Name: surgical procedures and methodology for a preclinical murine model of de novo mammary cancer metastasis
Uploaded: 2017-07-29T13:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis at JoVE.com

作者感谢Jo Hill的组织病理学帮助，John Gleysteen博士在外科技术方面的教学，Tessa Diebel的摄影辅助，Wong和Coussens实验室的所有成员进行重要的洞察和讨论，以及OHSU Knight Cancer Institute的财务支持。作者承认T32GM071388-10和T32CA106195-11支持CEG，NCI / NIH，国防部乳腺癌研究计划，苏珊·戈曼斯基金会，乳腺癌研究基金会，以及一个支持癌症 - Lustgarten基金会胰腺癌融合梦想团队转化研究基金（SU2C-AACR-DT14-14）向LMC提供MHW颁发基金奖的妇女健康圈，以及Brenden-Colson对MHW和LMC的胰腺健康中心。

以下协议中使用的动物由俄勒冈州健康与科学大学机构动物护理和使用委员会（IACUC）涵盖，该委员会旨在符合"动物福利法"条例和公共卫生服务（PHS）政策。 维持无菌条件:应使用灭菌仪器，小鼠之间应用无菌纱布擦拭，用PBS冲洗，然后用消毒剂70％乙醇灭菌至少15分钟。外科手术帽，面罩，长袍和手套应佩戴进行生存手术。用于生存手术的动物的手术前准备包括在以下方案中。有关试剂和设备的清单，请参阅表1。 1.从初级乳腺肿瘤中分离和制备单细胞悬液<ol><li>麻醉供体雌性100天龄转基因MMTV-PyMT（FVB / n）小鼠并维持在c通过麻醉面罩给予2％异氟烷的连续镇静。确认鼠标已被适当地麻醉，没有足部反射。 </li><li>在无菌环境中，通过使用无菌剪刀从上覆皮肤和周围脂肪组织和/或淋巴结中分离乳腺肿瘤，从100天龄的转基因雌性MMTV-PyMT（FVB / n）小鼠切除原发性乳腺肿瘤。通过颈椎脱位安乐死麻醉的小鼠。 </li><li> 用无菌剪刀或手术刀将手术切成小块（〜1.0 mm 3 ）。将肿瘤片放在3.0mg / mL胶原酶A和4.0U / mL DNase I中溶解于DMEM中。每1.0厘米直径肿瘤使用〜10毫升上述消化培养基。 <ol><li>用无菌搅拌棒在无菌的25mL瓶中以〜125rpm和37℃进行消化40分钟。 </li></ol></li><li>通过加入胎牛血清（FBS）至10％的最终稀释度来停止消化，并将整个混合物置于w上并保持冰。 </li><li>将消化的肿瘤悬浮液通过0.7μm尼龙过滤器过滤到50mL锥形管中，并丢弃残留在过滤器中的任何肿瘤。在4℃下以300xg离心上清液。 </li><li>将沉淀重悬于10ml DMEM / 1.0cm肿瘤中，并通过0.7μm尼龙过滤器再过滤。计数细胞浓度，然后在4℃下以300xg离心。 </li><li>将沉淀物重悬于10％二甲基亚砜与90％FBS浓度为2×10 7活细胞/ mL。将整个原发肿瘤的单细胞悬液储存在-80°C。 </li></ol> 2.原位乳头肿瘤注射<ol><li>部分解冻冷冻的原代肿瘤悬浮液在37°C，直到冷冻丸粒可以从低温管释放到20 mL DMEM中并计数细胞。在4℃下以300xg离心，并将细胞重悬于1:1 DMEM:生长因子减少的溶解基底膜制剂e以1×10 7个细胞/ mL的浓度从Engelbreth-Holm-Swarm（EHS）小鼠肉瘤中减量（参见材料表 ）。 </li><li>将麻醉的受体雌性同基因小鼠腹侧向上放置，并通过麻醉面罩施用2％异氟烷，保持连续镇静。 </li><li>使用雾化的70％乙醇灭菌右侧第4个乳腺注射部位，并用无菌棉签涂覆聚（乙烯基吡咯烷酮）碘。 </li><li>使用29 G 0.3 mL胰岛素注射器将100μL（来自冷冻原发性肿瘤悬浮液的1×10 6个活细胞）倾斜侧向上注入6至10周龄雌性FVB / n小鼠的未清除右侧第4个乳腺。 </li></ol> 3.