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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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, 而删除胸骨和暴露胸腔。解剖取出胸腔的拔牙时要小心。穿刺或损害肺部的表面会导致液体琼脂糖/A 介质泄漏, 肺部会被充气。
c) 在成像时删除肺部部分的多余介质.在显微镜下观察到肺部部分周围的过量液体介质会引起 "镜面" 效应。过量的液体可以反射来自肿瘤细胞的荧光光, 从而夸大图像中的荧光区域。去除多余的介质流体将会阻止图像中的此伪影。或者, 图像工件可以在图像的后期处理过程中被删除。
d) 光损伤的生物组织。当使用1光子显微镜的水银灯泡或激光器时, 一定要限制暴露在光线下的时间。光源的强度/功率百分比也可以降低。过度暴露于光源可能导致光损伤肺组织。配备发光二极管 (LED) 或2光子/多光子显微镜的显微镜是理想的, 因为它们在纵向成像研究中产生的光损伤较少。
美洲狮模型可以适应和修改, 以研究许多方面的肺部殖民化过程。此前体方法的另一个变体由 van Bijgaart 和同事21描述。在研究基因击倒或抗转移药物活性对肺肿瘤细胞生长的影响时, 建议从 5 x 105到 3 x 105中插入的单元格数, 因为可以屏蔽干预效果在大细胞 innoculum 的指数生长过程中。一些研究已经使用美洲狮模型研究肺转移进展的驱动程序4,5,8,22,23,24。各种荧光指示器染料或荧光报告基因可以用来标记肿瘤细胞, 以确定肺微环境中细胞生理学或基因表达8的变化。
美洲狮模型的一个局限性包括有限数量的兼容细胞系。与此检测兼容的已建立单元格线由门多萨和同事3列出。对于高、低转移性骨肉瘤细胞系, 无性相关细胞系的跟随对在美洲狮模型中生长并维持其转移倾向: 人 MG63.3 & MG63 细胞、MNNG & 143B、居屋细胞、小鼠 K7M2 和 K12细胞.研究人员必须通过经验主义的判断他们的细胞系是否能在 B 型介质中保持存活。另一个需要考虑的限制是肺组织可以保持体外的有限的时间长度。肺组织可以保留它的细胞成分30天, 在那以后, 并且肺的细胞组分开始死。因此, 研究肿瘤细胞与肺基质细胞之间的相互作用不应超过30天。
美洲狮模型提供了一种前所未有的方法来研究转移的 OS 细胞如何殖民肺组织。美洲狮模型最引人注目的方面来自以下事实: 几个低转移的 OS 细胞线 (居屋, MG63, K12), 其在体内表型在其他地方的特点25, 不能生长在美洲狮模型。相比之下, 无性相关的高度转移性 OS 细胞 (MNNG、MG63.3、K7M2) 更倾向于将肺组织体内25和美洲狮模型殖民。这表明, 尽管缺乏血液流动, 但在体内, 防止低转移的 OS 细胞株的肺微环境的细胞和细胞外成分在生长的过程中仍然保持在美洲狮模型中。换言之, 美洲狮模型的肺微环境仍然施加 "选择压力", 防止低转移的 OS 细胞在肺中生长。事实上, 研究高转移性的 OS 细胞生长在美洲狮是有用的, 以确定新的分子驱动因素转移的表型4,5。此外, 在高度转移的细胞中, 对上述靶点的遗传降调可以减少美洲狮模型和体内5的转移能力。对于候选的抗转移药物研究, 美洲狮模型可以用来确定哪些浓度范围可以减少肺组织中的转移性生长, 进而可以验证在体内。
该模型的未来应用应利用过量的商业可用的荧光染料和报告基因, 以深入研究的基础生物学的 OS 转移进展。例如, 2 '、7 '-dichlorofluorescin 双乙酸或 dihydroethidium 等荧光染料可以用来评估美洲狮模型中肿瘤细胞的氧化还原状态。荧光报告基因可以用来研究细胞生物学或评估启动者活动, 以确定哪些信号通路在高度转移的 OS 细胞在殖民过程中激活。这种方法可以用来可视化药物治疗是否有活动的肿瘤细胞在肺部生长。荧光显微镜平台, 产生较少的光损伤组织, 如 LED 设备显微镜或2光子/多光子共聚焦显微镜, 可用于研究如何转移的 OS 细胞迁移和侵入整个肺组织。此外, 肿瘤细胞与肺基质细胞之间的黏附相互作用可以通过荧光报告基因进行监测。
总结一下, 本文讨论了美洲狮模型的实用方面, 首先由门多萨和同事3开发。以低放大、widefield 荧光显微镜和高分辨率共焦显微镜为例, 评估高、低转移人 OS 细胞的生长情况。已经说明了议定书的几个关键步骤。此外, 还讨论了美洲狮模型的优点和局限性。本文提出了美洲狮模型在研究肺定植过程中的应用, 希望该模型能在骨肉瘤研究界得到更广泛的应用。

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>Table 2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture reagents for A-media, B-media, and complete media</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 &#38; B-media</td>
		</tr>
		<tr>
			<td>7.5% sodium bicarbonate solution</td>
			<td>Thermofisher</td>
			<td>25080094</td>
			<td>Component of A-media &#38; B-media</td>
		</tr>
		<tr>
			<td>Hydrocortizone</td>
			<td>Sigma-Alrich</td>
			<td>H6909</td>
			<td>Component of A-media &#38; B-media</td>
		</tr>
		<tr>
			<td>Retinol acetate-water soluable</td>
			<td>Sigma-Alrich</td>
			<td>R0635-5MG</td>
			<td>Component of A-media &#38; 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 &#38; 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 &#38; 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&#8217;s Phosphate Buffered Saline</td>
			<td>Thermofisher</td>
			<td>14190144</td>
			<td>Used in cell culture.</td>
		</tr>
		<tr>
			<td>Hank&#8217;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Table 3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for PuMA</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&#34;, 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Table 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments for PuMA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors 3.5&#34; Straight Sharp/Sharp</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td>For cutting lung sections</td>
		</tr>
		<tr>
			<td>4&#8221; (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&#8221; (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&#34; 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&#34; 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&#34; 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&#34; 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。

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

Nanyang Technological University

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

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

肝大肠癌转移的高级动物模型:成像技术和转移性克隆性质

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

科研

癌症研究

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

pT3N0M0 胃癌组织石蜡嵌入幻灯片上的弹性染色

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

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 ...

Perfusion and Inflation of the Mouse Lung for Tumor Histology

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform necropsies rapidly and efficiently from studies without sacrificing the quality of the tissues procured. The goal of this protocol is to present a method to rapidly perfuse, inflate, and fix mouse lungs for downstream histological analysis. This method does not standardize lung inflation; thus, it does not require any special procedures or equipment and instead simply instills fixative directly through the trachea following perfusion through the heart. This allows for sufficient estimation of tumor size, histology, and scoring. This also allows for the collection of frozen tissue prior to lung tissue fixation. This method is limited in that it does not allow for later morphometric quantification of the lung; however, it is more than sufficient for lung tumor analysis from genetically engineered mouse models (GEMMs), syngeneic models, as well as xenograft tumor and metastasis studies.

The purpose of this method is to present a simple and efficient method for the perfusion, inflation, and fixation of mouse lungs for the examination of lung tumor pathology and evaluation of metastases to the lung.

肿瘤病理学鼠肺的灌注和膨胀

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

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 ...

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. ...

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 ...