Work in Dr. Ogunwobi&#8217;s laboratory is supported by a Research Centers in Minority Institutions Program grant from the National Institute on Minority Health and Health Disparities (MD007599) of the National Institutes of Health. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIMHD or the NIH.

伦理声明：所有的动物研究批准了纽约市立大学亨特学院的机构动物护理和使用委员会（IACUC）。 1.预实验程序<ol><li>抵达后到的动物设施，最初容纳3 BALB / c小鼠用过滤的顶部和木片寝具一个笼子。确保鼠标可以免费获得食物，普瑞纳啮齿动物食物和水。床上用品和食物应每周更换两次，而网箱应每周清洁一次。 </li><li>使用与氧一起的异氟烷蒸发器以每分钟1升的恒定流速诱导小鼠全身麻醉。该系统是自力更生，包括精密蒸发器。 </li><li>位置动物在塑料盒中具有汽化器精度和气体/麻醉剂系统连接到它。最初设置的蒸发器，以2.5至动物是无意识的，在该点降低汽化器至2。 </li><li>此时，切换阀管线，它密封的框，并将其打开到鼻锥。 </li><li>移动到动物的准备区，用鼻锥体置于正确和连接的（用于维持麻醉）。 </li><li>准备5×10 6 BNL 1ME A.7R.1小鼠肝癌细胞每只小鼠植入。 BNL 1ME A.7R.1应生长在培养基（10％热灭活的FBS，1％青霉素/链霉素和2mM L-谷氨酰胺）在培养箱与空气和5％孵育在细胞培养皿在37℃下的 CO 2。 </li><li>用具有20G的针的注射器，含有5×10 6，BNL 1ME A.7R.1小鼠肝癌细胞到BALB / c小鼠的肝脏中的一个波瓣植入体100微升PBS，以产生原发肿瘤。植入后，房子在具有相同住宅要求植入前一个轿厢的BALB / c小鼠。 </li><li>在实验终点，肝癌的临床证据是观察到的显著减少身体状况分数（通常为4周后检测）。在这一点上，存在原发性肿瘤已经开发在肝脏而转移性沉积物形成在肺中的似然性。此后，开始循环肿瘤细胞的分离和传播的过程。 </li></ol> 2.收集全血的循环肿瘤细胞分离注：正确消毒手术区域，并确保它是整洁的。高压灭菌所有手术器械或根据制造商的建议浸泡在杀菌剂<ol><li>通过在实验终点人道安乐死（肿瘤发展的临床证据）牺牲小鼠。小鼠安乐死应包括二氧化碳窒息。观察小鼠正确，确认麻醉正确分配。二氧化碳应缓慢释放到经过一段5室 - 10分钟，造成呼吸停止。心内放血和cervic按照人脱位。5 </li><li>使用附连到1ml注射器收集全血地下25针。 Heparanize用0.01 ml肝素注射器。穿刺立即皮肤与针劣于xyphisternum在大约45°角。推进针头慢慢刺破心脏，然后放下针的角度。稳步举行的地方针头和注射器，并绘制出500微升 - 1000微升全血从心脏。 </li><li>取下注射器和针和血液转移至预标记的1.5 ml的无热原管。离心收集到1.5 ml的无热原管中的全血，在1167×g离心5分钟。 注：离心分离的全血样品分为三层：由红细胞，中间薄层的血沉棕黄层，以及一个顶血浆层底部或血细胞比容层。 </li><li>通过移液仔细清除血浆收集到一个单独的1.5毫升无热原管菲尔特呃实验诸如检测的无细胞核酸7。 </li><li>为循环肿瘤细胞（CTC的）的隔离，收集血沉棕黄层成在准备红细胞（RBC）裂解单独1.5 ml的无热原管。这是必要的，以除去残余的RBCs在血沉棕黄层层。 </li></ol> 3.配方的红细胞（RBC）裂解液<ol><li>使红细胞裂解缓冲液的1L溶液。 </li><li>使500毫升氯化铵和500毫升的Tris。然后混合以创建的RBC裂解缓冲液的1L溶液用氯化铵的155毫和的Tris 10mM的终浓度。 </li><li>缓冲溶液的pH为7.5。存储RBC裂解缓冲液在2至8℃，并带来至RT之前立即使用。 </li></ol>巴菲大衣4.红细胞裂解<ol><li>加入1毫升的RBC裂解缓冲液的无菌无热原1.5毫升管含有所收集的血沉棕黄层。 </li><li>通过混合管的内容物反相多次。孵育管的内容混合5分钟，在室温在板凳上。在1167离心混合内容，5分钟×g的。 </li><li>离心后，发白粒料将获得。弃去上清液微红缓慢而小心地将10％的漂白粉无沉淀任何中断。如果颗粒不发白，重新执行步骤3.1 - 3.5。增加RBC裂解缓冲液中温育时间为10 - 15分钟也可以是有帮助的。 </li><li>安全处置生物废料一旦在10％漂白的生物垃圾无害化处理完毕24小时后。 </li></ol> 5.传播在细胞培养注：肿瘤细胞通常是快速生长的细胞白血细胞，其丰富的细胞接种入培养皿的原始混合物。以下反复变化的介质中，和的CTC的后续的传代，所有的白血细胞被除去的CTC的相对较纯的群体存在。 <ol><li>经弃去上清成10％的漂白剂，在完全培养基中洗涤发白沉淀在1ml磷酸盐缓冲盐水（PBS）至少一次，重悬在细胞培养物中繁殖。 </li><li>一旦洗涤完成后，加入1 ml的完整BNL 1ME A.7R.1培养基（10％热灭活的FBS，1％青霉素/链霉素和2mM L-谷氨酰胺）中，以沉淀和重新悬浮在培养基中的小球。 </li><li>种子细胞的悬浮搅和成细胞培养皿。孵育细胞培养皿在37℃下在湿润的培养箱中与空气和5％的CO 2。 </li><li>每两至三天更换细胞培养基。后五至七天，将CTC的粘附在细胞培养皿，并开始增殖。这些细胞将保持存活超过25代和反复冻融循环 5之后。 </li></ol> 6.验证肝癌的循环肿瘤细胞系<ol><li>执行APolymerase链反应（PCR）为基础的方法中，以扩增小鼠β珠蛋白基因的一个具体的DNA片段，以确认新建立的四氯化碳细胞系是小鼠细胞像原来植入BNL 1ME A.7R.1肝癌线。方法最初是描述和Steube 等验证。5,8 </li><li>执行免疫染色的肝细胞特异性标记的cAMP反应性元件通过使用特定的抗体结合蛋白3样3（CREB3L3）。这将确认新成立的CTC确实是肝细胞像原来植入的BNL 1ME A.7R.1细胞系。5 </li></ol>

