结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担
Summary
所述方法将实验性尾静脉转移测定与活体动物成像相结合，除了对肺部转移数和大小进行定量外，还能够实时监测乳腺癌转移的形成和生长。
Abstract
转移是癌症相关死亡的主要原因，转移性疾病患者的治疗选择有限。鉴定和测试新的治疗靶点，这将有助于开发更好的治疗转移性疾病需要临床前在体内模型。这里演示的是一个合成小鼠模型，用于分析乳腺癌转移殖民化和后续生长。转移性癌细胞通过编码萤火虫荧光素酶和ZsGreen蛋白的病毒载体稳定地转导。然后，候选基因在荧光素酶 / ZsGreen 表达的癌细胞中稳定地纵，然后通过侧尾静脉将细胞注射到小鼠体内，以测定转移的殖民化和生长。然后，使用体内成像设备测量活体动物肿瘤细胞的生物发光或荧光，以量化转移性负担随时间的变化。荧光蛋白的表达允许在实验结束时对肺部转移的数量和大小进行量化，而无需切片或组织染色。该方法提供了一种相对快速、简便的方法，用于测试候选基因在转移性殖民化和生长中的作用，并且比传统的尾静脉转移测定提供了更多的信息。使用这种方法，我们表明，在乳腺癌细胞中同时敲除Yes相关蛋白（YAP）和带有PDZ结合图案（TAZ）的转录共激活剂可减轻肺部转移负担，这种减轻的负担是转移殖民化显著受损和减少转移生长的结果。
Introduction
癌症仍然是全球第二大死因1和转移是造成这些死亡大多数2，3。然而，对控制转移性殖民化和随后生长的分子机制的了解有限，阻碍了对转移性疾病的有效治疗的发展。确定新的治疗靶点需要一种测定，以测试候选基因的扰动表达或功能如何影响转移形成和生长。虽然自主鼠标模型有其优点，但它们的生成既耗时又昂贵，因此更适合于目标验证而不是目标发现。移植模型系统，其中候选基因在体外癌细胞中扰动，然后对转移电位的影响在体内被评估，比自体模型更便宜，吞吐量更高。此外，用于稳定传递RNAi、CRISPR/CAS9和转基因的病毒载体也随处可见，使得在癌细胞系中几乎容易干扰任何感兴趣的基因或基因。这种方法也可以用来通过将细胞移植到免疫功能低下或人化小鼠中来分析候选基因在转移性殖民化和人类癌细胞系生长中的作用。
用于测试移植癌细胞在体内形成的两种检测是自发转移测定和实验性转移测定。在自发转移测定4，5中，癌细胞被注射到小鼠体内，允许形成原发性肿瘤，然后对自发转移形成和随后的生长进行测定。此模型的强度是细胞必须完成转移过程的所有步骤，才能形成转移性肿瘤。然而，许多癌细胞系在自发转移模型中不能有效地转移，任何影响原发性肿瘤生长的细胞操作都可能混淆转移测定的结果。实验转移测定，其中癌细胞直接注射到循环，用于避免这些陷阱。常见的实验性转移测定包括尾静脉注射6，7，8（这里演示），心内注射9，和门户静脉注射10。
此处介绍的协议的目的是提供一个体内实验性转移测定，允许研究人员实时监测转移形成和生长，以及量化同一小鼠肺部的端点转移数和大小。为此，传统的实验尾静脉转移测定6、7、8与活体动物成像相结合，使用体内成像装置9、11、12、13、14。通过侧尾静脉将稳定表达荧光素酶和荧光蛋白的肿瘤细胞注射到小鼠体内，然后利用体内成像装置测量肺部转移性负担随时间的变化（图1）。然而，活体动物成像设备无法区分或测量单个转移的大小。因此，在实验结束时，使用荧光立体显微镜计算和测量肺部荧光转移的大小，而无需切片和组织学或免疫组织化学（图1）。该协议可用于测试改变候选基因的表达或功能如何影响转移形成和生长。潜在的治疗化合物，如小分子或功能阻断抗体也可以测试。
为了证明这种方法，我们首先进行了概念验证实验，其中在转移小鼠乳腺癌细胞中击倒了必要的复制因子，复制蛋白A3（RPA3）。研究表明，与注射控制细胞的小鼠相比，注射RPA3敲除细胞的小鼠在每个时间点的转移负担明显较少。对含转移的肺的分析表明，这种转移负担的减少是显著减少转移殖民化和损害形成转移的生长的结果。为了进一步演示此技术，我们测试了同时击倒 Yes 相关蛋白 （YAP） 和带有 PDZ 结合图案 （TAZ） 的转录共激活剂是否会损害转移性殖民化或后续生长。YAP 和 TAZ 是两个相关的转录共激活器，是 Hippo 路径的关键下游效应器。我们15，16和其他已经牵连YAP和TAZ在转移（在17，18，19），表明这些蛋白质是很好的治疗目标。一直，我们发现注射YAP/TAZ敲除细胞的小鼠显著减轻了转移负担。对肺部的分析表明，YAP/TAZ敲除细胞形成的转移要少得多，而确实形成的转移较小。这些实验演示了实验性转移测定如何让研究人员快速、廉价地测试候选基因在转移形成和生长中的作用。他们进一步展示了活体动物成像和整个肺部转移的荧光定量的组合使用如何使研究人员更好地了解转移殖民化过程中的步骤。
Protocol
该协议涉及使用小鼠和生物有害物质，需要获得相关机构安全委员会的批准。这里所有描述的体内工作都得到奥尔巴尼医学院动物护理和使用委员会（IACUC）的批准。 注：有关协议概述，请参阅图 1中的原理图。 1. 包装所有必需的逆转录病毒和慢病毒 注：所述协议使用慢病毒或逆转录病毒载体来稳稳地表达荧光?…
Representative Results
为了演示上述方法，我们进行了概念验证实验，其中关键的复制因子 RPA3 在转移性小鼠乳腺癌细胞系 （4T122） 中被击倒。虽然该协议描述了在基因操作之前用荧光素酶和荧光蛋白标记细胞，但我们使用了一种改良的方法，因为RNAi载体也提供ZsGreen（图2A）。首先，4T1细胞通过慢病毒结构稳稳地转导，编码萤火虫荧光素酶和湿霉素抗性（pHAGE-Lucife…
Discussion
方法的关键步骤
