JoVE Journal > Cancer Research
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Cancer Research

斑马鱼神经母细胞瘤转移模型

Published: March 14, 2021
doi:
10.3791/62416
, , , ,
1Department of Biochemistry and Molecular Biology,Mayo Clinic College of Medicine, Mayo Clinic Cancer Center

Summary

本文介绍了实时开发、表征和跟踪斑马鱼神经母细胞瘤模型肿瘤转移的方法，特别是在 MYCN 和 LMO1过表达的转基因斑马鱼品系中，该谱系自发地发生转移。

Abstract

斑马鱼已成为研究人类疾病，特别是癌症的重要动物模型。除了应用于斑马鱼建模的强大转基因和基因组编辑技术外，易于维护，高产量生产力和强大的实时成像功能使斑马鱼成为研究转移以及体内该过程背后的细胞和分子基础的有价值的模型系统。第一个斑马鱼神经母细胞瘤（NB）转移模型是通过在多巴胺-β-羟化酶（dβh）启动子的控制下过度表达两个癌基因MYCN和LMO1而开发的。共过表达的MYCN和LMO1导致神经母细胞发生率降低和外显率增加，以及肿瘤细胞的加速远处转移。这种新模型可靠地重申了人类转移性NB的许多关键特征，包括临床相关和转移相关遗传改变的参与;体内转移的自然和自发发展;和转移的保守部位。因此，斑马鱼模型具有剖析体内肿瘤转移复杂过程的独特优势。

Introduction

斑马鱼已被广泛使用并应用于多个研究领域，特别是在癌症方面。该模型提供了许多优点 – 例如其强大的繁殖，经济高效的维护以及肿瘤生长和转移的多功能可视化 – 所有这些都使斑马鱼成为研究和研究肿瘤发生和转移的细胞和分子基础的强大工具。用于大规模基因组图谱、转基因、基因过表达或敲除、细胞移植和化学筛选的新技术极大地增强了斑马鱼模型的力量1。在过去的几年中，已经开发了许多斑马鱼系来研究各种人类癌症的肿瘤发生和转移，包括但不限于白血病，黑色素瘤，横纹肌肉瘤和肝细胞癌2，3，4，5。此外，第一个斑马鱼神经母细胞瘤（NB）模型是通过在多巴胺-β-羟化酶（dβh）启动子的控制下在外周交感神经系统（PSNS）中过度表达MYCN（一种癌基因）而产生的。通过该模型，进一步证明激活的ALK可以与MYCN协同作用，以加速肿瘤发作并增加体内肿瘤外显率6。

NB源自神经嵴细胞的交感肾上腺谱系，是儿童中高度转移的癌症7。它占儿科癌症相关死亡的10%8。NB在诊断时广泛转移，临床上可表现为主要起源于交感神经节链和PSNS9，10的肾上腺髓链的肿瘤。 MYCN 扩增通常与 NB 患者的不良结局相关11，12。此外， LMO1 已被确定为高风险病例中的关键NB易感基因13，14。研究发现，在斑马鱼模型的PSNS中 ，MYCN 和 LMO1 的转基因共表达不仅促进了NB的早期发作，而且还诱导了与高危NB13患者常见的相似部位的组织和器官的广泛转移。最近，在较新的NB斑马鱼模型中也观察到了NB的另一种转移表型，其中编码RNA结合蛋白的 MYCN 和 Lin28B 在 dβh 启动子的控制下都过度表达16。

斑马鱼的稳定转基因方法通常用于研究目的基因的过表达是否有助于正常发育和疾病发病机制14，15。该技术已成功用于证明多个基因和途径对NB肿瘤发生的重要性6，16，17，18，19，20。本文将介绍如何在PSNS中产生过表达MYCN和LMO1的转基因鱼系，以及如何证明这两种癌基因的合作加速了NB肿瘤发生和转移的发生13。首先，通过将dβh-EGFP-MYCN构建体注射到野生型（WT）AB胚胎的单细胞阶段，开发了在dβh-EGFP-MYCN（指定为MYCN系）控制下过表达EGFP-MYCN的转基因系，如前所述6，17。通过将两个DNA构建体dβh-LMO1和dβh-mCherry在单细胞阶段13中共注射到WT胚胎中，开发了一种在PSNS（指定的LMO1系）中过表达LMO1的单独转基因系。先前已经证明，共注射的双DNA构建体可以共同整合到鱼类基因组中;因此，LMO1和mCherry在转基因动物的PSNS细胞中共表达。一旦注射的F0胚胎达到性成熟，它们就会与WT鱼杂交，以鉴定具有转基因整合的阳性鱼。简而言之，F1后代首先通过荧光显微镜筛选PSNS细胞中的mCherry表达。通过基因组PCR和测序进一步证实了MCherry阳性鱼中LMO1的种系整合。在成功鉴定各转基因品系后，将杂合MYCN和LMO1转基因鱼的后代杂交，生成表达MYCN和LMO1的复合鱼系（指定MYCN;改性活生物体1系）。含肿瘤的MYCN;通过荧光显微镜每两周监测一次LMO1鱼，以寻找远离原发部位的区域，即腺间区域（IRG，相当于人类肾上腺的斑马鱼）转移性肿瘤的证据13。确认MYCN中肿瘤的转移;应用改性活生物体1鱼组织学和免疫组化分析。

