使用快速和定量的细胞内胆碱膜模型发现转移调节器
Summary
这是一种有效的方法来筛选抑制剂或癌症转移的驱动因素。细胞，用表达库转导，被注射到鸡胆碱膜血管，形成转移菌落。已降低或增加侵入性的菌落被切除、扩大、重新注入以确认其表型，最后使用高吞吐量测序进行分析。
Abstract
癌症研究的最新进展说明了癌症转移的高度复杂性。发现多个基因或基因网络参与不同程度地调节癌症转移级联基因和基因产物，取决于癌症类型、组织和单个患者特征。这些是基因治疗和个性化医学方法的潜在重要目标。开发快速筛选平台对于确定这些遗传目标至关重要。
小鸡胆碱膜（CAM）是一种高度血管化、富含胶原蛋白的膜，位于蛋壳下，允许在发育中的胚胎中进行气体交换。由于CAM的位置和血管化，我们开发它作为一个活体人类癌症转移模型，允许强大的人类癌细胞异种化和实时成像癌细胞与胶原蛋白丰富的基质和血管的相互作用。
利用这种模型，设计了一个定量筛查平台，用于识别癌症转移的新驱动因素或抑制剂。我们用完整的人类基因组shRNA基因库转导了一池头部和颈部HEp3癌细胞，然后以低密度将细胞注入CAM血管。细胞增殖并形成单肿瘤细胞群落。无法侵入CAM组织的个体菌落被视为紧凑的菌落表型，并被切除，以识别细胞中存在的转导shRNA。对个别殖民地的入侵性进行了评估。进行了多轮选择，以降低误报率。个体、分离的癌细胞克隆或表达感兴趣基因的新设计的克隆受到原发性肿瘤形成检测或癌细胞血管共选分析。总之，我们提出了一个快速筛选平台，允许对动态和复杂的事件级联进行反转移目标识别和活性分析。
Introduction
转移是癌症患者死亡的主要原因1，2，3。转移性癌细胞利用不同的信号通路，取决于癌症的类型，在整个转移级联的五个步骤：局部入侵，内化，在循环中生存，奢侈和殖民地扩张在遥远的转移点。目前对这一转移过程的理解表明，有两个瓶颈步骤，一个是原发性肿瘤对癌细胞的定向侵入，二是建立远地点转移病变4、5、6。这两个步骤都需要癌细胞在初始入侵或遥远转移性病变形成的部位与胶原蛋白和血管积极相互作用。因此，转移性癌细胞必须能够附着在细胞上，重塑胶原纤维，并沿着血管壁定向侵入7。筛选模型，可以迅速识别抗金属治疗目标，以阻止癌细胞完成这些步骤是最重要的。现有的体外筛查模型并不能完全模拟复杂的活组织环境。鼠标型号既昂贵又耗时。因此，迫切需要提供复杂的活组织环境和快速识别目标的活体筛查平台。
在过去十年中，鸡胚胎已被确定为人类癌症转移8、9、10、11、12的强大且具有成本效益的模型。小鸡胆管素膜（CAM）组织是薄和半透明的，这使得它非常适合细胞和细胞群落行为在原发性肿瘤和/或转移位点12的活性显微成像。从多个人类癌细胞系的原发性肿瘤生长可以在微注射到CAM组织后几天内启动和转移。癌细胞可以通过多种方式管理到CAM组织，包括静脉注射、内膜或作为植物上的胶原蛋白，这种灵活性使研究人员能够专注于癌症进展的特定阶段，例如转移性病变形成、从原发性肿瘤中侵入或血管生成。
在这里，我们描述了一个定量筛选平台，可用于测量癌细胞建立侵入性转移性病变的能力。用表达库转导的癌细胞以低密度静脉注射到CAM血管中。转移菌落形成4-5天，然后评估由此产生的殖民地的入侵能力和血管相互作用。未入侵的个体菌落被切除、传播，其表型通过重新注射和量化菌落紧凑度和癌细胞血管接触在CAM中得到确认。负责单转移性群落突变表型的表达库结构通过高通量测序从分离的群落基因组DNA中识别出来。同一平台可进一步用于验证基因与观察到表型之间的因果关系，或对观察到的表型进行深入的机械研究。
Protocol
Representative Results
Discussion
在这里，我们描述了基于快速荧光显微镜的活性筛选方案，可用于重要的应用，如基因或药物候选屏幕。使用这种鹰凸轮模型快速筛选和量化使用感兴趣的表型的癌细胞，这些癌细胞已被遗传感兴趣的基因库转导或与单个表达结构发生感染。由于转导或转染协议因库类型而异，因此未包含在此程序中。表型相关的转移菌落从 CAM 中切除，扩展和 DNA 分离，用于高通量测序，以确定兴趣的表达结构。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了加拿大癌症协会研究所的资助，该研究所向JDL和KS#702849。刘易斯博士在艾伯塔省癌症基金会的支持下担任前列腺癌研究的弗兰克和卡拉·索琼基主席。
Materials
| 1 mL disposable syringes | BD | BD309659 | |
| 15 mL conical centrifuge tubes. | Corning | CLS430791-500EA | |
| 18 gauge x 1 1/2 BD precision needle | BD | BD305196 | we use 1.2mm x 40mm, it is possible to use shorter needles if preferred |
| 2.5% Trypsin solution | many sources are available | ||
| 4T1 mouse breast cancer | ATCC | CRL-2539 | |
| B16F10 mouse melanoma cell line | ATCC | CRL-6475 | |
| Benchtop centrifuge. | many sources are available | Any TC compatible centrifuge that can be used to spin down the cells is suitable | |