优化给定细胞系和小鼠菌株的细胞数量（步骤 3）至关重要，因为这将极大地影响形成转移的数量和实验的长度。如果注射的细胞过多或转移生长时间过长，转移可能难以计数，因此难以评估基因操作的影响。然而，如果注射的细胞太少，可能形成很少或没有转移。因此，应使用不同数量的细胞进行初步实验，以确定转移生长的最佳数量和时间长度。为了确保一个?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢艾米莉·诺顿协助病毒感染和批判性阅读手稿。我们还感谢Ryan Kanai帮助获取肺部图像，凯特E.Tubbesing帮助研究肺部绿色转移的图像分析。我们感谢动物研究设施的工作人员的支持和协助，在编写这个视频。这项工作得到了苏珊·科门职业催化剂奖的支持，该奖金授予J.M.L.（#CCR17477184）。
Materials
| 10% SDS-PAGE Gel | For western blot | ||
| 2.5% Trypsin | Gibco | 15090-046 | Trypsin for tissue culture |
| 96 well flat bottom white assay plate | Corning | 3922 | For measuring luciferase and renilla signal in cultured cells |
| Alcohol wipes | For sterolizing the injection site before tail vein injecitons | ||
| BALB/C mice (female, 6 weeks) | Taconic | BALB-F | For tail vein metastatic colonization and burden assays |
| BSA regular | VWR Ameresco | 97061-416 | For western blot |
| Cell lysis buffer | Cell Signaling | For collecting protien samples | |
| Celltreat Syringe Filters, PES 30mm, 0.45 μm | Celltreat | 40-229749-CS | For filtering viral supernatant |
| CO2 and euthanasia chamber | For euthanasing the mice | ||
| Dual-luciferase reporter assay kit | Promega | E1960 | For measuring luciferase and renilla signal in cultured cells |
| Dulbecco&39;s phosphate buffered saline | Himedia | TS1006 | For PBS |
| EDTA | VWR | 97061-406 | Used to dilute trypsin for tissue culture |
| FBS 100% US origin | VWR | 97068-085 | Component of complete growth media |
| Fujifilm LAS-3000 gel imager | Fujifilm | For western blot | |
| GAPDH(14C10) Rabibit mAb | Cell Signaling | 2118 | For western blot |
| Goat anti-rabbit IgG (H+L) Secondary Antibody, HRP conjugate | Thermo Scientific | 31460 | For western blot |
| Human embryonic kidney cells, HEK-293FT | Invitrogen | R70007 | Cell line used for packging virus |
| HyClone DMEM/High clucose | GE Healthcare life sciences | SH30243.01 | Component of complete growth media |
| Hygromycin B, Ultra Pure Grade | VWR Ameresco | 97064-810 | For antibiotic selection of infected cells |
| I3-P/i3 Multi-Mode Microplate/EA | Molecular devices | For measuring luciferase and renilla signal in cultured cells | |
| Imagej | Used for image analysis of lung metastases: threshold set to 25 & 100 | ||
| Immuno-Blot PVDF Membrane | Biorad | 1620177 | For western blot |
| Isoflurane | For mouse anesthesia | ||
| IVIS Lumina XRMS In Vivo Imaging System (in vivo live animal imaging device) | PerkinElmer | CLS136340 | For in vivo imaging of metastatic burden |
| Leica M205 FA & Lecia DCF3000 G (GFP and bright field filters) | Leica Microsystems | Microscope and camera for visualing, counting and taking pcitures of metastases in the lungs; 10X magnifacation, 3.5 sec exposure, 1.4 gain | |
| L-Glutamine | Gibco | 25030-081 | Component of complete growth media |