Protocol

所有使用斑马鱼和动物护理/维护的研究方法均按照梅奥诊所的机构指南进行。 1. PSNS中过表达的转基因构建体开发 转基因 构建体的制备和显微注射 为了开发 LMO1-pDONR221 进入克隆，使用PCR扩增从人细胞系获得的cDNA中人类 LMO1 的编码区域。 制作25μL反应，详见：2.5μL1标准Taq反应缓冲液，0.125μLTaq DNA聚合酶，0.5μL10mM dNTPs，2μLcDNA模板，0.5?…

Representative Results

为了确定LMO1是否与MYCN协同作用以影响NB发病机制，将驱动在dβh启动子控制的PSNS细胞中表达LMO1（dβh：LMO1和dβh：mCherry）或MYCN（dβh：EGFP-MYCN）表达的转基因构建体注射到斑马鱼胚胎中13。如图1A所示，在开发稳定的转基因品系并验证其基因型后，杂合MYCN和LMO1鱼进行了杂交。他们的后代分别…

Discussion

在过去的几十年里，斑马鱼一直被普遍用于研究，特别是在癌症研究中，原因显而易见，例如其易于维护，强大的繁殖以及 体内成像的 明显优势1，28。由于斑马鱼模型的外部受精和发育，它们可以很容易地在胚胎上纵，这与哺乳动物模式生物（如大鼠和小鼠）相辅相成，用于大规模的遗传研究 1 ， 2<s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家癌症研究所的R01 CA240323（S.Z.）资助;美国国防部（DoD）的拨款W81XWH-17-1-0498（S.Z.）;V癌症研究基金会（S.Z.）的V学者奖和梅奥生物医学发现中心（S.Z.）的平台资助;以及梅奥诊所癌症中心和个性化医学中心（S.Z.）的支持。

Materials

3,3’-Diaminobenzidine (DAB) Vector Kit Vector SK-4100
Acetic Acid Fisher Scientific / Acros Organic 64-19-7
Agarose GP2 Midwest Scientific 009012-36-6
Anti-Tyrosine Hydroxylase (TH) Antibody Pel-Freez P40101
Avidin/Biotin Blocking Kit Vector SP-2001
BOND Intense R Detection Leica Biosystems DS9263
BOND primary antibody diluent Leica Biosystems Newcastle, Ltd. AR9352
BOND-MAX IHC instrument Leica Biosystems Newcastle, Ltd. N/A fully automated IHC staining system
CH211-270H11 BAC clone BACPAC resources center (BRFC) N/A
Compound microscope equipped with DP71 camera Olympus AX70
Cytoseal XYL (xylene based mounting medium) Richard-Allan Scientific 8312-4
Eosin Leica 3801601 ready-to-use (no preparation needed)
Ethanol Carolina 86-1263
Expand Long Template PCR System Roche Applied Science, IN 11681834001
Gateway BP Clonase II enzyme mix Invitrogen, CA 11789-020
Gateway LR Clonase II enzyme mix Invitrogen, CA 11791-100
Goat anti-Rb secondary antibody (Biotinylated) Dako E0432
Hematoxylin Solution, Harris Modified Sigma Aldrich Chemical Company Inc. / SAFC HHS-32-1L
HRP Avidin D Vector A-2004
Hydrochloric Acid Aqua Solutions 4360-1L
Hydrogen Peroxide, 3% Fisher Scientific H324-500
I-SceI enzyme New England Biolabs, MA R0694L
Kanamycin sulfate Teknova, Inc. K2150
Kimberly-Clark Professional Kimtech Science Kimwipes Fisher Scientific 34133
Lithium Carbonate Sigma Aldrich Chemical Company Inc. / SAFC 554-13-2
Microtome for sectioning Leica Biosystems RM2255
One Shot TOP10 Chemically Competent E. coli Invitrogen C404006
p3E-polyA  Dr. Chi-Bin Chien, Univ. of Utah N/A a generous gift
(Please refer to webpage http://tol2kit.genetics.utah.edu/index.php/Main_Page to obtain material, which is freely distrubted as described.)
Parafin wax Surgipath Paraplast 39603002 Parrafin to parafin
Paraformaldehyde Alfa Aesar A11313
pDEST vector (modified destination vector containing I-SceI recognition sites) Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germany N/A a generous gift
pDONR 221 gateway donor vector Thermo Fisher Scientific 12536-017
pDONRP4-P1R donor vector  Dr. Chi-Bin Chien, Univ. of Utah N/A a generous gift
Phenol red, 0.5% Sigma Aldrich  P0290
Phosphate Buffered Saline (PBS), 10X BioRad 1610780
Picrosirrius red stain kit Polysciences 24901-250
pME-mCherry Addgene 26028 (DBH construct)
Proteinase K, recombinant, PCR Grade Roche 21712520
QIAprep Spin MiniPrep Kit Qiagen 27104
RDO Rapid Decalcifier Apex Enginerring RDO04
Sodium Azide (NaN3) Sigma Aldrich 26628-22-8
Stereo fluorescence microscope Leica MZ10F
Stereoscopic fluorescence microscope equipped with a digital sight DS-U1 camera for imaging Nikon SMZ-1500
Taq DNA Polymerase New England Biolabs, MA M0273L
Tissue-Tek VIP® 6 AI Vacuum Infiltration Processor Sakura N/A Model #: VIP-6-A1
Tricaine-S Western Chemical Incorporated 20513
Xylene Thermo Fisher Scientific X3P1GAL
DOWNLOAD MATERIALS LIST

