作者希望感谢明敏博士和雅安女士的技术支持。这项工作得到了台湾科技部 (103-2325-006-009, 最 104-2325 b-006-001, 最 105-2325-006-001, MOST-106-2320-B-006-068-MY3) 和卫生部 (MOHW106-TDU-B-211-144004) 的支持。我们也感谢国立中诚大学医学院核心研究实验室对其多光子共聚焦显微镜的支持。

所有老鼠的实验都是按照我研究所的指导方针 (NCKU 医学院的实验室动物护理和使用指南) 进行的。
1. 仪器、文化媒体和菜肴的准备
<ol><li>在启动实验协议之前, 获得无菌手术缝合, 1 毫升注射器与26克/0.5 厘米针 (用于尾静脉注射), 3 毫升注射器与24克/3 厘米针 (为肺灌注), Dulbecco 的改良鹰媒体 (DMEM), 胰蛋白酶-EDTA 溶液 (5胰蛋白酶和0.53 毫米), 胎牛血清 (血清), 1x PBS (137 毫米氯化钠, 2.7 毫米氯化钾, 10 毫米 Na2米 4, 和1.8 毫米, 2 米 4),和6厘米的文化菜肴.</li>
</ol>2. 悬浮液中肿瘤细胞的制备和恢复
<ol><li>准备无菌 Dulbecco 的改良鹰培养基 (DMEM), 含有10% 或20% 的血清和新鲜添加2毫米 l-谷氨酰胺, 1x PBS 和0.05% 胰蛋白酶-EDTA。</li>
	<li>培养细胞在 DMEM 补充与10% 的血清37摄氏度, 并种植它们, 直到有70-80% 融合在养殖皿。</li>
	<li>从菜肴中取出培养基 (DMEM), 然后用2毫升无菌 1x PBS 冲洗两次。</li>
	<li>加入1毫升的0.05% 胰蛋白酶-EDTA 进入菜肴, 彻底覆盖所有的细胞。立即删除 800 ul 的解决方案, 只留下 200 ul 在盘子里。在37摄氏度的三十年代至1分钟孵化菜肴 (视细胞类型而定), 等到大多数细胞处于悬浮状态。</li>
	<li>加入1毫升新鲜 DMEM, 其中10% 只血清直接进入菜肴, 以停止胰蛋白酶的酶活性, 并大力用1000µL 吸管将细胞悬浮在一起, 以制备单个细胞悬浮液。</li>
	<li>将细胞从盘子转移到一个1.5 毫升的离心管, 并在室温下以 162 x g 为3分钟旋转细胞。</li>
	<li>在1.5 毫升 DMEM 中吸出上清和并用重悬肿瘤细胞, 其中20% 只血清和端到端将悬浮液中的细胞以37°c 的2小时旋转。 注: 如果培养基在恢复过程中变黄, 则用含有20% 血清的新鲜培养基交换旧培养基。</li>
</ol>3. 羧基琥珀酰亚胺酯 (CFSE) 悬浮液中肿瘤细胞的荧光染色和分析
<ol><li>在悬浮细胞恢复后, 在 Neubauer 计数室中, 用台盼蓝计数合适的存活细胞数, 用于滴定荧光染色剂量和尾静脉注射。</li>
	<li>在室温下将 162 x g 的细胞向下旋转3分钟, 并用重悬1毫升灭菌的 1x PBS 中的颗粒细胞, 其次是室温离心, 162 x g 为3分钟。</li>
	<li>并用重悬500µL 中的颗粒细胞, 含有10% 的血清和 0, 5, 10, 20, 或40µM CFSE 剂量滴定, 并孵化细胞悬浮在 37 oC 10 分钟在黑暗中。</li>
	<li>在室温下将 162 x g 的细胞向下旋转3分钟, 并用重悬 DMEM 4 毫升的颗粒细胞, 含有1% 的血清。重复这洗涤步骤三次。并用重悬的颗粒细胞与4毫升的 DMEM 单独为最终洗涤。</li>
	<li>CFSE 标记细胞进行荧光活化细胞分选 () 分析, 以量化单个细胞或尾静脉注射的荧光强度。 注: 在进行药物分析或尾静脉注射之前, 请将细胞放在冰上。</li>
</ol>4. 肺定植试验