<ol>
	<li>Ashworth, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+of+cancer+in+which+cells+similar+to+those+in+the+tumors+were+seen+in+the+blood+after+death.">A case of cancer in which cells similar to those in the tumors were seen in the blood after death.</a> Australian Medical Journal. 1869, 146-147 (14).</li><li>van de Stolpe, A., den Toonder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Tumor+Cells:+What+Is+in+It+for+the+Patient?+A+Vision+towards+the+Future.">Circulating Tumor Cells: What Is in It for the Patient? A Vision towards the Future.</a> Cancers. 6, 1195-1207 (2014).</li><li>Sheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capture,+release+and+culture+of+circulating+tumor+cells+from+pancreatic+cancer+patients+using+an+enhanced+mixing+chip.">Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip.</a> Lab on a chip. 14, 89-98 (2014).</li><li>George, T. J., Ogunwobi, O. O., Sheng, W., Fan, Z. H., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#34;Tissue+is+the+issue&#34;:+circulating+tumor+cells+in+pancreatic+cancer.">&#34;Tissue is the issue&#34;: circulating tumor cells in pancreatic cancer.</a> Journal of gastrointestinal cancer. , (2014).</li><li>Ogunwobi, O. O., Puszyk, W., Dong, H. J., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+upregulation+of+HGF+and+c-Met+drives+metastasis+in+hepatocellular+carcinoma.">Epigenetic upregulation of HGF and c-Met drives metastasis in hepatocellular carcinoma.</a> PloS one. 8, e63765 (2013).</li><li>Cheng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent,+biocompatible+nanostructured+surfaces+for+cancer+cell+capture+and+culture.">Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture.</a> International journal of nanomedicine. 9, 2569-2580 (2014).</li><li>Yu, M., Stott, S., Toner, M., Maheswaran, S., Haber, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of cell biology. 192, 373-382 (2011).</li><li>Steube, K. G., Koelz, A. L., Drexler, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+verification+of+rodent+cell+lines+by+polymerase+chain+reaction.">Identification and verification of rodent cell lines by polymerase chain reaction.</a> Cytotechnology. 56, 49-56 (2008).</li><li>Pantel, K., Alix-Panabieres, C., Riethdorf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+micrometastases.">Cancer micrometastases.</a> Nature reviews. Clinical oncology. 6, 339-351 (2009).</li><li>Pantel, K., Brakenhoff, R. H., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells.">Detection, clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature reviews. Cancer. 8, 329-340 (2008).</li><li>Cristofanilli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells,+disease+progression,+and+survival+in+metastatic+breast+cancer.">Circulating tumor cells, disease progression, and survival in metastatic breast cancer.</a> The New England journal of medicine. 351, 781-791 (2004).</li><li>De Giorgi, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+and+bone+metastases+as+detected+by+FDG-PET/CT+in+patients+with+metastatic+breast+cancer.">Circulating tumor cells and bone metastases as detected by FDG-PET/CT in patients with metastatic breast cancer.</a> Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 21, 33-39 (2010).</li><li>Eloubeidi, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+ultrasound-guided+fine+needle+aspiration+biopsy+of+patients+with+suspected+pancreatic+cancer:+diagnostic+accuracy+and+acute+and+30-day+complications.">Endoscopic ultrasound-guided fine needle aspiration biopsy of patients with suspected pancreatic cancer: diagnostic accuracy and acute and 30-day complications.</a> The American journal of gastroenterology. 98, 2663-2668 (2003).</li><li>O'Toole, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+complications+of+EUS-guided+fine-needle+aspiration.">Assessment of complications of EUS-guided fine-needle aspiration.</a> Gastrointestinal endoscopy. 53, 470-474 (2001).</li></ol>

