为区域分数 (a), 由安德伍德18进一步描述。 肺肿瘤负担 = 转移病灶面积总和/肺切片总面积</li>
	<li>计算对照组和其余组肺部分的肺肿瘤负担。绘制每组 0, 3, 7 和14天的平均肺肿瘤负担。代表 widefield 荧光图片的高和低转移人 OS 细胞生长在美洲狮模型在渐进时间点显示 (图 4A)。显示的折线图显示的褶皱变化 (规范化到0天) 在转移肿瘤负担的百分比随着时间的推移, 表明 (图 4B)。</li>
</ol>4. 美洲狮模型的共焦荧光成像
注意: 对于共焦成像, 组织处理类似于前一节, 除了更大的肺切片被切割 (完整的横断面, 1-2 毫米厚), 以允许更多的 ROIs 被成像。
DAR4M 肺实质标签:
<ol><li>浸泡肺部切片在10μμM 的 DAR4M (在 HBSS) 45 分钟37摄氏度。DAR4M 在肺细胞内标记活性氮种类。</li>
	<li>45分钟的孵化后, 用新鲜的 HBSS 冲洗肺部切片。</li>
	<li>将肺切片放置在35毫米的玻璃杯底圆碟上, 并在共焦显微镜上进行图像处理。</li>
	<li>图 5中显示的示例图像的图像参数列在表 5中。</li>
	<li>为感兴趣的区域获取 Z 堆栈。使用建议的 Z 切片厚度进行采样。影片 1和补充影片 1中提供了一个显示 DAR4M-labeled 肺组织中绿色荧光 OS 单元格的3D 堆栈的代表性影片。</li>
</ol>对于图 5中显示的共焦图像示例, 成像会话是终端。如果需要纵向成像, 则建议使用2光子或多光子设备的共聚焦 LSM 显微镜进行成像, 因为活组织的光损伤少了 19, 20. 在美洲狮模型中表达 MG63 细胞的 RFP:
<ol><li>在步骤1和2中使用 MG63 细胞表达美图-RFP 结构, 执行美洲狮协议作为大纲。</li>
	<li>将肺切片放置在35毫米的玻璃杯底圆碟上, 并在共焦显微镜上进行图像处理。</li>
	<li>图 6中显示的示例图像的图像参数列在表 6中。影片 2和补充影片 2中提供了一个具有红色荧光线粒体的绿色荧光 OS 单元格的3D 堆栈的代表性影片。</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+spread+and+micrometastasis+development:+quantitative+approaches+for+in+vivo+models.">Cancer spread and micrometastasis development: quantitative approaches for in vivo models.</a> Bioessays. 24 (10), 885-893 (2002).</li><li>Entenberg, 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=A+permanent+window+for+the+murine+lung+enables+high-resolution+imaging+of+cancer+metastasis.">A permanent window for the murine lung enables high-resolution imaging of cancer metastasis.</a> Nat Methods. 15 (1), 73-80 (2018).</li><li>Kim, 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=Quantification+of+cancer+cell+extravasation+in+vivo.">Quantification of cancer cell extravasation in vivo.</a> Nat Protoc. 11 (5), 937-948 (2016).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Underwood, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantitative stereology. , (1970).</li><li>Tanaka, 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=In+vivo+optical+imaging+of+cancer+metastasis+using+multiphoton+microscopy:+a+short+review.">In vivo optical imaging of cancer metastasis using multiphoton microscopy: a short review.</a> Am J Transl Res. 6 (3), 179-187 (2014).</li><li>Prouty, A. M., Wu, J., Lin, D. T., Camacho, P., Lechleiter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+laser+scanning+microscopy+as+a+tool+for+Xenopus+oocyte+research.">Multiphoton laser scanning microscopy as a tool for Xenopus oocyte research.</a> Methods Mol Biol. 322, 87-101 (2006).</li><li>Bijgaart, R. J., Kong, N., Maynard, C., Plaks, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+Live+Imaging+of+Lung+Metastasis+and+Their+Microenvironment.">Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment.</a> J Vis Exp. (108), e53741 (2016).</li><li>Guha, 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=Mitochondrial+retrograde+signaling+induces+epithelial-mesenchymal+transition+and+generates+breast+cancer+stem+cells.">Mitochondrial retrograde signaling induces epithelial-mesenchymal transition and generates breast cancer stem cells.</a> Oncogene. 33 (45), 5238-5250 (2014).</li><li>Ren, L., Morrow, J. 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=+Positively+selected+enhancer+elements+endow+osteosarcoma+cells+with+metastatic+competence."> Positively selected enhancer elements endow osteosarcoma cells with metastatic competence.</a> . Nat Med. , (2018).</li><li>Ren, L., 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=+Metabolomics+uncovers+a+link+between+inositol+metabolism+and+osteosarcoma+metastasis."> Metabolomics uncovers a link between inositol metabolism and osteosarcoma metastasis.</a> Oncotarget. 8 (24), 38541-38553 (2017).</li><li>Ren, L., 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=Characterization+of+the+metastatic+phenotype+of+a+panel+of+established+osteosarcoma+cells.">Characterization of the metastatic phenotype of a panel of established osteosarcoma cells.</a> Oncotarget. 6 (30), 29469-29481 (2015).</li></ol>