References

  1. Veldman, M., Lin, S. Zebrafish as a developmental model organism for pediatric research. Pediatric Research. 64, 470-476 (2008).
  2. Feitsma, H., Cuppen, E. Zebrafish as a cancer model. Molecular Cancer Research. 6 (5), 694 (2008).
  3. Ethcin, J., Kanki, J. P., Look, A. T. Zebrafish as a model for the study of human cancer. Methods in Cell Biology. 105, 309-337 (2010).
  4. Benjamin, D. C., Hynes, R. O. Intravital imaging of metastasis in adult Zebrafish. BMC Cancer. 17 (1), 660 (2017).
  5. Kim, I. S., et al. Microenvironment-derived factors driving metastatic plasticity in melanoma. Nature Communications. 8, 14343 (2017).
  6. Zhu, S., et al. Activated ALK collaborates with MYCN in neuroblastoma pathogenesis. Cancer Cell. 21 (3), 362-373 (2012).
  7. Maris, J. M., Hogarty, M. D., Bagatell, R., Cohn, S. L. Neuroblastoma. Lancet. 369 (9579), 2106-2120 (2007).
  8. Park, J. R., et al. Children’s oncology group’s 2013 blueprint for research: neuroblastoma. Pediatric Blood and Cancer. 60 (6), 985-993 (2013).
  9. Hoehner, J. C., et al. A developmental model of neuroblastoma: differentiating stroma-poor tumors’ progress along an extra-adrenal chromaffin lineage. Laboratory Investigation: A Journal of Technical Methods and Pathology. 75 (5), 659-675 (1996).
  10. Tsubota, S., Kadomatsu, K. Origin and initiation mechanisms of neuroblastoma. Cell and Tissue Research. 372 (2), 211-221 (2018).
  11. Tolbert, V. P., Matthay, K. K. Neuroblastoma: Clinical and biological approach to risk stratification and treatment. Cell and Tissue Research. 372 (2), 195-209 (2018).
  12. Maris, J. M. Recent advances in neuroblastoma. New England Journal of Medicine. 362 (23), 2202-2211 (2010).
  13. Zhu, S., et al. LMO1 Synergizes with MYCN to promote neuroblastoma initiation and metastasis. Cancer Cell. 32, 310-323 (2017).
  14. Patton, E. E., Zon, L. I. The art and design of genetic screens: zebrafish. Nature Reviews Genetics. 2 (12), 956-966 (2001).
  15. Lieschke, G. J., Currie, P. D. Animal models of human disease: zebrafish swim into view. Nature Reviews Genetics. 8 (5), 353-367 (2007).
  16. Tao, T., et al. LIN28B regulates transcription and potentiates MYCN-induced neuroblastoma through binding to ZNF143 at target gene promotors. Proceedings of the National Academy of Sciences of the United States of America. 117 (28), 16516-16526 (2020).
  17. Ung, C. Y., Guo, F., Zhang, X., Zhu, Z., Zhu, S. Mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis. Journal of Visualized Experiments. (97), e52567 (2015).
  18. Zhang, X., et al. Critical role for GAB2 in neuroblastoma pathogenesis through the promotion of SHP2/MYCN cooperation. Cell Reports. 18 (12), 2932-2942 (2017).
  19. Zimmerman, M. W., et al. MYC drives a subset of high-risk pediatric neuroblastomas and is activated through mechanisms including enhancer hijacking and focal enhancer amplification. Cancer Discovery. 8 (3), 320-335 (2018).
  20. Koach, J., et al. Drugging MYCN oncogenic signaling through the MYCN-PA2G4 binding interface. Cancer Research. 79 (21), 5652-5667 (2019).
  21. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. Stages of embryonic development of the zebrafish. Developmental Dynamics. 203 (3), 253-310 (1995).