The authors declare that they have no competing financial interests.

In this study, how to isolate and propagate CTCs from the whole blood of a syngeneic mouse with HCC were described. The objective of this work is to enhance the ongoing studies of the mechanisms of cancer metastasis. 
A factor that contributes to the poor prognosis of many cancers is the lack of timely detection and consequent widespread dissemination of the malignancy3. CTCs originate from primary cancers and spread via the blood stream to distant organs. As such, CTCs are importan...

The cancer research community has known of the existence of circulating tumor cells (CTCs) since first being observed by Thomas Ashworth in 18691. Since then, CTCs have been shown to be important in tumor metastasis and disease progression2-5. Today, solid tumors are a major cause of morbidity and mortality worldwide. CTCs are rare cells that originate from primary tumors and travel through the blood stream to different organs of which only a small fraction ultimately develop into metastasis2-5. Notably, there is positive correlation between tumor size and CTC number3,4. 
An understanding of CTC biology can contribute to the search for targeted therapy. Furthermore, CTCs may have diagnostic applications. To achieve these potential clinical applications, one needs to overcome some current challenges to studying CTCs. One challenge is related to the fact that CTCs may be present as single cells or as clusters and they may even be able to change their phenotype in response to the blood microenvironment2. Moreover, detection can be very challenging, in part, due to the low count of CTCs (a few to hundreds per milliliter) among one billionhematologic cells per milliliterin the blood6. Nevertheless, in recent years, research into the potential clinical applications of CTCs from solid organ cancers has intensified.
Despite these efforts, the challenges of studying and understanding the role of CTCs persist due to the rarity of CTCs and the inadequacy of the technological tools currently available. Despite these challenges, the tremendous potential for clinical applications continues to be an incentive to pursue research into the role of CTCs in cancer metastasis.
We were recently successful in isolating and propagating in cell culture CTCs from an orthotopic syngeneic mouse model of hepatocellular carcinoma (HCC) metastasis5. The purpose of the current paper is to describe in detail all aspects of the successful methodology. The significance of this methodology lies in the fact that this approach may be modified in order to successfully isolate and propagate in culture human CTCs, thus enhancing the possibility of in vitro studies of CTC biology. 
There are multiple potential clinical applications for the use of CTCs. CTCs may be useful for prognosis, response monitoring, screening, dynamic monitoring of tumor molecular alterations, and personalized therapy4. Therefore, a better understanding of the biology of CTCs has high potential for clinical impact.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Heparin Sodium Salt 1G</td>
			<td>VWR</td>
			<td>89508-852</td>
			<td></td>
		</tr>
		<tr>
			<td>BTX Tube Micro 1.5mL Clear NS</td>
			<td>VWR</td>
			<td>89511-254</td>
			<td>1.5mL&#160; pyrogen-free eppendorf Tubes</td>
		</tr>
		<tr>