<ol><li>准备标记有20µM CFSE 的单元格 (请参阅步骤3.1 到 3.4)。 注: 根据 CFSE 的滴定结果, 确定肿瘤细胞标签的工作剂量 (参照步骤 3.3)。</li>
	<li>在老鼠的基础上, 用一只小鼠的 C57BL/6, 用一盏卤素灯为5-10 分钟预热4-6 周大的雄性小鼠 (6 只老鼠) 的尾巴, 以扩张老鼠的尾脉。</li>
	<li>彻底混合和吸入 CFSE 标记的肿瘤细胞与1毫升 (26Gx1/2) 注射器, 避免任何气泡在细胞悬浮 (与最终细胞密度 5 x 106细胞/毫升)。</li>
	<li>仔细注射 1 x106 CFSE 标记肿瘤细胞200µL DMEM 到鼠标尾静脉, 应在鼠标抑制剂。 注: 在注射过程中, 如果针头直接放在尾静脉的腔内, 肿瘤细胞就应该很容易地传递到循环中, 而不需要用力推动注射器。如果很难推动肿瘤细胞通过, 它们很可能被注射到尾静脉外, 进入皮下空间。</li>
	<li>将静脉接受 CFSE 标记肿瘤细胞的小鼠分为两组: 一组在肺定植试验中采用共聚焦显微术和 ImageJ 定量法进行肺灌注, 另外一个将单独留在4-5周的长期肺定植试验。</li>
	<li>在 20, 38, 或 45 h 和与 1x 106肿瘤细胞静脉注射在时间路线-和剂量依赖性滴定, 麻醉瘤小鼠与50-75 毫克/千克 zoletil 50, 这是一个公认的和适当的 anesthetizer23。 注意: 在小鼠眼中使用兽医软膏, 以防止麻醉时的干燥。</li>
	<li>用手术剪刀从腹部纵向切开皮肤和皮下组织。用手术剪刀和镊子打开胸腔, 充分暴露心脏和肺部。 注意: 小心避免切断血管。</li>
	<li>用无菌手术缝线系上腔静脉 (SVC) 和下腔静脉, 防止肺灌注时灌注液回流。</li>
	<li>用手术剪刀切开左心室壁, 打开裂缝 (约2-4 毫米), 从肺部排出灌注液。</li>
	<li>用3毫升注射器将 1x PBS 注入右心室, 并在肺灌注过程中取出持续吸入的排泄液, 直到肺部从红色变为完全苍白。</li>
</ol>5. 肺定植肿瘤细胞的共焦显微成像与分析
<ol><li>从胸腔中取出现在苍白的肺部, 用手术剪刀和钳24切除气管和结缔组织韧带, 将五裂片分开。</li>
	<li>准备一个担架状的肺支架, 如图 3B所示, 通过围绕两个金属棒缠绕尼龙缝合, 以产生网状纹理, 在两个杆之间放置肺裂片的空间。把肺支架放在6厘米的盘子里。 注: 肺支架将用于稳定的肺裂片下方的目标透镜的共聚焦显微镜。</li>
	<li>将肺叶固定在肺支架的网状纹理上。用 1x PBS 覆盖并滋润肺裂片。</li>
	<li>主题的6厘米培养皿窝藏肺瓣在肺部持有者共聚焦显微镜捕捉荧光图像记录肺殖民肿瘤细胞。</li>
	<li>对于成像, 使用物镜 (5X, MPLN, NA: 0.1, WD:20, 空气)。</li>
	<li>旋转过滤轮到 NIBA (激发/发射; 470-495 nm/510-550 nm), 并使用 488 nm 激光激发 CFSE, 以清晰地可视化的荧光点图像的肺裂片。</li>
	<li>将过滤轮旋转到 R690 (用于埋汰 HP DeepSee 激光器) 以2.0 µs/像素扫描速度扫描, 并优化高压、增益和偏移电平。</li>
	<li>使用具有10µs/像素扫描速度的显微镜软件捕获荧光图像 (512 x 512 像素)。</li>
	<li>使用 ImageJ 和 GraphPad 软件对显微图像进行量化和统计分析, 如前所述21。</li>
</ol>6. 长期肺定植测定
注意: 重复步骤4.1 到4.5。
<ol><li>在肿瘤细胞接种4-5 周后牺牲老鼠, 用 Bouin 的液体25 1 到2天来修复肺部。</li>
	<li>用软件21对小鼠肺部肿瘤结节进行定量分析和统计分析。</li>
</ol>

