同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率
1Department of Molecular and Comparative Pathobiology,The Johns Hopkins University School of Medicine, 2Sidney Kimmel Comprehensive Cancer Center,Johns Hopkins University, 3Department of Surgery,Johns Hopkins University School of Medicine, 4Department of Coloproctological Surgery,Juntendo University Faculty of Medicine, 5Program for Comparative Medicine, Gene Therapy Program, Perelman School of Medicine,University of Pennsylvania, 6Tuskegee College of Veterinary Medicine, 7Department of Human Pathology,Juntendo University Faculty of Medicine, 8Department of Surgery, Juntendo University Shizuoka Hospital,Juntendo University School of Medicine, 9Department of Urology,The Johns Hopkins School of Medicine, 10Department of Gastroenterology and Minimally Invasive Surgery,Juntendo University Faculty of Medicine
Summary
在这里，描述了同源原位植入术，然后是伴有自发性肺转移的骨肉瘤的截肢手术，可用于转移生物学的临床前研究和新疗法的开发。
Abstract
骨肉瘤（OS）治疗的最新进展发生在20世纪80年代，当时与单独手术相比，多药化疗被证明可以提高总生存率。为了解决这个问题，该研究的目的是通过全面的组织学，成像，生物学，植入和截肢手术方法来完善大鼠OS的鲜为人知的模型，以延长生存期。我们使用免疫功能正常的，异种的Sprague-Dawley（SD），同源大鼠模型，植入UMR106 OS细胞系（来自SD大鼠）与原位胫骨肿瘤植入物到3周大的雄性和雌性大鼠中来模拟儿科OS。我们发现大鼠发展为可重复的原发性和转移性肺肿瘤，并且在植入后3周截肢可显着降低肺转移的发生率并防止意外死亡。从组织学上讲，大鼠的原发性和转移性OS与人类OS非常相似.使用免疫组织化学方法，研究表明大鼠OS被巨噬细胞和T细胞浸润。对OS细胞的蛋白质表达调查显示，这些肿瘤表达ErbB家族激酶。由于这些激酶在大多数人类操作系统中也高度表达，因此该大鼠模型可用于测试ErbB途径抑制剂的治疗。
Introduction
骨肉瘤（OS）是儿童，青少年和年轻人中最常见的原发性骨肿瘤。OS治疗的最新进展发生在20世纪80年代，当时与单独手术相比，多药化疗被证明可以提高总生存率1。OS 在骨快速生长期间发展，通常发生在长管骨中，如股骨、胫骨和肱骨。它们的特征在于溶骨，成骨细胞或混合外观，伴有显着的骨膜反应2。化疗和手术切除可以改善65%的患者5年生存期患者的预后2，3。不幸的是，患有转移性疾病的高等级OS患者的生存率为20%。OS侵犯区域性，主要转移到肺部或其他骨骼，在男性中更为普遍。对这些年轻患者最迫切的需求是一种预防和消除远处转移可行性的新疗法。
OS临床前模型已经过审查4、5、6、7 和少数可用的使用截肢原位OS的免疫功能正常的模型已经开发出来。2000年，使用具有原位同源OS和截肢8的BALB / c小鼠开发了一个重要模型。与该小鼠模型相比，大鼠模型基于遗传异种和10倍大的动物，从而具有一些优势。大鼠UMR106模型是从Sprague Dawley（SD）大鼠中的32P诱导OS开发的，该OS衍生到细胞系9中。2001年，UMR106-01的原位植入首次在无胸腺小鼠的植入胫骨中描述，这些胫骨具有快速，一致的原发性肿瘤发展和放射学，组织学特征与人类OS相同。肺转移发展并依赖于UMR106的原位放置到骨微环境中10。2009年，Yu等人11在较大的雄性SD大鼠中使用UMR106细胞建立了可重复的原位股骨OS大鼠模型。未截肢大鼠肿瘤植入的成功率和肺转移率与此处提供的数据相似。在这项研究中，使用年轻大鼠对模型进行了额外的截肢，这表明原发性肿瘤切除的时间对于建模OS至关重要，特别是与转移进展有关。通过这种改进，截肢和体内成像改进了该模型，用于OS新药评估的临床前研究。
Protocol
所有涉及大鼠的程序和实验都是根据约翰霍普金斯动物护理和使用委员会批准的协议进行的。 1.SD大鼠OS细胞系UMR-106细胞培养方案 在DMEM中生长细胞，补充10%（v / v）FBS，青霉素（10 U / mL）-链霉素（10 U / mL）在37°C在加湿的5%CO2 气氛中。使用具有2-8个传代的细胞进行实验12。 2. 胫骨内注射OS细胞方案 …
Representative Results