作者没有什么可透露的。

以下技术文章描述了美洲狮模型在研究 OS 中肺定植的一些实用方面。在《议定书》中, 研究人员应格外注意的一些关键步骤包括以下几个方面:
a) 气管插管。气管在解剖周围的肌肉和结缔组织时容易受损。此外, 导管针可以很容易地通过气管。在插入套管时要密切注意针的斜角如何进入气管。
b) 刺穿或损害肺部的表面。肺可以很容易刺穿或损坏的解剖剪刀...

改善儿童转移性骨肉瘤 (OS) 患者的预后仍然是一个关键的未满足的临床需要1。这突出了发展新的分子靶向治疗的重要性。常规化疗靶向肿瘤细胞增殖没有证明是有效的治疗转移性疾病, 因此, 新的战略必须针对转移过程本身2。本文讨论了一种相对较新型的体外肺转移模型、门多萨和同事 3开发的肺转移试验 (彪马) 的实际应用, 为发现新的操作系统中肺转移进展的分子驱动程序4,5。然而, 在继续之前, 应该谨慎地简单地接触几种目前的转移模型, 以及美洲狮模型如何比传统的体外检测提供了一些优势。
大多数用于研究转移的实验模型包括体外和体内系统, 它概括了转移级联的特定步骤或几个步骤。这些步骤包括: 1) 肿瘤细胞从原发肿瘤转移到附近的血管 (血液或淋巴) 和转运内的中转, 3) 在第二部位的拘捕, 4) 在次生部位的渗出和存活, 5) 形成淋巴结和 6) 成长为血管化转移 (图 1)。体外模型的转移可以包括2维 (2D) 迁移和3维 (3D) 胶入侵检测, 并在其他地方详细审查6。对于体内模型, 两种常用的模型系统包括: 1)自发性转移模型是将肿瘤细胞原位注射到特定组织类型中, 形成局部肿瘤, 自发地脱落转移细胞。到遥远的地点;2)实验性转移模型是将肿瘤细胞注入靶器官上游血管的地方。例如, 肿瘤细胞的尾静脉注射导致发展肺转移瘤5,7,8。其他实验性转移模型包括将肿瘤细胞注入脾脏或肠系膜静脉, 导致肝转移的发展9,10。韦尔奇11详细讨论了这些体内模型的实际注意事项。另一个用于研究小儿肉瘤转移的体内模型是肾肾包膜下肿瘤植入模型, 它导致局部肿瘤形成和自发转移到肺部12, 13.更技术上苛刻的技术, 如活体 videomicroscopy 可以直接可视化, 实时, 转移癌细胞与转移部位 (即肺或肝脏) 的微血管的相互作用, 如麦克唐纳14和 Entenberg15, 或胚膜中的癌细胞渗出, 如 Kim 16所述。
美洲狮模型是一个前体,肺组织外植体, 封闭培养系统, 其中荧光肿瘤细胞的生长可以通过荧光显微镜在一个月内纵向观察 (见图 2A)。该模型概括肺定植的初始阶段 (步骤3至 5) 在转移级联。美洲狮模型优于传统的体外模型的一些主要优势是: 1) 它提供了一个机会, 纵向测量转移癌细胞生长在3D 微环境中, 保留许多特点的肺部微环境在体内 3;2) 美洲狮允许研究人员评估在3D 肺微环境下, 候选基因或药物治疗的击倒是否具有抗转移活性;3) 美洲狮模型具有多种类型的荧光显微平台 (图 2B) 的灵活性, 如 widefield 荧光显微镜或激光扫描共焦显微术, 其中的示例显示在图 2C & D中,分别.本文将讨论如何使用美洲狮模型获得的纵向成像数据的转移生长的增强绿色荧光蛋白 (eGFP) 表达, 人高和低转移性骨肉瘤细胞 (MNNG 和居屋细胞, 分别) 使用低放大 widefield 荧光。用共聚焦激光扫描显微镜对美洲狮模型中的线粒体进行标记的荧光染料的成像实例, 并对其进行了研究。