作者没有什么可透露的。

结合长期的肺定植试验, 我们在这里使用的短期方法, 以评估在体肺殖民化的 CTCs 在遥远的器官清楚地揭示和区分 polyFN 的具体作用聚集在 CTCs 的殖民肺, 然后导致渗出和转移的过程18,19,20,21。虽然标记细胞与长期细胞追踪器 CFSE 允许我们跟踪静脉注射注射 CTCs 在肺灌注前长达三天, 并保留足够的?...

转移是癌症死亡的主要原因1,2。从初级组织中获得的肿瘤细胞进入悬浮循环, 并经受了各种血流挑战,例如脱落凋亡、免疫攻击和因血压或几何约束而造成的抗剪应力损伤, 在它们被能够殖民遥远的器官, 一个关键步骤, 成功转移3,4,5,6。因此, 目前正在大力研究循环肿瘤细胞 (CTCs), 并将这些特征与肿瘤恶性肿瘤、转移和癌症患者存活率7、8,9. 由于癌症转移的过程具体描述了活体活动, 动物模型是唯一的方法, 捕捉整个系统过程的转移10,11,12.
CTCs 成为转移性肿瘤组织通过多细胞事件包括1,2遥远的器官的殖民化。然而, 最常用的转移分析13,14,15没有提供一种方法来观察反恐委员会远距离器官的殖民化。因此, 迫切需要对反恐委员会的定植可视化进行体内检测设计。虽然在体内和体外短期肺定植试验的设计, 问题和缺点仍然存在。例如, 虽然绿色荧光蛋白 (GFP)-overexpressing 肿瘤细胞已用于这些化验22,23, 它需要时间稳定染和克隆肿瘤细胞与充分的 GFP 荧光强度下显微镜。同样, 虽然肿瘤细胞的瞬态染色与长期细胞示踪 CFSE 已被用于取代 GFP 表达的肿瘤细胞, 它仍然难以判断是否附有 CFSE 标记的肿瘤细胞或仅仅存在于切除的遥远的器官的脉管16,17。
在 CTCs 表面组装的高分子纤维连接蛋白 (polyFN) 已被发现对转移性肿瘤组织的最终建立有重要贡献18、19、20、21、22.在这里, 我们进行了短期肺定植试验, 其中悬浮 Lewis 肺癌细胞 (LLCs) 稳定表达 FN-shRNA (shFN) 或 shRNA (shScr) 和预先标记 CFSE 被静脉注射到 C57BL/6 小鼠。2-3 天后, 小鼠肺先灌入磷酸盐缓冲盐水 (PBS), 以完全去除血管内的未连接 CTCs, 然后再进行共焦显微术和定量的肺殖民 LLCs。我们清楚地表明, 与 shScr LLCs 相比, 肺殖民化 shFN LLCs 和肿瘤结节的数量明显减少, 大大证实 polyFN 在 CTCs 中的作用, 促进 CTCs 的殖民化和生长。肺。我们的研究需要进一步研究 polyFN 在肿瘤转移中的作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;(BSA)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#39;s Fluid</td>
			<td>MCC(medical chemical corporation)/POISON</td>
			<td>456-A-1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE Proliferation Dye</td>
			<td>ebiosciences</td>
			<td>65-0850-85</td>
			<td>Full name: Carboxyfluorescein succinimidyl ester</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media (DMEM)&#160;</td>
			<td>(Gibco)ThermoFisher Scientific</td>
			<td>12100-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-6381-92-6</td>
			<td>For prepared trypsin-EDTA solution( Final concentration: 0.53mM )&#160;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>(Gibco)ThermoFisher Scientific</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Lewis lung carcinoma (LLC)</td>
			<td>ATCC, Manassas, VA, USA</td>
			<td>CRL-1642</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine, USP&#160;</td>
			<td>(Gibco)ThermoFisher Scientific</td>
			<td>21051-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-7447-40-7</td>
			<td>For prepared 1X PBS ( Final concentration: 2.7mM )</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-7778-77-0</td>
			<td>For prepared 1X PBS ( Final concentration: 1.8mM )</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-7647-14-5</td>
			<td>For prepared 1X PBS ( Final concentration: 137mM )</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Cyrusbioscience (Taipei, Taiwan)</td>
			<td>101-10039-32-4</td>
			<td>For prepared 1X PBS ( Final concentration: 10mM )</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma Aldrich</td>
			<td>T6146</td>
			<td>0.5 g mix with 100 mL 1X PBS</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma Aldrich</td>
			<td>T4799</td>
			<td>For prepared trypsin-EDTA solution ( Final concentration: 5g/L )</td>
		</tr>
		<tr>
			<td>Zoletil 50</td>
			<td>Virbac</td>
			<td></td>
			<td>To dilute with 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Tabletop Centrifuge 2420</td>
			<td>KUBOTA Co.</td>
			<td>2420</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish (6cm)</td>
			<td>Wuxi NEST Biotechnology Co.</td>
			<td>705001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringe (with needle)</td>
			<td>Perfect Medical Industry Co.</td>
			<td></td>
			<td>24G/3 cm;3 ml &#38; 26G/0.5 cm;1 ml</td>
		</tr>
		<tr>
			<td>End over end mixer</td>
			<td>C.T.I YOUNG CHENN</td>
			<td>TS-20</td>
			<td>For suspended cells recovery&#160;</td>
		</tr>
		<tr>
			<td>FACSCalibur (FACS)</td>
			<td>BD biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dimeda</td>
			<td>10.102.14</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Direct Heat CO2 incubator</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>HEPA CLASS 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse restrainer (Cylindrical Restrainer 15-30 gm)</td>
			<td>Stoelting</td>
			<td>51338</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiphoton Confocal Microscope BX61WI</td>
			<td>Olympus</td>
			<td>FV1000MPE</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Marienfeld-Superior</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Dimeda</td>
			<td>08.370.11</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures&#160;</td>
			<td>UNIK SURGICAL SUTURES MFG. CO.</td>
			<td>NO. 0034</td>
			<td>Black Braided silk; non-absorbable (25YD; U.S.P. 4/0)</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Wuxi NEST Biotechnology Co.</td>
			<td>615001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Greiner tube</td>
			<td>Greiner bio-one</td>
			<td>188271</td>
			<td></td>
		</tr>
	</tbody>
</table>

