用于测试（新）辅助疗法的不完全切除软组织肉瘤的小鼠模型
Summary
在该协议中，我们描述了一个小鼠模型，不完整的手术切除软组织肉瘤测试（新）辅助疗法。
Abstract
手术通常是许多实体肿瘤的第一次治疗。然而，局部复发经常发生在原发性肿瘤切除后，尽管辅助或新佐剂疗法。当手术边缘不足无肿瘤，导致残余癌细胞时，将发生这种情况。从生物学和免疫学的角度来看，手术不是一个空事件;伤口愈合环境已知能诱导亲肿瘤和抗肿瘤通路。因此，旨在预防局部复发的药物开发临床前模型在测试新的（新）辅助疗法时应纳入手术切除，以模拟接受手术治疗的患者的临床设置。
在这里，我们描述了一个小鼠模型不完全手术切除的 WEHI 164 软组织肉瘤，允许测试（新）辅助疗法在伤口愈合反应的设置。在此模型中，50% 或 75% 的肿瘤被切除，留下一些原位癌组织，在临床环境中模拟手术后的粗残病。此模型允许在手术环境中测试疗法，同时考虑伤口愈合反应，这可能会影响（新）辅助治疗的疗效。不完全的手术切除导致肿瘤在没有辅助治疗的情况下在所有小鼠中可重现。带检查点封锁的辅助治疗可减少肿瘤再生。因此，该模型适用于在脱毛手术及其相关的伤口愈合反应的背景下测试疗法，并可以扩展到其他类型的固体癌症。
Introduction
手术仍然是许多实体肿瘤1的主要治疗选择，包括软组织肉瘤2，3。2,尽管癌症手术技术有所改善，并结合（新）辅助疗法，在原发性肿瘤切除4，5,后癌症复发和转移的风险仍然很高。在软组织肉瘤中，复发特别多发，在手术现场，导致发病率和死亡率增加。在临床环境中，可能很难获得足够宽的边距（例如，由于解剖限制），导致不完全切除和随后的肿瘤复发6。手术压力和随后的伤口愈合过程已知创造一个免疫抑制肿瘤微环境有利于肿瘤复发7,7，8。因此，软组织肉瘤，特别是免疫疗法的新疗法的发现和发展，最好考虑手术伤口愈合反应。
大多数临床前研究佐剂疗法最初使用皮下合成或异种移植小鼠模型，没有纳入手术压力和伤口愈合反应9,9，10。因此，我们开发了一个合成皮下小鼠软组织肉瘤模型，结合了不完整的手术切除。WEHI 164纤维肉瘤细胞在皮下接种，一旦肿瘤建立，我们去除50-75%的肿瘤体积（图1A-E）。肿瘤不断从剩余的肿瘤重新生长。此模型允许测试辅助疗法，同时考虑手术压力和伤口愈合的效果。多个小组在多项研究中都使用了类似的手术模型，发现这些模型可重复有效，包括11、12、13。,12,13在这里，我们提供了此协议的详细说明。
Protocol
这些实验中使用的动物是从动物资源中心（西澳大利亚州珀斯）获得的。在Har harryPerkins医学研究所生物资源北设施（西澳大利亚州珀斯）的条件下饲养动物。所有实验都是按照哈利·帕金斯医学研究所动物伦理委员会批准的议定书进行的。在这些实验中使用了8-12周大的BALB/c小鼠。WEHI 164 纤维瘤细胞系来自澳大利亚细胞银行（新南威尔士州韦斯特米德）。 1. 细胞接种 ?…
Representative Results
肿瘤生长到50 mm2的大小是部分脱虫的理想尺寸。50 mm2 肿瘤的不完全手术切除导致肿瘤在没有辅助免疫治疗的情况下 100% （n=5） 可再生（图 4A）。接下来，我们使用该模型测试辅助免疫疗法，使用抗体对检查点分子细胞毒性T淋巴细胞相关蛋白4（CTLA-4）和编程死亡受体1（PD-1）。对抗CTLA-4或抗PD-1小鼠的治疗，治愈率分别达…
Discussion
我们为软组织肉瘤的不完全手术切除小鼠模型提供一种方案，以测试术内疗法。我们还标准化了手术切口，以便评估治疗后小鼠之间的伤口愈合情况。
肿瘤放置是此协议的重要组成部分。我们选择了皮下肿瘤模型，以便于手术访问肿瘤位点，并管理局部疗法，同时对小鼠负担最小。同样重要的是，确保肿瘤生长在皮下空间，而不是在近膜内，这可能导致意外的发病率和死亡?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由袜子它到萨科马的赠款支持！基金会、澳大利亚和新西兰肉毒病协会、儿童白血病和癌症研究基金会和永久慈善组织。W.J.L由西蒙·李奖学金和来自国家健康和医学研究理事会和西澳大利亚州癌症理事会的奖学金支持。
Materials
| 26 gauge 0.5 mL insulin syringe | Becton Dickinson, Australia | 326769 | None |
| 2-Mercaptoethanol | Life Technologies Australia Pty Ltd | 21985023 | None |
| Anaestetic gas machine | Darvall Vet, Australia | SKU: 2848 | None |
| Anti-CTLA-4 | BioXcell, USA | BE0164 | None |
| Anti-PD-1 | BioXcell, USA | BP0273 | None |