| Circular coverslips, 22 mm. | Fisher Scientific | 12-545-101 | |
| Collagenase | Sigma | C0130-100MG | |
| Confocal microscope | We use Nikon A1r | ||
| cotton swabs | many sources are available | must be sterilized before use | |
| Culture media appropriate for the cell lines used | many sources are available | We grow HT1080, HEp3 and b16 cell lines in DMEM, 10% FBS media | |
| Egg incubator | many sources are available | An exact model that is necessary depends on the scale of the screen. Available sources are MGF Company Inc., Savannah, GA, or Lyon Electric Company Inc., Chula Vista, CA | |
| eppendor tubes , 1.5ml | Sigma | T4816-250EA | |
| Fertilized White Leghorn eggs | any local supplyer | ||
| fine forceps | many sources are available | must be sterilized begfore use | |
| Hemocytometer | Millipore-Sigma | MDH-4N1-50PK | |
| HT1080 human fibrosarcoma cell line | ATCC | CCL-121 | |
| Image analysis software | We use Nikon Elements | ||
| Lectin Lens Culinary Agglutinin (LCA) conjugated with Fluorescein or Rhodamine | Vector Laboratories | RL-1042, FL-1041 | Dilute stock (5mg/ml) 50-100x depending on the microscope sensetivity. Must be a different color from the color of cell line used for screening |
| MDA-MB-468 human breast cancer | ATCC | HTB-132 | |
| PBS (1x) | many sources are available | ||
| Plastic weighting dishes | Simport | CA11006-614 | dimensions are 78x78x25mm; many other sources are available |
| small surgical scissors | many sources are available | must be sterilized before use | |
| Sodium borosilicate glass capillary tubes, outer diameter 1.0 mm, inner diameter 0.58 mm, 10 cm length | Sutter Instrument | BF100-58-10 | |
| Square petri dishes (used as lids for the weighting dishes). | VWR | CA25378-115 | dimensions are 100x100x15mm; many other sources are available |
| Stereo fluorescent microscope | We use Zeiss Lumar v12 | ||
| Tygon R-3603 laboratory tubing | Cole-Parmer | AAC00001 | 1/32 in inner diameter, 3/32 in. outer diameter, 1/32 in. wall thickness |
| U-118 MG human glioblastoma | ATCC | HTB-15 | |
| U-87 MG human glioblastoma | ATCC | HTB-14 | |
| Vertical pipette puller | many sources are available | we use David Kopf Instruments, Tujunga, CA; Model 720 |
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