| Lipofectamine 3000 | Life technologies | L3000008 | For YAP/TAZ-TEAD reporter transfection |
| Living Image 3.2 (image software program) | PerkinElmer | Software for IVIS | |
| Mouse breast cancer cells, 4T1 | Karmanos Cancer Institute | Aslakson, CJ et al.,1992 | Mouse metastatic breast cance cell line |
| Multi-Gauge version 3.0 | Fujifilm | Software for quantifying western blot band intensity | |
| Opti-MEM (transfection buffer) | Gibco | 31985-062 | For packaging virus and transfection |
| Penicillin Streptomycin | Gibco | 15140-122 | Component of complete growth media |
| Pierce BCA protein assay kit | Thermo Scientific | 23225 | For quantifying protein concentration |
| Pierce Phosphatase Inhibitor Mini Tablets | Thermo Scientific | A32957 | Added to cell lysis buffer |
| Pierce Protease Inhibitor Mini Tablets | Thermo Scientific | A32953 | Added to cell lysis buffer |
| Polybrene (hexadimethrine bromide) | Sigma-Aldrich | 45-H9268 | For infection |
| Puromycin | Sigma-Aldrich | 45-P7255 | For antibiotic selection of infected cells |
| Rodent restrainer | For restraining mice during tail vein injeciton | ||
| SDS-PAGE running buffer | For western blot | ||
| TAZ (V3886) Antibody | Cell Signaling | 4883 | For western blot |
| TBST buffer | For western blot | ||
| TC20 automated cell counter | Bio-Rad | For counting cells | |
| Vectors | See Table 1 for complete list of vectors | ||
| VWR Inverted Fluorescence Microscope | VWR | 89404-464 | For visualizing fluorescence in ZSGreen labeled cells |
| Western transfer buffer | For western blot | ||
| XenoLight D-Luciferin K+ Salt | PerkinElmer | 122799 | Substrate injected into mice for in vivo bioluminescent IVIS images |
| X-tremeGENE 9 DNA transfection reagent (lipid solution for transfection) | Roche | 6365787001 | For packaging virus |
| YAP (D8H1X) XP Rabbit mAb | Cell Signaling | 14074 | For western blot |
References
- Gupta, G. P., Massague, J. Cancer metastasis: building a framework. Cell. 127 (4), 679-695 (2006).
- Chaffer, C. L., Weinberg, R. A. A perspective on cancer cell metastasis. Science. 331 (6024), 1559-1564 (2011).
- Wendt, M. K., Molter, J., Flask, C. A., Schiemann, W. P. In vivo dual substrate bioluminescent imaging. Journal of Visualized Experiments. (56), (2011).
- Moret, R., et al. Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis. Journal of Visualized Experiments. (147), (2019).
- Lizardo, M. M., Sorensen, P. H. Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay. Journal of Visualized Experiments. (133), (2018).