| Buprenorphine Hydrochloride Injection, 0.3mg/mL | RB healthcare UK Limited, UK | 55175 | Prescription order |
| Chlorhexidine Surgical Scrub 4% | Perigo Australia, Australia | CHL01449F(scrub | None |
| Fetal Bovine serum | CellSera, Australia | AU-FBS-PG | None |
| Forceps Fine 10.5 cm | Surgical house, Western Australia | CC74110 | None |
| Forceps Fine 12 cm Serrated | Surgical house, Western Australia | CC74212 | None |
| Forceps Halsted 14 cm | Surgical house, Western Australia | CD01114 | None |
| Heating chamber | Datesand Ltd, UK | Mini-Thermacage | None |
| HEPES (1M) | Life Technologies Australia Pty Ltd | 15630080 | None |
| Isoflurane | Henry Schein Animal Health, Australia | SKU: 29405 | Prescription order |
| Lubricating Eye Ointment | Alcon | n/a | None |
| Penicillin/streptomycin 1000X | Life Technologies Australia Pty Ltd | 15140122 | None |
| Phosphate Buffered Solution 10x | Life Technologies Australia Pty Ltd | 70013-032 | None |
| Reflex 7mm Clips | Able scientific, Australia | AS59038 | None |
| Reflex 7mm Wound Clip Applicator | Able scientific, Australia | AS59036 | None |
| Reflex Wound Clip Remover | Able scientific, Australia | AS59037 | None |
| Rodent Qube Anesthesia Breathing Circuit | Darvall Vet, Australia | #7885 | None |
| Roswell Park Memorial Institute (RPMI) 1640 Medium + L-glutamine | Life Technologies Australia Pty Ltd | 21870092 | None |
| Scissors Iris STR 11 cm | Surgical house, Western Australia | KF3211 | None |
| Scissors Iris STR 9 cm | Surgical house, Western Australia | JH4209 | None |
| Small Induction Chamber | Darvall Vet, Australia | SKU: 9630 | None |
| TrypLE express 1x | Life Technologies Australia Pty Ltd | 12604-021 | None |
References
- Orosco, R. K., et al. Positive Surgical Margins in the 10 Most Common Solid Cancers. Scientific Reports. 8 (1), 5686 (2018).
- Haas, R. L., et al. Perioperative Management of Extremity Soft Tissue Sarcomas. Journal of Clinical Oncology. 36 (2), 118-124 (2018).
- Brennan, M. F., Antonescu, C. R., Moraco, N., Singer, S. Lessons learned from the study of 10,000 patients with soft tissue sarcoma. Annals of Surgery. 260 (3), 416-421 (2014).
