Name: a "patient-like" orthotopic syngeneic mouse model of hepatocellular carcinoma metastasis
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Description: Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

道德守则 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）。 注意:八只BALB / c小鼠在本实验过程中使用。五只BALB / c小鼠植入5×10 6，BNL 1ME A.7R.1小鼠肝癌细胞产生的原发肿瘤。三只BALB / c小鼠，没有植入用作对照。 BNL 1ME A.7R.1是从鼠非致瘤性肝细胞细胞系，BNL CL.2衍生由化学转变该鼠肝癌细胞株。 BNL 1ME A.7R.1从美国典型培养物保藏中心（ATCC）获得。 1.预手术<ol><li>确保小鼠全部存活手术是健康的。为了确保小鼠是健康的，由皮肤的隆起检查脱水的迹象。不要用小鼠疾病或D的任何证据istress在这些实验中。 </li><li>称取小鼠以确保它们不减持。确保重量至少16-20克。 </li><li>准备5×10 6 BNL 1ME A.7R.1小鼠肝癌细胞每只小鼠植入: <ol><li>抽吸媒体从10ml的60％ - BNL 1ME A.7R.1小鼠肝癌细胞70％汇合细胞培养皿。用5ml PBS洗涤两次。加2ml胰蛋白酶的菜和在培养箱与空气和5％的CO 2 5分钟在37℃。 </li><li>接着，移液管加入10μl胰蛋白酶消化细胞分化成在显微镜下在10倍的放大倍率的手动血细胞计数用于细胞计数。算在四个大角落正方形中发现的细胞的总数和由细胞的总数乘以稀释因子。然后，通过平方计数的数分裂并通过10 6乘以为细胞浓度（每毫升的细胞）。 </li><li>使用的计算，收集每只小鼠在灭菌的5百万个细胞1.5ml微量管。标签的1.5 ml离心管中。 </li></ol></li><li>重悬在100μl磷酸盐缓冲盐水（PBS）中的5×10 6，BNL 1ME A.7R.1小鼠肝癌细胞。保持这些细胞在冰上，直到植入手​​术。 </li><li>进行必要的准备小鼠外科手术如麻醉诱导和头发修剪远离消毒设备和材料，以避免污染的活动。 </li><li>使用与氧一起的异氟烷蒸发器以1L每分钟（约2.1％）的恒定浓度流速诱导小鼠通过吸入异氟烷麻醉。注意:该系统是自立，和包括一个精密汽化器，氧的供应和链接到所需要的气/麻醉剂清除呼吸回路一活性炭罐。 </li><li>确保麻醉足以防止动物从手术过程中经历的疼痛。执行脚趾捏，观察动物的非responsiveness确认麻醉。 </li><li>此时，切换阀管线，密封的框和开放到鼻锥。 </li><li>只要鼠标被麻醉，敷眼软膏的一个微小的量，以角膜为从干燥和损坏防患于未然。 </li><li>接着，将动物以​​制备区域，用鼻锥体正确地放置和连接的（用于维持麻醉）。准备手术切口进行手术通过脱毛的部位，在这个准备区。 </li><li>确保手术的区域是在不含所有其他活动的位置。 </li><li>确保工作表面坚固，并适当消毒，未弄脏的，易用的手术前。放置一个无菌的一块亚麻或手术托盘在它的。 </li><li>确保手术视野仅可用于进行外科手术的人员。确保手术室进行清洗和清扫用合适的清洁剂，并用适当的disinfectant。 </li><li>确保它也位于远离门窗作为气流可以传播粉尘导致现场的污染。 </li><li>执行生存手术无菌器械。或者，根据制造商的建议，或高压釜浸泡仪器消毒剂。使用基本无菌物品是可以使用了。 </li><li>根据功能的不同工具，以帮助防止无菌区域受到污染。 </li><li>剃周围的切口（正中切口）站点约1英寸访问动物的肝脏。从动物手术的部位取下的头发了。 </li><li>剃须后，擦洗肥皂消毒剂（优碘外科洗手）的区域，并用无菌PBS液清洗多余的消毒液了。执行这几次，与整个磨砂工艺处理大约持续5分钟。卸下并丢弃用过的手套。 </li><li>后来，改为防护服，如surgic人长衫，磨砂顶和面罩。接着，把手术手套前，双手消毒用适当的溶液擦洗。注:这是一个必要的步骤，以防止脱落，污染手术部位与潜在的污染物。 </li></ol> 2.手术<ol><li>提供一个外部热源温暖整个麻醉的整个时期。动物可能会遇到一些低体温是因为麻醉导致体温调节障碍;因此，维持体温。 </li><li>莱鼠标在整个手术的背面。 </li><li>修复前肢，后肢和鼠标尾部的手术台与手术磁带件。 </li><li>做一个直线切口立即逊于xyphisternum。 </li><li>使用无菌的自固手术用牵引器具，以暴露肝脏。 </li><li>与具有注射器20G针，缓慢而小心地注入含有5×10 6，BNL 1ME A.7R.1 100微升PBS小鼠肝癌细胞到肝脏的右叶。 </li><li>除去针后，应用消毒棉签至少1分钟，以停止任何潜在的出血由于肝脏高血管。 </li><li>关闭皮下组织与可吸收缝合线之后皮肤缝合手术用夹子。随后，优碘擦洗一次清理伤口。注意:这将确保手术伤口闭合。 </li><li>皮下给予100微升生理盐水。 </li></ol> 3.后手术<ol><li>立即手术后，鼠标转移到一个干净的笼子与手术后的身份证，以通知的鼠标状态照料。提供食物和水。注:根据我们的经验，刚刚动过手术的境遇最好的，如果单独住用湿的食物和水的最初24小时内术后鼠标。 </li><li>保持动物温暖，热灯产生的热量不超过38ºC。 ü瑟温度计来测量温度，直到鼠标完全恢复。 </li><li>众议院的动物关在笼子里最好用纸张的床上用品，以确保舒适。 </li><li>初步恢复后，监测动物的不适，出血，疼痛和痛苦的迹象。给予丁丙诺啡（0.05 -0.1毫克/千克），以减轻疼痛。密切监控活动，行为和外观，以及水和食物消耗量为约2小时。如果动态，将动物回区域老鼠经常正常的住房，并持续监控在6-12小时，间隔24小时后手术。如果需要执行重量检查。 </li><li>但是，如果动物是有问题休养，采取适当的支持治疗措施。如果小鼠无法恢复，人道安乐死通过CO 2窒息和颈椎脱位鼠标。按照安乐死认可机构的指导方针。 </li><li>手术后，检查肝癌的临床证据通常是observab乐为显著减少，体况评分。注意:在这一点上，初级肿瘤可能已经开发了在肝脏和转移性沉积物在肺部有可能开发的。 </li><li>此时，牺牲参与本实验由人道的安乐死所有小鼠（实验和对照）。若使小鼠二氧化碳窒息:非常缓慢地释放二氧化碳进入室在一段5 -10分钟。这将导致呼吸骤停。 </li></ol>

