使用生物发光监测小鼠乳腺癌生长和转移集落形成
Summary
在这里，我们描述了一种涉及荧光素酶和绿色荧光蛋白在各种乳腺癌细胞系中表达的非侵入性监测方法。该协议提供了一种在小鼠中实时监测肿瘤形成和转移定植的技术。
Abstract
乳腺癌是一种常见的异质性恶性肿瘤，也是女性死亡的第二大原因，主要是由于远处的器官转移。已经产生了几种动物模型，包括广泛使用的原位小鼠模型，其中癌细胞被注射到乳腺脂肪垫中。然而，这些模型不能帮助监测肿瘤生长动力学和转移定植。在小鼠中实时监测癌细胞的尖端工具将显着促进对肿瘤生物学的理解。
在这里，建立了稳定表达荧光素酶和绿色荧光蛋白（GFP）的乳腺癌细胞系。具体而言，该技术包含两个连续的步骤，通过在 体外 测量荧光素酶活性而启动，然后将癌细胞植入非肥胖严重联合免疫缺陷（NOD-SCID）小鼠的乳腺脂肪垫中。注射后，通过无创生物发光成像系统实时监测肿瘤生长和转移定植。然后，通过荧光显微镜检查肺部表达GFP转移的定量，以验证观察到的生物发光结果。这种复杂的系统结合了荧光素酶和基于荧光的检测工具，可评估体内的癌症转移 ， 这在乳腺癌治疗和疾病管理中具有巨大的应用潜力。
Introduction
乳腺癌是全世界常见的癌症类型，美国每年诊断出约250，000例新病例1。尽管发病率很高，但一套新的抗癌药物显着改善了乳腺癌患者的预后2。然而，这些治疗仍然不足，因为许多患者经历疾病复发和转移扩散到重要器官2，这是患者发病和死亡的主要原因。因此，乳腺癌研究的主要挑战之一是确定调节远端转移形成的分子机制，以开发抑制其发展的新方法。
癌症转移是一个动态过程，其中细胞从原发肿瘤中分离并通过血液循环侵入邻近组织。因此，其中细胞经历类似转移级联反应的动物模型可以促进识别控制该过程的机制3，4。此外，这些 体内 模型对于开发乳腺癌治疗剂5，6是必不可少的。然而，这些原位模型不能指示实际的肿瘤生长动力学，因为效果仅在终止时确定。因此，我们建立了一种基于荧光素酶的工具，以实时检测肿瘤发展和转移定植。此外，这些细胞表达GFP以检测转移集落。这种方法相对简单，不涉及任何侵入性操作3。因此，结合荧光素酶和荧光检测是推进乳腺癌治疗和疾病管理的临床前研究的有用策略。
Protocol
所有小鼠实验均在希伯来大学机构动物护理和使用委员会批准的协议MD-21-16429-5下进行。此外，希伯来大学还获得了实验动物护理评估和认证协会（AAALAC）的认证。 1. 细胞系维持 注意：本方案中使用了人乳腺癌细胞系（MCF-7，MDA-MB-468和MDA-MB-231）。 在Dulbecco的改良Eagle培养基（DMEM）中培养所有乳腺癌细胞系，并在37°C下在加湿的二氧化碳（5%CO 2）培?…
Representative Results
我们生成了表达GFP和荧光素酶载体的乳腺癌细胞系（MDA-MB-231，MCF-7和MDA-MB-468）。具体来说，这是通过顺序感染实现的。首先，乳腺癌细胞系感染了表达荧光GFP的慢病毒载体。GFP阳性细胞（GFP+）在感染后2天进行分类（图1A，B）并感染pLX304荧光素酶-V5载体。然后，使用杀菌素选择荧光素酶以产生指示的（GFP +，Luc +）细胞。为了验证 体?…
Discussion
基于动物的实验是癌症研究的必要条件7，8，9，实际上已经开发了许多方案3，6，10，11，12，13，14。然而，这些研究中的大多数仅在实验结束时确定了…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢Y.D.S.实验室的成员。我们要感谢耶路撒冷哈达萨医学中心的Wohl转化医学研究所提供小动物成像设施。这项研究得到了以色列癌症研究基金会的研究 职业发展奖 的支持。
Materials
| 1.7 mL eppendorf tubes | Lifegene | LMCT1.7B-500 | |
| 10 µL tips | Lifegene | LRT10 | |
| 1000 µL tips | Lifegene | LRT1000 | |
| 15 mL tubes | Lifegene | LTB15-500 | |
| 200 µL tips | Lifegene | LRT200 | |
| 6 well cell culture plate | COSTAR | 3516 | |
| 96 well Plates BLACK flat bottom | Bar Naor | BN30496 | |
| Automated Cell Counters | Thermofisher | A50298 | |
| BD FACSAria III sorter | BD | ||
| BD Microlance 3 Needles 27 G (3/4'') | BD | 302200 | |
| BD Plastipak Syringes 1 mL x 120 | BD | 303172 | |
| Corning 100 mm x 20 mm Style Dish | CORNING | 430167 | |
| Corning 150 mm x 20 mm Style Dish | CORNING | 430599 | |
| Countess cell counting chamber slides | Thermofisher | C10228 | |
| Dulbecco's modified Eagle's medium (DMEM), high glucose, no glutamine | Biological Industries | 01-055-1A | |
| Eclipse 80i microscope | Nikon | ||
| eppendorf Centrifuge 5810 R | Sigma Aldrich | EP5820740000 | |
| Fetal Bovine Serum (FBS) | Biological Industries | 04-127-1A | |
| FUW GFP | Gifted from Dr. Yossi Buganim's lab (Hebrew University of Jerusalem) | ||
| HEK293T | Gifted from Dr. Lior Nissim's lab (Hebrew University of Jerusalem) | ||
| Isoflurane, USP Terrell | Piramal | NDC 66794-01-25 | |
| IVIS Spectrum In Vivo Imaging System | Perkin Elmer | 124262 | |
| L-Glutamine Solution | Biological industries | 03-020-1A | |
| Living Image Software | PerkinElmer | bioluminescence measurement | |
| MCF-7 | ATCC | ATCC HTB-22 | |
| MDA-MB-231 | ATCC | ATCC HTB-26 | |
| MDA-MB-468 | ATCC | ATCC HTB-132 | |
| Pasteur pipettes | NORMAX | 2430-475 | |
| PBS | Hylabs | BP655/500D | |
| pCMV-dR8.2-dvpr | Addgene | #8455 | Provided by David M. Sabatini’s lab (Whitehead institute, Boston, USA) |
| pCMV-VSV-G | Addgene | #8454 | Provided by David M. Sabatini’s lab (Whitehead institute, Boston, USA) |
| Penicillin-Streptomycin Solution | Biological Industries | 03-031-1B | |