			<td>Needle Sterile Disp BD 25GX1IN</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
		</tr>
		<tr>
			<td>Slp Tip SRNG 1ml 200 each per pack</td>
			<td>VWR</td>
			<td>BD309659</td>
			<td>1mL syringe tip</td>
		</tr>
		<tr>
			<td>Syringe 1ml leur lok Pk 100</td>
			<td>VWR</td>
			<td>BD309628</td>
			<td>1 mL syringe</td>
		</tr>
		<tr>
			<td>VWR Forceps Tissue 6</td>
			<td>VWR</td>
			<td>82027-446</td>
			<td>Forceps</td>
		</tr>
		<tr>
			<td>Cyromold intrm 15X15X5MM PK 100</td>
			<td>VWR</td>
			<td>25608-924</td>
			<td>Cyromold</td>
		</tr>
		<tr>
			<td>Cryo-oct compund 4oz</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Oct compound</td>
		</tr>
		<tr>
			<td>VWR Slide sprfrst 25X75MM PK72</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td>Slides</td>
		</tr>
		<tr>
			<td>VWR Cover Glass #2 22X5oMM OZ</td>
			<td>VWR</td>
			<td>48-382-128</td>
			<td>Cover Glass</td>
		</tr>
		<tr>
			<td>VWR Slide Box True North Fm Pu</td>
			<td>VWR</td>
			<td>89140-278</td>
			<td>Slide Box</td>
		</tr>
		<tr>
			<td>Super HT PAP Pen</td>
			<td>VWR</td>
			<td>89427-058</td>
			<td>PAP pen</td>
		</tr>
		<tr>
			<td>Water RNASe and DNAse free 2L</td>
			<td>VWR</td>
			<td>101454-204</td>
			<td>Nuclease Free Water</td>
		</tr>
		<tr>
			<td>Buffer Tris Ultra Pure Grade 500G</td>
			<td>VWR</td>
			<td>97061-796</td>
			<td>Tris Buffer</td>
		</tr>
		<tr>
			<td>Ammonium Chloride ACS Grade 2&#160; 5KG</td>
			<td>VWR</td>
			<td>97062-048</td>
			<td>Ammonium Chloride Buffer</td>
		</tr>
		<tr>
			<td>Falcon Tissue Culture Dish 60 x 15mm Style polystyrene</td>
			<td>VWR</td>
			<td align="right">353002</td>
			<td>Tissue Culture Dish</td>
		</tr>
		<tr>
			<td>Clorox&#174; Germicidal Bleach, Regular</td>
			<td>VWR</td>
			<td>89501-620</td>
			<td>Clorox Bleach</td>
		</tr>
		<tr>
			<td>PBS, 1X (Phosphate-Buffered Saline) without calcium &#38; magnesium (500mL)</td>
			<td>Thermo Fischer Scientific</td>
			<td>21-040-CV</td>
			<td>PBS, 1X</td>
		</tr>
		<tr>
			<td>DMEM, 1X&#160; with 4.5 g/L glucose &#38; L-glutamine without sodium pyruvate</td>
			<td>Thermo Fischer Scientific</td>
			<td>10-017-CV</td>
			<td>DMEM 1X media for BNL 1ME A.7R.1 cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Seru8m</td>
			<td>Thermo Fischer Scientific</td>
			<td>35-010-CV</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Solution, 100X</td>
			<td>Thermo Fischer Scientific</td>
			<td>30-002-Cl</td>
			<td>Penicillin Streptomycin</td>
		</tr>
		<tr>
			<td>Sorvall Biofuge pico</td>
			<td>Thermo Fischer Scientific</td>
			<td align="right">75002411</td>
			<td>13000rpm Centrifuge</td>
		</tr>
	</tbody>
</table>

isolation and propagation of circulating tumor cells from a mouse cancer model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

此处描述的方法已经表明的CTC可被分离。小鼠安乐死在实验终点。在参与过程中二氧化碳窒息，心内放血，和颈椎脱位。的过程的关键步骤的示意图示出在图2中。由心脏内放血收集的全血样品使用上述协议处理。之后，离心法确定进行的RBC裂解血沉棕黄层层。然后将其后跟另一个离心步骤以收集在PBS中洗涤该发白沉淀，并再悬浮于完全细胞培养基用于在细胞培养物繁殖。可能会出现?...

Watch this Scientific Journal Video about Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model at JoVE.com

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

isolation-propagation-circulating-tumor-cells-from-mouse-cancer

Research

JoVE Journal

Medicine

11.9K 次观看.  Hunter College of The City University of New York. Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.