<table><tbody><tr><td>&lt;strong&gt;Table 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell culture reagents for A-media, B-media, and complete media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MNNG-HOS</td><td>ATCC</td><td>CRL-1547</td><td>highly metastatic OS cell line</td></tr><tr><td>HOS</td><td>ATCC</td><td>CRL-1543</td><td>poorly metastatic OS cell line</td></tr><tr><td>MG63.3</td><td>Amy LeBlanc Laboratory (NCI)</td><td>N/A</td><td>highly metastatic OS cell line</td></tr><tr><td>MG63</td><td>ATCC</td><td>CRL-1427</td><td>poorly metastatic OS cell line</td></tr><tr><td>10X M199 media</td><td>Thermofisher</td><td>11825015</td><td>Base media for A-media and B-media</td></tr><tr><td>Distilled Water (sterilized)</td><td>Thermofisher</td><td>15230-147</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>7.5% sodium bicarbonate solution</td><td>Thermofisher</td><td>25080094</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>Hydrocortizone</td><td>Sigma-Alrich</td><td>H6909</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>Retinol acetate-water soluable</td><td>Sigma-Alrich</td><td>R0635-5MG</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>Penicillin/Streptomycin 10X concentrated (10000 U/ml) solution</td><td>Thermofisher</td><td>15140122</td><td>Component of A-media &amp;amp; B-media, complete media.</td></tr><tr><td>Bovine insulin solution (10mg/ml)</td><td>Sigma-Alrich</td><td>I0516-5ML</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>DMEM, high glucose</td><td>Thermofisher</td><td>11965092</td><td>Base media of Complete Media</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Thermofisher</td><td>25030081</td><td>Component of Complete Media</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher</td><td>16000044</td><td>Component of Complete Media</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Thermofisher</td><td>14190144</td><td>Used in cell culture.</td></tr><tr><td>Hank&amp;rsquo;s Buffered Salts Solution, no calcium, no magnesium, no phenol red</td><td>Thermofisher</td><td>14175095</td><td>Used to resuspend cell pellet prior to injection</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermofisher</td><td>25200114</td><td>Used in cell culture.</td></tr><tr><td>DAR4M</td><td>Enzo</td><td>ALX-620-069-M001</td><td>Used to label lung parenchyma.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Table 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials for PuMA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Zeiss 710 Confocal LSM</td><td>Zeiss</td><td>N/A</td><td>Upright LSM confocal microscope</td></tr><tr><td>Zeiss 780 Confocal LSM</td><td>Zeiss</td><td>N/A</td><td>Inverted LSM confocal microscope</td></tr><tr><td>SCID mice</td><td>Charles River</td><td>N/A</td><td>NOD.CB17-Prkdcscid/NcrCrl, female, age 6-8 weeks</td></tr><tr><td>GelFoam</td><td>Harvard Apparatus</td><td>59-9863</td><td>Used as a support for lung tissue sections.</td></tr><tr><td>SeaPlaque Agarose</td><td>Lonza</td><td>50100</td><td>Used during insufflation of the lung.</td></tr><tr><td>1 ml syringe with 27 gauge needle</td><td>Fisherscientific</td><td>14-826-87</td><td>Used for tail vein injection.</td></tr><tr><td>10 ml syringe</td><td>BD</td><td>309604</td><td>Used for insufflation of the lung.</td></tr><tr><td>20 gauge catheter</td><td>Terumo</td><td>SR-OX2032CA</td><td>Used during insufflation of the lung.</td></tr><tr><td>Abbott IV extension set (30&quot;, Sterile)</td><td>Medisca</td><td>8342</td><td>Used during insufflation of the lung.</td></tr><tr><td>Alcohol swabs</td><td>BD</td><td>326895</td><td>For wiping tail vein before injection</td></tr><tr><td>Sterile surgical gloves</td><td>Fisherscientific</td><td>Varies with size</td><td>Asceptic handing of mouse lungs</td></tr><tr><td>30 cm ruler</td><td>Staples</td><td></td><td>Used for insufflation of the lung.</td></tr><tr><td>Support stand for ruler</td><td>Pipette.com</td><td>HS29022A</td><td>Used for insufflation of the lung.</td></tr><tr><td>35 mm glass-bottomed culture dish</td><td>Ibidi</td><td>81158</td><td>Used during imaging of lung slices</td></tr><tr><td>Absorbent Underpads with Waterproof Moisture Barrier</td><td>VWR</td><td>56617-014</td><td>Used to line the sterile work area in the biological hood.</td></tr><tr><td>Catgut Plain Absorbable Suture</td><td>Braun</td><td>N/A</td><td>Used to tie off cannulated trachea.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Table 4&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Surgical instruments for PuMA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micro Dissecting Scissors 3.5&quot; Straight Sharp/Sharp</td><td>Roboz</td><td>RS-5910</td><td>For cutting lung sections</td></tr><tr><td>4&amp;rdquo; (10 cm) Long Serrated Straight Extra Delicate 0.5mm Tip</td><td>Roboz</td><td>RS-5132</td><td>For manipulating/holding lung sections.</td></tr><tr><td>4&amp;rdquo; (10 cm) Long Serrated Slight Curve 0.8mm Tip</td><td>Roboz</td><td>RS5135</td><td>For manipulating/holding lung sections.</td></tr><tr><td>Thumb Dressing Forceps; Serrated; Delicate; 4.5&quot; Length; 1.3 mm Tip Width</td><td>Roboz</td><td>RS-8120</td><td>For general dissection.</td></tr><tr><td>Thumb Dressing Forceps 4.5&quot; Serrated 2.2 mm Tip Width</td><td>Roboz</td><td>RS-8100</td><td>For general dissection.</td></tr><tr><td>Extra Fine Micro Dissecting Scissors 3.5&quot; Straight Sharp/Sharp, 20mm blade</td><td>Roboz</td><td>RS-5880</td><td>For general dissection.</td></tr><tr><td>Knapp Scissors; Straight; Sharp-Blunt; 27mm Blade Length; 4&quot; Overall Length</td><td>Roboz</td><td>RS-5960</td><td>For general dissection.</td></tr></tbody></table>