| Petri dish 90 mm (90×15) | MINI PLAST | 820-090-01-017 | |
| Pipettes 10ml | Lifegene | LG-GSP010010S | |
| Pipettes 25ml | Lifegene | LG-GSP010050S | |
| Pipettes 5ml | Lifegene | LG-GSP010005S | |
| pLX304 Luciferase-V5 blast plasmid | Addgene | #98580 | |
| Polybrene | Sigma Aldrich | #107689 | |
| Prism 9 | GraphPad | ||
| Reagent Reservoirs | Bar Naor | BN20621STR200TC | |
| SMZ18 Stereo microscopes | Nikon | ||
| Sodium Chloride | Bio-Lab | 190359400 | |
| Syringe filters | Lifegene | LG-FPV403030S | |
| Trypan Blue 0.5% solution | Biological industries | 03-102-1B | |
| Trypsin EDTA Solution B (0.25%), EDTA (0.05%) | Biological Industries | 03-052-1a | |
| Vacuum driven Filters | SOFRA LIFE SCIENCE | SPE-22-500 | |
| Virusolve | disinfectant | ||
| VivoGlo Luciferin, In Vivo Grade | Promega | P1043 | |
| X-tremeGENE HP DNA Transfection Reagent | Sigma Aldrich | #6366236001 |
Referências
- Waks, A. G., Winer, E. P. Breast cancer treatment: A review. JAMA. 321 (3), 288-300 (2019).
- Jin, X., Mu, P. Targeting breast cancer metastasis. Breast Cancer: Basic and Clinical Research. 9, 23-34 (2015).
- Saha, D., et al. In vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model. Journal of Visualized Experiments: JoVE. (56), e3175 (2011).
- Rashid, O. M., et al. Is tail vein injection a relevant breast cancer lung metastasis model. Journal of Thoracic Disease. 5 (4), 385-392 (2013).
- Fantozzi, A., Christofori, G. Mouse models of breast cancer metastasis. Breast Cancer Research. 8 (4), 212 (2006).
- Kocatürk, B., Versteeg, H. H. Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth. Journal of Visualized Experiments: JoVE. (96), e51967 (2015).
- Baker, M. The whole picture. Nature. 463 (7283), 977-979 (2010).
- Wang, Y., Tseng, J. -. C., Sun, Y., Beck, A. H., Kung, A. L. Noninvasive imaging of tumor burden and molecular pathways in mouse models of cancer. Cold Spring Harbor Protocols. 2015 (2), 135-144 (2015).
- Kim, J. E., Kalimuthu, S., Ahn, B. -. C. In vivo cell tracking with bioluminescence imaging. Nuclear Medicine and Molecular Imaging. 49 (1), 3-10 (2015).
- Paschall, A. V., Liu, K. An orthotopic mouse model of spontaneous breast cancer metastasis. Journal of Visualized Experiments: JoVE. (114), (2016).
- Morten, B. C., Scott, R. J., Avery-Kiejda, K. A. Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines. Journal of Visualized Experiments. (115), e54040 (2016).
- Zimmerman, M., Hu, X., Liu, K. Experimental metastasis and CTL adoptive transfer immunotherapy mouse model. Journal of Visualized Experiments: JoVE. (45), e2077 (2010).
- Lv, X., et al. Orthotopic transplantation of breast tumors as preclinical models for breast cancer. Journal of Visualized Experiments: JoVE. (159), e61173 (2020).
- Cheng, R. Y. S., et al. Studying triple negative breast cancer using orthotopic breast cancer model. Journal of Visualized Experiments: JoVE. (157), e60316 (2020).
- Bajikar, S. S., et al. Tumor-suppressor inactivation of GDF11 occurs by precursor sequestration in triple-negative breast cancer. Developmental Cell. 43 (4), 418-435 (2017).
- Khatib, A., et al. The glutathione peroxidase 8 (GPX8)/IL-6/STAT3 axis is essential in maintaining an aggressive breast cancer phenotype. Proceedings of the National Academy of Sciences of the United States of America. 117 (35), 21420-21431 (2020).
Citar este artigo
Solaimuthu, B., Hayashi, A., Khatib, A., Shaul, Y. D. Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence. J. Vis. Exp. (177), e63060, doi:10.3791/63060 (2021).