- Mohanty, S., Xu, L. Experimental metastasis assay. Journal of Visualized Experiments. (42), (2010).
- Welch, D. R. Technical considerations for studying cancer metastasis in vivo. Clinical and Experimental Metastasis. 15 (3), 272-306 (1997).
- Lim, E., et al. Monitoring tumor metastases and osteolytic lesions with bioluminescence and micro CT imaging. Journal of Visualized Experiments. (50), (2011).
- Goddard, E. T., Fischer, J., Schedin, P. A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer. Journal of Visualized Experiments. (118), (2016).
- Cordero, A. B., Kwon, Y., Hua, X., Godwin, A. K. In vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer. Journal of Visualized Experiments. (42), (2010).
- Ozawa, T., James, C. D. Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging. Journal of Visualized Experiments. (41), (2010).
- Lim, E., Modi, K. D., Kim, J. In vivo bioluminescent imaging of mammary tumors using IVIS spectrum. Journal of Visualized Experiments. (26), (2009).
- Oshima, G., et al. Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones. Journal of Visualized Experiments. (117), (2016).
- Lamar, J. M., et al. SRC tyrosine kinase activates the YAP/TAZ axis and thereby drives tumor growth and metastasis. Journal of Biological Chemistry. 294 (7), 2302-2317 (2019).
- Lamar, J. M., et al. The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain. Proceedings of the National Academy of Sciences U S A. 109 (37), 2441-2450 (2012).
- Warren, J. S. A., Xiao, Y., Lamar, J. M. YAP/TAZ Activation as a Target for Treating Metastatic Cancer. Cancers (Basel). 10 (4), (2018).
- Janse van Rensburg, H. J., Yang, X. The roles of the Hippo pathway in cancer metastasis. Cell Signal. 28 (11), 1761-1772 (2016).
- Harvey, K. F., Pfleger, C. M., Hariharan, I. K. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell. 114 (4), 457-467 (2003).
- Journal of Visualized Experiments Science Education Database. Basic Methods in Cellular and Molecular Biology. The Western Blot. Journal of Visualized Experiments. , (2019).
- Wong, W., Farr, R., Joglekar, M., Januszewski, A., Hardikar, A. Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs. Journal of Visualized Experiments. (98), (2015).
- Aslakson, C. J., Miller, F. R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Ressearch. 52 (6), 1399-1405 (1992).
- Fellmann, C., et al. Functional identification of optimized RNAi triggers using a massively parallel sensor assay. Molecular Cell. 41 (6), 733-746 (2011).
- Reticker-Flynn, N. E., et al. A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis. Nature Communications. 3, 1122 (2012).
- Sun, Z., et al. Prognostic Value of Yes-Associated Protein 1 (YAP1) in Various Cancers: A Meta-Analysis. Public Library of Science One. 10 (8), 0135119 (2015).
- Feng, J., Ren, P., Gou, J., Li, Z. Prognostic significance of TAZ expression in various cancers: a meta-analysis. Onco Targets and Therapy. 9, 5235-5244 (2016).
- Ge, L., et al. Yes-associated protein expression in head and neck squamous cell carcinoma nodal metastasis. Public Library of Science One. 6 (11), 27529 (2011).
- Zhang, X., et al. The Hippo pathway transcriptional co-activator, YAP, is an ovarian cancer oncogene. Oncogene. 30 (25), 2810-2822 (2011).
- Vlug, E. J., et al. Nuclear localization of the transcriptional coactivator YAP is associated with invasive lobular breast cancer. Cellular Oncology (Dordrecht). 36 (5), 375-384 (2013).
- Kim, T., et al. MRTF potentiates TEAD-YAP transcriptional activity causing metastasis. European Molecular Biology Organization Journal. 36 (4), 520-535 (2017).
- Nallet-Staub, F., et al. Pro-invasive activity of the Hippo pathway effectors YAP and TAZ in cutaneous melanoma. Journal of Investigative Dermatology. 134 (1), 123-132 (2014).
- Hsu, Y. L., et al. Angiomotin decreases lung cancer progression by sequestering oncogenic YAP/TAZ and decreasing Cyr61 expression. Oncogene. 34 (31), 4056-4068 (2015).
- Lau, A. N., et al. Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis. European Molecular Biology Organization Journal. 33 (5), 468-481 (2014).