手术切除原位乳腺肿瘤<ol><li>肿瘤细胞注射后38-60天，安乐死不显示体积范围在172至450mm 3之间的原位肿瘤体积的长度x（宽度2 </sup&gt;）/ 2]（约75％的注射小鼠）。 </li><li>通过麻醉面罩给予2％异氟烷，将连续镇静下的适当肿瘤大小腹侧放置麻醉小鼠，并确认小鼠用不足的脚捏反射适当地麻醉。在镇静时应用兽药软膏防止干燥。 </li><li>喷雾70％乙醇，对原发肿瘤周围的手术区进行消毒，然后用无菌棉签涂敷聚（乙烯基吡咯烷酮）碘。 注意:手术部位的脱毛导致〜5％小鼠的皮肤剥离和/或感染，因此被排除在该步骤之外（数据未显示）。 </li><li> 如图1C所示，使用钝刀中间尾部对肿瘤进行初始皮肤切口。 </li><li>接下来，使肿瘤内侧的皮肤（ 图1C ）更好地切除。注意灼烧任何脉管系统的需要在延伸切口之前，将位于皮肤上的肿瘤编织。 </li><li>继续皮肤切口（肿瘤后），然后进行优异的皮肤切除（肿瘤外侧）和内侧切除（优于肿瘤）（ 图1C ）。 </li><li>皮肤周围切除肿瘤周围（ 图1C ）后，使用镊子将附着于肿瘤的覆盖皮肤抬起，同时将肿瘤从腹壁肌肉组织中分离出来，并保持乳腺不受影响。 </li><li>识别（通过钝器解剖）和烧灼通过第 4和第 5乳腺运行大型船只。 </li><li>在烧灼部位消耗大约一半的第4和第 5 个乳腺，以释放肿瘤，覆盖的皮肤和乳腺的部分（ 图1C ）。 </li><li>出血时，积极发现出血立即烧灼他们。如果超过250μL的血液丢失，请将小鼠从学习中排除并安乐死。 </li><li>使用伤口夹具将伤口夹紧闭切割部位（ 图1C ）。 注意:手术后10天用伤口夹去除剂取下伤口夹。 </li><li> 皮下施用温暖的无菌生理盐水（占动物体重的4％），并用热灯保持动物温暖。在麻醉恢复期间每5-10分钟检查动物。 注意:施用布比卡因或利多卡因会增加术后死亡率（数据未显示）。手术后1小时，出现疼痛迹象（臀部，不愿意移动，无法修复）的小鼠安乐死。 <ol><li>一旦鼠标完全恢复，返回到其他老鼠的公司。 </li></ol></li></ol>血液和肺的分离和处理用于流式细胞术和组织学 &lt;OL&gt; <li>在研究终点，如果需要，准备小鼠进行各种组织病理学评估。在安乐死前90分钟，给予每只小鼠腹膜内注射浓度为6.25μg/μL的1×PBS中的溴脱氧尿苷（BrdU，50μg/ g小鼠重量）。 注意:冷冻储存的溶解的溴脱氧尿苷在制备后1个月内使用。 </li><li>在安乐死前10分钟，麻醉小鼠，使用肝素化毛细管收集后蹄血（&gt; 500μL），随后转移到涂有冰上的EDTA二钾管。 </li><li>为了去除肺部和残留的乳腺组织，用剪刀从下腹部到口部进行中线切口以暴露胸腔和腹腔（ 图2A ），并横向剥离皮肤，还暴露剩余的右侧第 4和第 5 个乳腺腺体。 </li><li>消除剩余的乳腺组织，并检查我吨通过苏木精和伊红（H＆E）染色（ 补充 图1）评估串行切片福尔马林固定的石蜡包埋的（FFPE）组织，以排除原发性肿瘤再生长。 </li><li>为了清除肺，用剪刀打开隔膜的腹壁，然后通过切开腹主动脉来安乐死动物，在灌注肺之前排出血液（ 图2B -2C ）。 </li><li>从腹部方法切割隔膜，确保避免肺和心脏。然后，通过切穿肋骨的侧面暴露胸部（ 图2D ）。 </li><li>在20 mL注射器上使用23号针头，通过心脏右心室灌注〜5.0 mL DPBS（〜10 mL / min）的肺，直到肺完全变白（ 图2E -2F ）。立即切断主体的心脏因此，血液不会重新灌注到肺部。 </li><li>对于待组织的肺组织进行组织病理学评估，使用23号针头（ 图2G - 2H ），将4℃下的〜1.0mL 10％福尔马林注射到暴露的气管斜面侧朝向肺。一旦肺完全扩张并充满固定剂，停止注射（ 图2I ）。 </li><li>根据标准组织病理学程序，从气管切除肺叶，并将中性缓冲福尔马林的肺组织固定在随后的石蜡包埋或OCT-冷冻培养基中。 </li><li>通过FFPE肺组织的连续切片定量评估肺转移负荷，并通过H＆E染色（ 图1B ）评估切片切片（100μm，十三个切片）。 </li></ol>

