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><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;(BSA)</td><td>Cyrusbioscience (Taipei, Taiwan)</td><td>101-9048-46-8</td><td></td></tr><tr><td>Bouin&#039;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&#039;s Modified Eagle Media (DMEM)&amp;nbsp;</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 )&amp;nbsp;</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&amp;nbsp;</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 (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</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 (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</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&amp;nbsp;</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;Equipment&lt;/strong&gt;</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 &amp;amp; 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&amp;nbsp;</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 CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;nbsp;</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 ...

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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

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

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 ...

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 ...

医学

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 ...

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 ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

多维共同培养系统模拟肺鳞癌进展

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

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 ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

循环肿瘤细胞系:基础和转化研究的创新工具

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

CTC Immunofluorescence Assay : A Technique to  Characterize Circulating Tumor Cells

Source: Garcia, J., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/59873/semi-automatic-pd-l1-characterization-enumeration-circulating-tumor">Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence.</a>&#160;J. Vis. Exp.&#160;(2019)
This video describes the Circulating Tumor Cells (CTC) Immunofluorescence assay, a technique to characterize the biomarkers on circulating tumor cells in non-small cell lung cancer patient samples. This assay could be used to monitor dynamic changes of the tumor and immune response, which may reflect the response to chemotherapy, radiation, and possible immunotherapy treatments.

CTC 免疫荧光检测:一种表征循环肿瘤细胞的技术

Source: Garcia, J., et al. Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by ...