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
Summary
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.
Abstract
肝转移和进展的速度缓慢的有限数量的患者可以用局部治疗成功治疗方法1,2。然而，知之甚少肝转移的异质性，并且需要能够评估个体转移性集落的发育的动物模型。这里，我们提出，提供定量可视个体肿瘤克隆的发展，在肝脏和估计它们的生长动力学和移效率的能力肝转移的一种先进的模型。我们产生与荧光素酶和tdTomato和拥有不同生长特性稳定标记HCT116人结肠癌细胞的单克隆衍生物的面板。用脾注射后跟脾切除，大多数这些克隆能够产生肝转移，但与定植的不同的频率和不同的生长率。使用体内成像SYSTE米（IVIS），它是可能的可视化和量化转移发展与体内的发光和离体荧光成像。此外，漫射发光成像断层扫描（DLIT）提供肝转移体内三维分布。 体外收获肝脏荧光成像提供个别肝脏转移菌落定量测量，从而允许肝定植的频率的评估和生长动力学的转移。由于该模型类似于临床上观察到的肝脏转移，它可以作为用于检测与肝脏转移相关基因和用于肝脏转移性疾病测试潜在烧蚀或辅助治疗的一种模式。
Introduction
从初级结直肠癌（CRC）肝转移患者被预后不良表征。初级非转移性的CRC的5年生存率（阶段I – III）中被估计为75 – 88％的3,4-，而肝转移患者（IV期）具有的只有8 5年生存率- 12％5 6。然而，转移性患者是一组异质性，不同数量的转移和复发的不同时代的呈现。临床观察表明，转移（其可以是成比例的定植能力或定植频率）和任何单转移（正比于本地生长速率）的大小的数目是独立的预后因素1,7。换句话说，转移克隆定植肝脏的成功取决于两个主要的性能：它们生长的能力和它们的传播，并在肝微生存能力。
该设计成功的临床模型与捕获和定量转移克隆的性能可显着提高我们的肝转移生物学的理解，并提供的潜在的治疗方法的设计的有效工具的能力。实验肝转移模型已经先前报道8,9，但是他们都没有提供定量捕获，并描述在体内和离体个体转移性克隆的性质的能力。
在这里，我们提出肝转移，其中包括肿瘤克隆具有不同的肝脏定植效率和生长特性的产生一个新的，先进的模式。我们采用与荧光素酶和tdTomato荧光蛋白与具有在转移能力固有差异单克隆细胞系的产生癌细胞双标记的组合。在此实验模型中，数据表明，开发肝转移可以在定植频率方面和倍增时间（Td），这是与临床观察结果一致进行说明。该模型的定量性质使得它易于采用用于药物发现和诊断的目的。
Protocol
Representative Results
Discussion
在目前的报告中提出的动物模型是基于两种主要方法。首先，为了确保观察具有不同倾向转移克隆定殖并在肝脏增殖的能力，高度异质的单克隆细胞系的组建立了，而不是建立普通癌细胞系12,13。单克隆办法转移发展受到最近的基因组数据14合理的，以前被成功地用于转移过程10,15,16建模。第二，通过萤光素酶和tdTomato标记的亲代细胞系的双标记，以便提供转移性生长在?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢杰弗里·格林L.博士（芝加哥大学）的Luc2-tdTomato质粒和HCT116细胞系，安仁索兰奇先生（动物资源中心）的小鼠管理，拉拉莱尼博士的协助与DLIT。荧光灯和发光强度Quantifications在综合小动物成像研究资源在芝加哥上IVIS频谱大学（珀金埃尔默，霍普金顿，MA）进行。这项工作是由弗吉尼亚和DK路德维希基金会为癌症研究的支持，肺癌症研究基金会（LCRF），前列腺癌基金会（PCF）和癌症中心支援津贴（P30CA014599）。该资助者在研究设计，数据收集和分析，决定发表或准备手稿中没有作用。
Materials
| IVIS Spectrum In Vivo Imaging System | Caliper Life Sciences | 124262 | In vivo imaging system |
| LivingImage 4.0 Software | Caliper Life Sciences | 128165 | Imaging software |
| VAD-MGX Research Anesthetic Machine | Vetamac | VAD-MGX | Inhalation anesthesia machine |
| DMEM | Gibco | 11965-118 | Cell culture reagents |
| DPBS | Gibco | 14190250 | Cell culture reagents |
| Penicillin-Streptomycin, liquid (10,000 units penicillin;10,000 μg streptomycin) | Invitrogen | 15140163 | Cell culture reagents |
| HBSS | ThermoFisher | 24020117 | Cell culture reagents |
| Buprenex Injection (0.3mg/mL) | Reckitt Benckiser Healthcare Ltd. | 12496-0757-5 | Buprenorphine hydrochloride |
| Gemini Cautery System | Braintree Scientific | GEM 5917 | Hand-held cautery for splenectomy |
| Micro Clip; Straight; 70 Grams Pressure; 1.5mm Clip Width; 10mm Jaw Length | Roboz Surgical Instrument | RS-5426 | Hemoclip: Hemostasis instruments after spleen injection |
| D-luciferin, potassium salt | Goldbio Technology | LUCK-1G | Luciferin potassium salt |
| Opti-MEM I Reduced Serum Medium | Gibco | 31985062 | Reduced Serum Medium |
| TC20 Automated Cell Counter | BIO-RAD | 1450102 | Automatic cell counter |
| JMP10 software | SAS Institute | Data analysis software | |
| BD FACSAria II cell sorter | BD Biocsiences | Cell sorter |
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