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作者没有披露与本文提供的数据的冲突。

修改和故障排除: 当钝器从腹壁解剖肿瘤时，肿瘤可能仍然粘附在腹壁上。在&lt;5％注射肿瘤的小鼠中观察到这种情况（数据未显示）。对于具有粘附于腹壁的肿瘤的小鼠，应当对小鼠进行安乐死，因为切除难度而没有原发性肿瘤再生长。 模型/技术的限制: 除了肺外，其他研究者报告除了肺外，分布在肝脏?...

北美地区的女性患乳腺癌的终生风险约为12％ 2 ;大多数这些个体将通过手术去除原发性肿瘤，并且依赖于癌症亚型，然后将在佐剂设置中接受靶向的内分泌，化疗和/或放射治疗3 。诊断为接受抗雌激素治疗的激素受体阳性癌症妇女和接受HER2靶向治疗的HER2阳性肿瘤的妇女与放射治疗/化疗相关的实例包括:三重阴性肿瘤尚无靶向治疗3 。尽管在放疗，化疗，个性化和基于激素疗法的进步是补充手术切除，复发的疾病诊断为II期或III期疾病有4名妇女的30％-70％，由于治疗是在远处器官转移根除疾病很大程度上是无效的，包括肺，骨，b雨和/或肝脏5 。这是特别重要的，因为当没有原发性肿瘤再生长时发生转移性疾病时，这意味着在确定性手术时传播的恶性细胞可能已经存在于二级器官中。因此迫切需要能够消除或减缓转移性肿瘤生长的治疗方法。 虽然乳腺癌发生的从头鼠模型在揭示调节肿瘤进展1的机制方面已经非常显着，但现有模型也有一些限制。其中之一是事实， 从头转基因模型通常在多个乳腺中发展原发性肿瘤，其中原发性肿瘤负担限制了研究的持续时间。虽然原发性肿瘤细胞逃逸和转移性种子可能在这些模型的肿瘤进展早期发生，但是转移性肿瘤的坦率发展发生在晚期，而且依赖于n小鼠模型和应变背景，经常部分渗透1 。这进一步限制了从头模型发现调节继发器官转移的分子的效用，以及用于评估治疗剂在辅助治疗中的临床前功效。 为了规避这些局限性，我们开发了一种乳腺癌转移到肺的从头自体模型。携带晚期从头乳腺肿瘤的父母转基因雌性（ 即 ，本文所述用于研究的FVB / n株系背景上的MMTV-PyMT）老化至〜100天6 ，此时，其原发性肿瘤经手术切除并酶解解为单细胞悬液悬浮液（1×10 6个细胞）又原位移植到6-7周龄的受体同基因雌性小鼠中，其中单一原发性乳腺肿瘤发生在38至60天的时间段3 ）处，将受体小鼠麻醉并手术切除原发性肿瘤，使得在手术部位的肿瘤再生长被最小化，与女性的手术一致（ 补充图1 ）。在FVB / n株系背景下，小鼠在手术后〜115天内以45％的外显率在肺中发展组织学检测的转移灶（ 图1B ）。随着转移性肿瘤生长的这种延长的延迟，该模型在辅助治疗递送中是独特的，并且用于阐明和评估影响原发性肿瘤手术切除后的转移进展的潜在生物学。