- Smith, H. G., et al. Patterns of disease relapse in primary extremity soft-tissue sarcoma. British Journal of Surgery. 103 (11), 1487-1496 (2016).
- Uramoto, H., Tanaka, F. Recurrence after surgery in patients with NSCLC. Translational Lung Cancer Research. 3 (4), 242-249 (2014).
- Stojadinovic, A., et al. Analysis of the prognostic significance of microscopic margins in 2,084 localized primary adult soft tissue sarcomas. Annals of Surgery. 235 (3), 424-434 (2002).
- Krall, J. A., et al. The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy. Science Translational Medicine. 10 (436), (2018).
- Bakos, O., Lawson, C., Rouleau, S., Tai, L. H. Combining surgery and immunotherapy: turning an immunosuppressive effect into a therapeutic opportunity. Journal for ImmunoTherapy of Cancer. 6 (1), 86 (2018).
- Predina, J. D., et al. Characterization of surgical models of postoperative tumor recurrence for preclinical adjuvant therapy assessment. American Journal of Translational Research. 4 (2), 206-218 (2012).
- Talmadge, J. E., Singh, R. K., Fidler, I. J., Raz, A. Murine models to evaluate novel and conventional therapeutic strategies for cancer. American Journal of Pathology. 170 (3), 793-804 (2007).
- Khong, A., et al. The efficacy of tumor debulking surgery is improved by adjuvant immunotherapy using imiquimod and anti-CD40. BMC Cancer. 14, 969 (2014).
- Broomfield, S., et al. Partial, but not complete, tumor-debulking surgery promotes protective antitumor memory when combined with chemotherapy and adjuvant immunotherapy. Cancer Research. 65 (17), 7580-7584 (2005).
- Predina, J. D., et al. A positive-margin resection model recreates the postsurgical tumor microenvironment and is a reliable model for adjuvant therapy evaluation. Cancer Biology & Therapy. 13 (9), 745-755 (2012).
- Tsukamoto, A., Serizawa, K., Sato, R., Yamazaki, J., Inomata, T. Vital signs monitoring during injectable and inhalant anesthesia in mice. Experimental Animals. 64 (1), 57-64 (2015).
- Overwijk, W. W., Restifo, N. P. B16 as a mouse model for human melanoma. Current Protocols in Immunology. , (2001).
- Predina, J., et al. Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery. Proceedings of the National Academy of Sciences of the United States of America. 110 (5), E415-E424 (2013).
- Endo, M., Lin, P. P. Surgical margins in the management of extremity soft tissue sarcoma. Chinese Clinical Oncology. 7 (4), 37 (2018).
- Liu, J., et al. Improved Efficacy of Neoadjuvant Compared to Adjuvant Immunotherapy to Eradicate Metastatic Disease. Cancer Discovery. 6 (12), 1382-1399 (2016).
- Park, C. G., et al. Extended release of perioperative immunotherapy prevents tumor recurrence and eliminates metastases. Science Translational Medicine. 10 (433), (2018).
- Tai, L. H., et al. A mouse tumor model of surgical stress to explore the mechanisms of postoperative immunosuppression and evaluate novel perioperative immunotherapies. Journal of Visualized Experiments. (85), e51253 (2014).
- Gast, C. E., Shaw, A. K., Wong, M. H., Coussens, L. M. Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis. Journal of Visualized Experiments. (125), (2017).
- Qiu, W., Su, G. H. Development of orthotopic pancreatic tumor mouse models. Methods in Molecular Biology. 980, 215-223 (2013).
- Erstad, D. J., et al. Orthotopic and heterotopic murine models of pancreatic cancer and their different responses to FOLFIRINOX chemotherapy. Disease Models & Mechanisms. 11 (7), (2018).
Cite This Article
Rwandamuriye, F. X., Weston, B. J., Johns, T. G., Lesterhuis, W. J., Zemek, R. M. A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies. J. Vis. Exp. (161), e60882, doi:10.3791/60882 (2020).