practical considerations in studying metastatic lung colonization in osteosarcoma using the pulmonary metastasis assay

肺转移试验 (彪马) 是一种体外肺外植体和封闭细胞培养系统, 允许研究人员通过荧光显微镜研究骨肉瘤 (OS) 的肺定植生物学. 本文详细描述了该协议, 并讨论了使用 widefield 或共聚焦荧光显微镜平台获取转移生长图像数据的示例。美洲狮模型的灵活性使研究人员不仅可以研究在肺部微环境中的 OS 细胞的生长, 而且还可以评估随着时间推移抗转移治疗的效果。共焦显微镜允许前所未有的高分辨率成像的 OS 细胞相互作用的肺实质。此外, 当美洲狮模型与荧光染料或荧光蛋白基因记者相结合时, 研究人员可以对转移的 OS 细胞的肺微环境、细胞和亚型结构、基因功能和启动子活动进行分析。美洲狮模型提供了一个新的工具, 骨肉瘤研究人员发现新的转移生物学和评估活动的新型抗转移, 靶向治疗。

低放大 widefield 荧光显微术
对于美洲狮肺切片的 widefield 荧光显微镜, 有代表性的图像和量化数据显示在图 2C中,图 4A和B。转移倾向为高和低转移的细胞线在视觉上是明显的在进步时间点。MNNG 细胞可以有效地对肺组织进行殖民, 而居屋细胞根本不能生?...

Watch this Scientific Journal Video about Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay at JoVE.com

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

本文的目的是为肺转移试验 (美洲狮) 的协议提供详细的描述。该模型允许研究人员使用 widefield 荧光或共聚焦激光扫描显微镜研究肺组织中的转移性骨肉瘤 (OS) 细胞生长。

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 ...

practical-considerations-studying-metastatic-lung-colonization

Research

JoVE Journal

Cancer Research

10.0K 次观看.  National Institutes of Health. 本文的目的是为肺转移试验 (美洲狮) 的协议提供详细的描述。该模型允许研究人员使用 widefield 荧光或共聚焦激光扫描显微镜研究肺组织中的转移性骨肉瘤 (OS) 细胞生长。

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

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

实验转移的含量

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

医学

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进<em&gt;在原位</em灌注肺组织和X-gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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 ...

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

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

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 ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...