免疫功能正常的SD杂交大鼠用于这些OS研究，这提供了一个具有完整免疫系统的动物模型。我们使用来自ATCC的UMR106细胞系，该细胞系由最初从SD大鼠的OS中分离出来的细胞开发而成。我们将细胞植入SD大鼠体内，从而为OS提供了同源模型，UMR106细胞被植入3周龄雄性和雌性SD大鼠的胫骨中，模拟儿科OS模型。此外，UMR106细胞的原位植入胫骨干骺/骺骨骨骺中提供了相关的肿瘤微环境。 <p class="jove_content…
Discussion
具有OS胫骨植入物的大鼠在植入后3周内出现可测量的肿瘤。如果植入后3周截肢肿瘤，肺转移的发生率显着降低。操作系统既是溶骨细胞又是成骨细胞。没有截肢的大鼠出现多个且大小不一的肺转移，通过X线照相观察或在植入后7周进行尸检观察。EGFR，ErbB2和ErbB4在大鼠UMR106 OS中表达，类似于人类OS 16，17，18。CD3 T细胞和巨噬细?…
Declarações
The authors have nothing to disclose.
Acknowledgements
NIH通过国家癌症研究所资助，拨款编号CA228582。Shun Ishiyama目前正在接受东丽医疗株式会社的资助。
Materials
| AKT | Cell Signaling TECHNOLOGY | 4685S | |
| absorbable suture | Ethicon | J214H | |
| β-actin | SANTA CRUZ BIOTECHNOLOGY | sc-47778 | |
| β2-AR antibody | SANTA CRUZ BIOTECHNOLOGY | sc-569 | replaced by β2-AR (E-3): sc-271322 |
| Bis–Tris gels | Thermo Fisher | NP0321PK2 | |
| Buprenorphine SR Lab | ZooPharm | IZ-70000-201908 | |
| CD3 antibody | Dako | #A0452 | |
| CD68 antibody | eBioscience | #14-0688-82 | |
| Chemiluminescent substrate | cytiva | RPN2232 | |
| CL-Xposure film | Thermo Fisher | 34089 | |
| Complete Anesthesia System | EVETEQUIP | 922120 | |
| diaminobenzidine | VECTOR LABORATORIES | SK-4100 | |
| Doxorubicin | Actavis | NDC 45963-733-60 | |
| EGFR antibody | SANTA CRUZ BIOTECHNOLOGY | sc-03 | replaced by EGFR (A-10): sc-373746 |
| ERBB2 antibody | SANTA CRUZ BIOTECHNOLOGY | sc-284 | replaced by Neu (3B5): sc-33684 |
| ERBB4 antibody | SANTA CRUZ BIOTECHNOLOGY | sc-283 | replaced by ErbB4 (C-7): sc-8050 |
| ERK antibody | SANTA CRUZ BIOTECHNOLOGY | sc-514302 | |
| eye lubricant | PHARMADERM | NDC 0462-0211-38 | |
| Hamilton syringe (100 µL) | Hamilton | Model 1710 SN SYR | |
| horseradish peroxidase-linked secondary antibody | cytiva | NA934 | |
| HRP polymer detection kit | VECTOR LABORATORIES | MP-7401 | |
| HRP polymer detection kit | VECTOR LABORATORIES | MP-7402 | |
| isoflurane | BUTLER SCHEIN | NDC 11695-6776-2 | |
| isoflurane vaporizer | EVETEQUIP | 911103 | |
| UMR-106 cell | ATCC | CRL-1661 | |
| X-ray | Faxitron | UltraFocus | |
| X-ray processor | Hope X-Ray Peoducts Inc | MicroMax X-ray Processor | Hope Processors are not available in USA anymore |
| wound clips | BECTON DICKINSON | 427631 |
Referências
- Link, M. P., et al. The effect of adjuvant chemotherapy on relapse-free survival in patients with osteosarcoma of the extremity. New England Journal of Medicine. 314 (25), 1600-1606 (1986).