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The authors declare that they have no competing financial interests.

在这篇文章中进行了深入描述的方法的是考虑到最近报道Ogunwobi和成功地建立了肝癌转移的原位同基因小鼠模型的同事（图2）3。肿瘤的接受率在此过程中普遍偏高。我们先前观察到的100％3肿瘤接受率。然而，接受率可以根据研究者的能力是可变的。在由下一个训练新研究生完成的最新的实验中，将肿瘤接受率较低，为40％。尽管大多数实验终止于4 - 由于在体况评分?...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table><tbody><tr><td>Micro Dissecting Tweezer&amp;nbsp;</td><td>Roboz</td><td>(RS-5040)</td><td>Tip 0.20 x 0.12 mm</td></tr><tr><td>Graefe Micro Dissecting Forceps, serrated curved tip</td><td>Roboz</td><td>(RS-5111)</td><td>1 X 2 teeth, curved tip width 0.6 mm</td></tr><tr><td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6501)</td><td>Blunt 3 X 3 prongs, depth 4 mm, spread 25 mm</td></tr><tr><td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6503)</td><td>Blunt, 3 X 3 prongs, depth 4 mm, spread 19 mm</td></tr><tr><td>Jameson Caliper (Measures tumors)</td><td>Roboz</td><td>(RS-6466)</td><td>80 mm/3 inch scale, chrome plated&amp;nbsp;</td></tr><tr><td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td><td>Roboz</td><td>(RS-5852)</td><td>23 mm blades, length 4 inches, flat shanks and large rings</td></tr><tr><td>Scalpel with blades, for delicate dissecting procedures</td><td>Roboz</td><td>(RS-9861-36)</td><td></td></tr><tr><td>Scalpel handle&amp;nbsp;</td><td>Roboz</td><td>(RS-9884)</td><td>Solid</td></tr><tr><td>Littauer Stitch Sissors</td><td>Roboz</td><td>(RS-7074)</td><td>Length 4.5 inches</td></tr><tr><td>Brown needle holder, for easy suture tying</td><td>Roboz</td><td>(RS-7960)</td><td>Convex jaw, fine serrations</td></tr><tr><td>Reflex 7MM wound clips with reflex 7 clip applier</td><td>Roboz</td><td>(RS-9262)</td><td>safe, secure alternative method of wound closure</td></tr><tr><td>Instrument tray and lid</td><td>Roboz</td><td>RT-1350S</td><td></td></tr><tr><td>Mini-clipper with detachable blade</td><td>Roboz</td><td>(RS-5903)</td><td></td></tr><tr><td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td><td>Roboz</td><td>Ds-400, Ds-401, DS-501</td><td>For fast decontamination of micro-dissecting instruments.&amp;nbsp;&amp;nbsp; Instruments decontaminate within 15 sec.&amp;nbsp;</td></tr><tr><td>Microinjection needles</td><td>VWR</td><td>BD305125</td><td>25G Needle</td></tr></tbody></table>

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.

Balb / c小鼠的肝脏植入5×10 6，BNL 1ME A.7R.1小鼠肝癌细胞。肝癌发展的临床证据是观察到63天后手术。因此，将小鼠安乐死。尸体剖检在安乐死的小鼠进行的。肺和肝脏切除和仔细肉眼检查以鉴定宏观肿瘤。在一个鼠标，浅表肿瘤观察到一些附着到腹壁（图1A）的肝脏的表面上。在第二个鼠标，异常好看肝脏进行了观察。异常非常强烈地看起来像一个浅表肿瘤（图1B?...

Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

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

癌症的转移扩散是癌症相关死亡的主要原因。我们提供了一个深入的描述，我们赖以生存的手术方法，为建立"以病人为像"原位同基因小鼠模型系统研究的实质器官肿瘤转移的机制。

"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 ...

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12.2K 次观看.  Hunter College of the City University of New York. 癌症的转移扩散是癌症相关死亡的主要原因。我们提供了一个深入的描述，我们赖以生存的手术方法，为建立"以病人为像"原位同基因小鼠模型系统研究的实质器官肿瘤转移的机制。 

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

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

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

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

肝大肠癌转移的高级动物模型:成像技术和转移性克隆性质

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

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

结直肠癌原位小鼠模型

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

生物学

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

在肝炎症和纤维化环境中产生的肝细胞癌的致癌肝细胞诱导正交小鼠模型

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

缺血再灌注损伤中转移性肝肿瘤的模样

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

医学

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

肝细胞癌原位小鼠模型中的无创PET/MR成像

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝转移的临床前小鼠模型

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.