- Gu, J. J., et al. Inactivation of ABL kinases suppresses non-small cell lung cancer metastasis. Journal of Clinical Investigation Insight. 1 (21), 89647 (2016).
- Diepenbruck, M., et al. Tead2 expression levels control the subcellular distribution of Yap and Taz, zyxin expression and epithelial-mesenchymal transition. Journal of Cell Science. 127, 1523-1536 (2014).
- Han, S., et al. Suppression of miR-16 promotes tumor growth and metastasis through reversely regulating YAP1 in human cholangiocarcinoma. Oncotarget. 8 (34), 56635-56650 (2017).
- Yin, K., et al. Netrin-1 promotes metastasis of gastric cancer by regulating YAP activity. Biochemical Biophysical Research Communications. 496 (1), 76-82 (2018).
- Guo, L., et al. Knockdown of TAZ modifies triple-negative breast cancer cell sensitivity to EGFR inhibitors by regulating YAP expression. Oncology Reports. 36 (2), 729-736 (2016).
- Liu, Y. N., et al. Loss of Androgen-Regulated MicroRNA 1 Activates SRC and Promotes Prostate Cancer Bone Metastasis. Molecular and Cellular Biology. 35 (11), 1940-1951 (2015).
- Bartucci, M., et al. TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells. Oncogene. 34 (6), 681-690 (2015).
- Wang, T., et al. YAP promotes breast cancer metastasis by repressing growth differentiation factor-15. Biochimica et Biophysica Acta Molecular Basis of Disease. 1864, 1744-1753 (2018).
- Wang, J., Rouse, C., Jasper, J. S., Pendergast, A. M. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling. Science Signaling. 9 (413), (2016).
- Sun, S., Irvine, K. D. Cellular Organization and Cytoskeletal Regulation of the Hippo Signaling Network. Trends in Cell Biology. 26 (9), 694-704 (2016).
- Sharif, G. M., et al. Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling. Oncogene. 34 (48), 5879-5889 (2015).
- Li, C., et al. A ROR1-HER3-lncRNA signalling axis modulates the Hippo-YAP pathway to regulate bone metastasis. Nature Cell Biology. 19 (2), 106-119 (2017).
- Pei, T., et al. YAP is a critical oncogene in human cholangiocarcinoma. Oncotarget. 6 (19), 17206-17220 (2015).
- Qiao, Y., et al. YAP Regulates Actin Dynamics through ARHGAP29 and Promotes Metastasis. Cell Reports. 19 (8), 1495-1502 (2017).
- Zhou, W., Li, X., Premont, R. T. Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes. Journal of Cell Science. 129 (10), 1963-1974 (2016).
- Haemmerle, M., et al. Platelets reduce anoikis and promote metastasis by activating YAP1 signaling. Nature Communcations. 8 (1), 310 (2017).
- Liu, Y., et al. Increased TEAD4 expression and nuclear localization in colorectal cancer promote epithelial-mesenchymal transition and metastasis in a YAP-independent manner. Oncogene. 35 (21), 2789-2800 (2016).
- Hiemer, S. E., et al. A YAP/TAZ-Regulated Molecular Signature Is Associated with Oral Squamous Cell Carcinoma. Molecular Cancer Research. 13 (6), 957-968 (2015).
- Lee, H. J., et al. Fluid shear stress activates YAP1 to promote cancer cell motility. Nature Communications. 8, 14122 (2017).
- Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. M. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. Journal of Virology. 70 (8), 5701-5705 (1996).
- Mahoney, W. M., Hong, J. H., Yaffe, M. B., Farrance, I. K. The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members. Biochemical Journal. 388, 217-225 (2005).
- Zhao, H., et al. Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. Journal of Biomedical Optics. 10 (4), 41210 (2005).
- Chen, M. B., Lamar, J. M., Li, R., Hynes, R. O., Kamm, R. D. Elucidation of the Roles of Tumor Integrin beta1 in the Extravasation Stage of the Metastasis Cascade. Cancer Resesearch. 76 (9), 2513-2524 (2016).
- Labelle, M., Begum, S., Hynes, R. O. Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell. 20 (5), 576-590 (2011).
Cite This Article
Warren, J. S. A., Feustel, P. J., Lamar, J. M. Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs. J. Vis. Exp. (154), e60687, doi:10.3791/60687 (2019).