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>

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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 important in cancer metastasis and have potential for multiple clinical applications, such as for diagnosis3,9,10. This is supported by data from several clinical studies that have showed that CTCs have a strong correlation with the progression of the disease in various cancer types such as pancreatic, prostate and breast, among others3,11,12. Despite the fact that CTCs may be very useful for cancer diagnosis, isolating and characterizing CTCs are challenging because very few are present among billions of blood cells3,7.
A significance of isolating CTCs using our technique is that it provides a reproducible model which can be optimized for potential utility in a clinical setting. This may ultimately lead to the possibility of using this method to replace current biopsy procedures that are more invasive and associated with side effects3,13. Modifications of this method, such as peripheral intravenous blood collection, would be expected to utilize it in a clinical setting. Of course, it is acknowledged that isolating CTCs from humans is a much more challenging endeavor due to general low yield4,14. But, in many cases such as in pancreatic cancer, histo-pathologic or cytological validation using tissue biopsies is fruitless in spite of multiple invasive attempts4. Isolating CTCs has been described as &#8220;liquid biopsy&#8221; or &#8220;live biopsy&#8221; of a tumor which enables noninvasive cancer diagnosis and real-time monitoring of therapeutic response3. This methodology models the isolation of CTCs in a mouse model. Thus far, only one FDA- cleared assay, such as Veridex CellSearch System, has been approved to detect CTCs. In one study, this technology identified CTCs in only 11 of 26 patients. This system is associated with some inefficiency, limited purity and high cost4. Moreover, it lacks the important feature of being able to collect viable CTCs whether they are EpCAM-expressing or not for extensive analyses using multiple in vitro and in vivo methods.
An important feature of the described method is that it preserves the viability of the isolated CTCs3. Therefore, using this method allows for the subsequent extensive downstream analysis of both the molecular and functional characteristics of CTCs using a variety of techniques. 
In conclusion, a methodology for isolating and propagating CTCs that are viable and can be characterized molecularly and functionally is presented. There is potential that this method can be optimized for clinical applications.

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><tbody><tr><td>Heparin Sodium Salt 1 g</td><td>VWR</td><td>89508-852</td><td></td></tr><tr><td>BTX Tube Micro 1.5 ml Clear NS</td><td>VWR</td><td>89511-254</td><td>1.5 ml&amp;nbsp; pyrogen-free eppendorf Tubes</td></tr><tr><td>Needle Sterile Disp BD 25 G x 1 in</td><td>VWR</td><td>BD305125</td><td>25 G Needle</td></tr><tr><td>Slp Tip SRNG 1 ml 200 each per pack</td><td>VWR</td><td>BD309659</td><td>1 ml syringe tip</td></tr><tr><td>Syringe 1 ml 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 15 x 15 x 5 mm PK 100</td><td>VWR</td><td>25608-924</td><td>Cyromold</td></tr><tr><td>Cryo-oct compund 4 oz</td><td>VWR</td><td>25608-930</td><td>Oct compound</td></tr><tr><td>VWR Slide sprfrst 25 x 75 mm PK72</td><td>VWR</td><td>48311-703</td><td>Slides</td></tr><tr><td>VWR Cover Glass #2 22 x 50 mm</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 2 L</td><td>VWR</td><td>101454-204</td><td>Nuclease Free Water</td></tr><tr><td>Buffer Tris Ultra Pure Grade 500 g</td><td>VWR</td><td>97061-796</td><td>Tris Buffer</td></tr><tr><td>Ammonium Chloride ACS Grade 2, 5 Kg</td><td>VWR</td><td>97062-048</td><td>Ammonium Chloride Buffer</td></tr><tr><td>Falcon Tissue Culture Dish 60 x 15 mm Style polystyrene</td><td>VWR</td><td>353002</td><td>Tissue Culture Dish</td></tr><tr><td>Clorox 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 &amp;amp; magnesium (500 ml)</td><td>Thermo Fischer Scientific</td><td>21-040-CV</td><td>PBS, 1x</td></tr><tr><td>DMEM, 1x&amp;nbsp; with 4.5 g/L glucose &amp;amp; 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 Serum</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>75002411</td><td>13,000 rpm 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。可能会出现的一个问题是，得到的粒料可能不清晰的颜色。通过执行它附加次15分钟，或者可能 - 这个问题可以通过它增加到10修改RBC裂解缓冲液中的温育时间很容易地得到解决。的CTC的代表图像被传播中培养来自三个不同的小鼠显示在图1相比，BNL 1ME A.7R.1细胞。快速增殖和形态将帮助确定是否细胞接种的检验中心。然而，如PCR和免疫染色在步骤5.1中描述的验证技术 - 需要5.2到确认建立可行的CTC线。 <img alt="图1" src="/files/ftp_upload/52861/52861fig1.jpg" /> 图1.从传播的原位同源小鼠模型的循环肿瘤细胞（检验中心）在细胞培养的肝癌转移。细胞中繁殖15毫米×60 MM细胞培养皿中BNL 1ME A.7R.1培养基。增殖的肿瘤细胞粘附至菜和从三个独立的小鼠被示出。肿瘤细胞通过不断的通道仍然是可行的。 CTCOL1MEA081211，CTCOL1MEACTC08291的相衬图像1 CTCOLUF2419是在比较BNL 1ME A.7R.1细胞（40倍）采取40倍的放大倍率（物镜）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52861/52861fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/52861/52861fig2.jpg" /> 图2.的隔离和传播循环肿瘤细胞从老鼠肝癌模型系统的过程中，当肝癌的临床证据被观察到显著减少，体况评分，全血是从小鼠收集。经过多次离心，红细胞溶解，和几个洗涤步骤，该检验中心接种于细胞培养皿繁殖。肿瘤细胞的增殖，经过几天观察， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52861/52861fig2large.jpg" target="_blank">请点击他</a>再查看该图的放大版本。

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

12.0K 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.

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

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 ...

科研

癌症研究

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 ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

从小鼠横纹肌瘤分离的原发性肿瘤细胞的肿瘤球衍生和治疗

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

遗传学

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 ...

小鼠的引导肿瘤接种和多模式成像

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

作者聚焦:整合高分辨率活体成像和 MRI 以增强立体定向放射治疗计划

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

医学

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

"以病人为像"肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

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此视频中的章节

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

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

从小鼠横纹肌瘤分离的原发性肿瘤细胞的肿瘤球衍生和治疗

循环肿瘤细胞系:基础和转化研究的创新工具

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

背侧皮褶窗室肿瘤小鼠模型在转化癌症研究中用于联合活体显微镜和磁共振成像

原发灶和MEF细胞分离，研究肺转移

"以病人为像"肝癌原位同源小鼠模型的癌转移

的肿瘤特异性内皮细胞的分离与培养扩展