the establishment of a lung colonization assay for circulating tumor cell visualization in lung tissues

转移是导致癌症死亡的主要原因。研究了循环肿瘤细胞 (CTCs) 在促进肿瘤转移中的作用, 其中 CTCs 肺定植对早期肺转移过程有重要影响。因此, 动物模型是唯一的方法, 捕捉整个系统的转移过程。鉴于以前的实验设计中出现了一些问题, 用于检查 CTCs 对血管渗出的贡献, 我们建立了一种体内肺定植试验, 其中长期荧光细胞示踪剂, 羧基琥珀酰亚胺酯 (CFSE), 用于标记悬浮瘤细胞和肺灌注被执行清除非特别被困的 CTCs 在肺切除之前, 共焦成像和定量。聚合纤维连接蛋白 (polyFN) 组装在反恐委员会的表面已经发现, 以调解肺的殖民化最终建立的转移性肿瘤组织。在这里, 为了具体测试 polyFN 组装对 CTCs 肺定植和渗出的要求, 我们进行了短期肺定植试验, 其中悬浮 Lewis 肺癌细胞 (LLCs) 稳定表达 FN-shRNA (shFN) 或shRNA (shScr) 和预先标记的20微米的 CFSE 被静脉注射注射到 C57BL/6 小鼠。我们成功地证明, 与 shScr 有限责任公司细胞相比, shFN llc 细胞对小鼠肺部的殖民化能力显著降低。因此, 这一短期方法可以广泛应用于具体证明 CTCs 在循环内的能力, 以殖民肺部。

在进行体内肺定植试验之前, 如图 1A所示, 以测试悬浮肿瘤细胞的 polyFN 是否介导肺定植和/或渗出在促进转移, 我们首先滴定不同的CFSE 的浓度, 一种荧光化合物, 它是细胞渗透性和共价键结合胞内胺的分子26,27在悬浮肿瘤细胞, 并保持在细胞相当长一段时间。我们发现肿瘤细胞以剂量依赖性的方式有效?...

Watch this Scientific Journal Video about The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues at JoVE.com

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

需要一个动物模型来破译循环肿瘤细胞 (CTCs) 在肿瘤转移过程中促进肺定植的作用。在这里, 我们建立并成功地进行了体内检测, 专门测试了高分子纤维连接蛋白 (polyFN) 组装对 CTCs 肺定植的要求。

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 ...

the-establishment-lung-colonization-assay-for-circulating-tumor-cell

Research

JoVE Journal

Cancer Research

9.2K 次观看.  National Cheng Kung University. 需要一个动物模型来破译循环肿瘤细胞 (CTCs) 在肿瘤转移过程中促进肺定植的作用。在这里, 我们建立并成功地进行了体内检测, 专门测试了高分子纤维连接蛋白 (polyFN) 组装对 CTCs 肺定植的要求。

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

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

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

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 colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

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 ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

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 ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image analysis. Digital scanning is a fast way to scan a stack of slides and produce digital images with high quality. It can produce concordant results with conventional light microscopy (CLM) by pathologists. Furthermore, the availability of digital images makes it possible that the same slide can be concurrently observed by multiple people. Moreover, digital images of slides can be stored in a database, which means the long-term deterioration of glass slides is avoided. The limitations of this technique are as follows. First, it needs high-quality prepared tissue and the original immunohistochemistry (IHC) slides without any damage or excess sealant residue. Second, tumor or nontumor areas should be specified by experienced pathologists before the analysis using software, in order to avoid any confusion about the tumor or nontumor areas during scoring. Third, the operator needs to control the color reproduction throughout the digitization process in whole-slide imaging.

ZW10 interacting protein (ZWINT) participates in the mitotic spindle checkpoint and the pathogenesis of carcinoma. Here, we introduce a methodology of the immunostaining of ZWINT in human lung cancer tissues, followed by the digital scanning of whole slides and image analysis. This methodology can provide high-quality digital images and reliable results.

人肺组织中ZW10相互作用蛋白免疫染色的数字化分析

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

免疫荧光对非小细胞肺癌患者循环肿瘤细胞的半自动PD-L1特征化及枚举

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...