  22. DuBois, S. G., et al. Metastatic sites in stage IV and IVS neuroblastoma correlate with age, tumor biology, and survival. Journal of pediatric hematology/oncology. 21 (3), 181-189 (1999).
  23. Wattrus, S. J., Zon, L. I. Stem cell safe harbor: The hematopoietic stem cell niche in zebrafish. Blood Advances. 2 (21), 3063-3069 (2018).
  24. Menke, A. L., Spitsbergen, J. M., Wolterbeek, A. P., Woutersen, R. A. Normal anatomy and histology of the adult zebrafish. Toxicologic Pathology. 39 (5), 759-775 (2011).
  25. Renshaw, S. A., Trede, N. S. A model 450 million years in the making: Zebrafish and vertebrate immunity. Disease Models and Mechanisms. 5 (1), 38-47 (2012).
  26. Junqueira, L. C., Cossermelli, W., Brentani, R. Differential staining of collagens type I, II and III by Sirius Red and polarization microscopy. Archivum histologicum Japonicum (Nihon Soshikigaku Kiroku). 41 (3), 267-274 (1978).
  27. Sweat, F., Puchtler, H., Rosenthal, S. I. Sirius red F3BA as a stain for connective tissue. Archives of Pathology. 78, 69-72 (1964).
  28. Ignatius, M. S., Hayes, M., Langenau, D. M. In vivo imaging of cancer in zebrafish. Advances in Experimental Medicine and Biology. 916, 219-237 (2016).
  29. Howe, C. K., et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 496 (7446), 498-503 (2013).
  30. Fazio, M., Ablain, J., Chuan, Y., Langenau, D. M., Zon, L. I. Zebrafish patient avatars in cancer biology and precision cancer therapy. Nature Reviews Cancer. 20 (5), 263-273 (2020).
  31. Yoganantharjah, P., Gibert, Y. The Use of the zebrafish model to aid in drug discovery and target validation. Current Topics in Medicinal Chemistry. 17 (18), 2041-2055 (2018).
  32. Ignatius, M. S., et al. In vivo imaging of tumor-propagating cells, regional tumor heterogeneity, and dynamic cell movements in embryonal rhabdomyosarcoma. Cancer Cell. 21 (5), 680-693 (2012).
  33. Stoletov, K., Montel, V., Lester, R. D., Gonias, S. L., Klemke, R. High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 104 (44), 17406-17411 (2007).
  34. Ahmed, S., et al. Neuroblastoma with orbital metastasis: ophthalmic presentation and role of ophthalmologists. Eye. 20 (4), 466-470 (2006).
  35. Papaioannou, G., McHugh, K. Neuroblastoma in childhood: review and radiological findings. Cancer Imaging Society. 5 (1), 116-127 (2005).
  36. Langenau, D. M., et al. Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic Zebrafish. Oncogene. 27 (30), 4242-4248 (2008).
  37. Amores, A., et al. Zebrafish hox clusters and vertebrate genome evolution. Science. 282 (5394), 1711-1714 (1998).
  38. Postlethwait, J. H., et al. Vertebrate genome evolution and the zebrafish gene map. Nature Genetics. 18 (4), 345-349 (1998).
  39. Opazo, J. C., et al. Whole-genome duplication and the functional diversification of teleost fish hemoglobins. Molecular Biology and Evolution. 30 (1), 140-153 (2013).

Tags

Keywords: ZebrafishNeuroblastomaMetastasisIn Vivo ImagingTumor DisseminationMYCNLMO1TransgenicEGFPFluorescence MicroscopeTumor ScreeningTumor-bearing FishTumor Cell Migration
check_url/62416?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Her, Z. P., Yeo, K. S., Howe, C., Levee, T., Zhu, S. Zebrafish Model of Neuroblastoma Metastasis. J. Vis. Exp. (169), e62416, doi:10.3791/62416 (2021).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image