- Bielack, S. S., et al. Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. Journal of Clinical Oncology. 20 (3), 776-790 (2002).
- Botter, S. M., Neri, D., Fuchs, B. Recent advances in osteosarcoma. Current Opinion in Pharmacology. 16, 15-23 (2014).
- Ek, E. T. H., Dass, C. R., Choong, P. F. M. Commonly used mouse models of osteosarcoma. Critical Reviews in Oncology Hematology. 60 (1), 1-8 (2006).
- Guijarro, M. V., Ghivizzani, S. C., Gibbs, C. P. Animal models in osteosarcoma. Frontiers Oncology. 4, 189 (2014).
- Janeway, K. A., Walkley, C. R. Modeling human osteosarcoma in the mouse: From bedside to bench. Bone. 47 (5), 859-865 (2010).
- Mohseny, A. B., Hogendoorn, P. C., Cleton-Jansen, A. M. Osteosarcoma models: from cell lines to zebrafish. Sarcoma. 2012, 417271 (2012).
- Khanna, C., et al. An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential. Clinical & Experimental Metastasis. 18 (3), 261-271 (2000).
- Martin, T. J., et al. Parathyroid hormone-responsive adenylate cyclase in induced transplantable osteogenic rat sarcoma. Nature. 260 (5550), 436-438 (1976).
- Fisher, J. L., Mackie, P. S., Howard, M. L., Zhou, H., Choong, P. F. The expression of the urokinase plasminogen activator system in metastatic murine osteosarcoma: an in vivo mouse model. Clinical Cancer Research. 7 (6), 1654-1660 (2001).
- Yu, Z., et al. Establishment of reproducible osteosarcoma rat model using orthotopic implantation technique. Oncology Reports. 21 (5), 1175-1180 (2009).
- Zhang, P., et al. Homologous mesenchymal stem cells promote the emergence and growth of pulmonary metastases of the rat osteosarcoma cell line UMR-106. Oncology Letters. 8 (1), 127-132 (2014).
- Gabrielson, K., et al. Heat shock protein 90 and ErbB2 in the cardiac response to doxorubicin injury. Pesquisa do Câncer. 67 (4), 1436-1441 (2007).
- Sysa-Shah, P., et al. Bidirectional cross-regulation between ErbB2 and β-adrenergic signalling pathways. Cardiovascular Research. 109 (3), 358-373 (2016).
- Wachtman, L. M., Browning, M. D., Bedja, D., Pin, S., Gabrielson, K. L. Validation of the use of long-term indwelling jugular catheters in a rat model of cardiotoxicity. Journal of American Association Laboratory Animal Science. 45, 55-64 (2006).
- Abdou, A. G., et al. The Prognostic role of Ezrin and HER2/neu expression in osteosarcoma. Applied Immunohistochemistry & Molecular Morphology. 24 (5), 355-363 (2016).
- Hughes, D. P., Thomas, D. G., Giordano, T. J., McDonagh, K. T., Baker, L. H. Essential erbB family phosphorylation in osteosarcoma as a target for CI-1033 inhibition. Pediatric Blood & Cancer. 46 (5), 614-623 (2006).
- Wen, Y. H., et al. Epidermal growth factor receptor in osteosarcoma: expression and mutational analysis. Human Pathology. 38 (8), 1184-1191 (2007).
- Khanna, C., et al. Toward a drug development path that targets metastatic progression in osteosarcoma. Clinical Cancer Research. 20 (16), 4200-4209 (2014).
Citar este artigo
Ishiyama, S., Kissel, C., Guo, X., Howard, A., Saeki, H., Ito, T., Sysa-Shah, P., Orita, H., Sakamoto, K., Gabrielson, K. A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate. J. Vis. Exp. (171), e62139, doi:10.3791/62139 (2021).