<table><tbody><tr><td>Isofluorane</td><td>Piramal Healthcare</td><td>N/A</td><td>Prescription order</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>11088793001</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>12634010</td><td></td></tr><tr><td>25 mL Pyrex bottle</td><td>Sigma-Aldrich</td><td>CLS139525</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Bio&amp;nbsp;</td><td>S11150</td><td></td></tr><tr><td>0.7 &amp;micro;m nylon strainer&amp;nbsp;</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>50 mL conical tube&amp;nbsp;</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Growth factor-reduced Matrigel&amp;nbsp;</td><td>BD</td><td>354230</td><td>Growth factor-reduced solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma</td></tr><tr><td>Poly(vinylpyrrolidone)&amp;ndash;Iodine complex</td><td>Sigma-Aldrich</td><td>PVP1</td><td></td></tr><tr><td>29 gauge 0.3 mL insulin syringe</td><td>BD</td><td>324702</td><td></td></tr><tr><td>Small Vessel Cauterizer Kit</td><td>FST</td><td>18000-00</td><td></td></tr><tr><td>Wound clips</td><td>Texas Scientific</td><td>205016</td><td></td></tr><tr><td>AutoClip wound clip applier&amp;nbsp;</td><td>BD</td><td>427630</td><td></td></tr><tr><td>AutoClip wound clip remover&amp;nbsp;</td><td>BD</td><td>427637</td><td></td></tr><tr><td>Bromodeoxyuridine</td><td>Roche</td><td>10280879</td><td></td></tr><tr><td>Heparinized capillary tubes&amp;nbsp;</td><td>Fisher</td><td>22362566</td><td></td></tr><tr><td>Microtainer tubes with dipotassium EDTA&amp;nbsp;</td><td>BD</td><td>365974</td><td></td></tr><tr><td>20 mL syringe&amp;nbsp;</td><td>BD</td><td>309661</td><td></td></tr><tr><td>DPBS</td><td>Thermo-Fisher</td><td>14190-250</td><td></td></tr><tr><td>OCT-freezing medium&amp;nbsp;</td><td>VWR</td><td>25608930</td><td></td></tr><tr><td>Formalin, Buffered, 10% (Phosphate Buffer)</td><td>Fisher</td><td>SF100-4</td><td></td></tr><tr><td>23g needle</td><td>Fisher</td><td>14-826-6B</td><td></td></tr><tr><td>FVB/n mouse</td><td>Jackson Laboratories</td><td>001800</td><td></td></tr></tbody></table>

surgical procedures and methodology for a preclinical murine model of de novo mammary cancer metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

从衍生自MMTV-PyMT小鼠的原发乳腺肿瘤中接受1×10 6个细胞的大于75％的受体小鼠在38-60天内开发尺寸为172至450mm 3的单个乳腺腺癌（数据未显示）。符合随机分组的小鼠然后在手术切除原发性肿瘤后纳入研究组（ 图1C ）。在不到2％的原发肿瘤手术切除的小鼠中鉴定到原发性肿瘤再生长（ 补充 图1 ）。?...

Watch this Scientific Journal Video about Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis at JoVE.com

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

评估靶向乳腺癌转移的辅助治疗的临床前模型缺乏。为了解决这个问题，我们开发了从头肺乳腺癌转移，其特征在于，在辅助设定（后手术切除原发肿瘤的）给药的治疗可以为疗效影响先前接种的肺转移进行评估的鼠模型。

临床前小鼠模型的外科手术和方法<em&gt; De Novo</em&gt;乳腺癌转移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

surgical-procedures-methodology-for-preclinical-murine-model-de-novo

Research

JoVE Journal

Cancer Research

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

13.8K 次观看.  Oregon Health & Science University. 评估靶向乳腺癌转移的辅助治疗的临床前模型缺乏。为了解决这个问题，我们开发了从头肺乳腺癌转移，其特征在于，在辅助设定（后手术切除原发肿瘤的）给药的治疗可以为疗效影响先前接种的肺转移进行评估的鼠模型。 

视频: Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

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 ...

科研

癌症研究

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

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 ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

医学

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, ...

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 ...

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.

自发性乳腺癌转移的原位小鼠模型

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

小鼠原位转移性乳腺癌模型的建立及小鼠根治性乳房切除术

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

临床前小鼠模型的外科手术和方法

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