Name: a mouse model of incompletely resected soft tissue sarcoma for testing (neo)adjuvant therapies
Uploaded: 2020-07-28T13:00:00
Description: Watch this Scientific Journal Video about A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies at JoVE.com

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。

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
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<ol>
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R., Moraco, N., Singer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+learned+from+the+study+of+10,000+patients+with+soft+tissue+sarcoma.">Lessons learned from the study of 10,000 patients with soft tissue sarcoma.</a> Annals of Surgery. 260 (3), 416-421 (2014).</li><li>Smith, H. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+disease+relapse+in+primary+extremity+soft-tissue+sarcoma.">Patterns of disease relapse in primary extremity soft-tissue sarcoma.</a> British Journal of Surgery. 103 (11), 1487-1496 (2016).</li><li>Uramoto, H., Tanaka, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrence+after+surgery+in+patients+with+NSCLC.">Recurrence after surgery in patients with NSCLC.</a> Translational Lung Cancer Research. 3 (4), 242-249 (2014).</li><li>Stojadinovic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+prognostic+significance+of+microscopic+margins+in+2,084+localized+primary+adult+soft+tissue+sarcomas.">Analysis of the prognostic significance of microscopic margins in 2,084 localized primary adult soft tissue sarcomas.</a> Annals of Surgery. 235 (3), 424-434 (2002).</li><li>Krall, J. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B16+as+a+mouse+model+for+human+melanoma.">B16 as a mouse model for human melanoma.</a> Current Protocols in Immunology. , (2001).</li><li>Predina, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+local+tumor+microenvironment+in+recurrent+cancers+may+explain+the+failure+of+vaccines+after+surgery.">Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (5), E415-E424 (2013).</li><li>Endo, M., Lin, P. 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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>26 gauge 0.5 mL insulin syringe</td>
			<td>Becton Dickinson, Australia</td>
			<td>326769</td>
			<td>None</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>21985023</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Anaestetic gas machine</td>
			<td>Darvall Vet, Australia</td>
			<td>SKU: 2848</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Anti-CTLA-4</td>
			<td>BioXcell, USA</td>
			<td>BE0164</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Anti-PD-1</td>
			<td>BioXcell, USA</td>
			<td>BP0273</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection, 0.3mg/mL</td>
			<td>RB healthcare UK Limited, UK</td>
			<td>55175</td>
			<td>Prescription order</td>
		</tr>
		<tr>
			<td>Chlorhexidine Surgical Scrub 4%</td>
			<td>Perigo Australia, Australia</td>
			<td>CHL01449F(scrub</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>CellSera, Australia</td>
			<td>AU-FBS-PG</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Forceps Fine 10.5 cm</td>
			<td>Surgical house, Western Australia</td>
			<td>CC74110</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Forceps Fine 12 cm Serrated</td>
			<td>Surgical house, Western Australia</td>
			<td>CC74212</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Forceps Halsted 14 cm</td>
			<td>Surgical house, Western Australia</td>
			<td>CD01114</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Heating chamber</td>
			<td>Datesand Ltd, UK</td>
			<td>Mini-Thermacage</td>
			<td>None</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>15630080</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health, Australia</td>
			<td>SKU: 29405</td>
			<td>Prescription order</td>
		</tr>
		<tr>
			<td>Lubricating Eye Ointment</td>
			<td>Alcon</td>
			<td>n/a</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin 1000X</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>15140122</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution 10x</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>70013-032</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Reflex 7mm Clips</td>
			<td>Able scientific, Australia</td>
			<td>AS59038</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Reflex 7mm Wound Clip Applicator</td>
			<td>Able scientific, Australia</td>
			<td>AS59036</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Reflex Wound Clip Remover</td>
			<td>Able scientific, Australia</td>
			<td>AS59037</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Rodent Qube Anesthesia Breathing Circuit</td>
			<td>Darvall Vet, Australia</td>
			<td>#7885</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 Medium + L-glutamine</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>21870092</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Scissors Iris STR 11 cm</td>
			<td>Surgical house, Western Australia</td>
			<td>KF3211</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Scissors Iris STR 9 cm</td>
			<td>Surgical house, Western Australia</td>
			<td>JH4209</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Small Induction Chamber</td>
			<td>Darvall Vet, Australia</td>
			<td>SKU: 9630</td>
			<td>None</td>
		</tr>
		<tr>
			<td>TrypLE express 1x</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>12604-021</td>
			<td>None</td>
		</tr>
	</tbody>
</table>

a mouse model of incompletely resected soft tissue sarcoma for testing (neo)adjuvant therapies

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。

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...

Watch this Scientific Journal Video about A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies at JoVE.com

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies

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。

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

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant ...

该协议旨在模拟肿瘤切除手术后残留疾病的临床设置，因此使我们能够在术后伤口愈合的背景下测试新的癌症疗法。这种技术的优点是，它允许伤口的愈合量和肿瘤的量保持标准化。这项技术对于临床前研究尤其重要，因为临床前研究可以确定癌症的新疗法，它在当地复发，例如软组织肉瘤。这个视觉演示对于演示反转技术至关重要，它使我们能够暴露贴在皮肤侧的肿瘤。从小鼠中准备好的癌细胞的皮下接种开始，如文本协议中所述。第12天，接种后，当肿瘤达到约50平方毫米的大小时，在手术前30分钟，在颈部擦伤中，用100微升的丁丙诺啡皮下给小鼠服用剂量。设置手术区，用覆盖着长凳涂层的热垫设置鼻锥进行麻醉。安排干净的手术设备，以便他们很容易到达，包括氯沙丁，拭子，纱布，眼凝胶，两个弯曲的钳子，剪刀，夹子施用器，夹子去除剂，和夹子填充。将加热室加热至37摄氏度，并设置另一个加热垫进行回收。请注意，此过程需要两个人。助理必须在手术过程中密切监视鼠标的呼吸。对文本协议中描述的小鼠进行麻醉后，执行捏合测试和角膜反射测试，以确保小鼠在开始手术前完全麻醉。用少量的眼部凝胶盖住老鼠的眼睛，以避免眼睛干燥。开始这个手术，用氯环丙胺擦拭手术区。用钳子和一把剪刀沿着后侧做一厘米的直切口，离肿瘤三毫米远。不要直接切开覆盖肿瘤的皮肤，因为它不能愈合良好。避免使用手术刀切肿瘤，并使用钳子铲块。使用钳子，拉开肿瘤和内膜之间的筋膜和皮下脂肪组织。皮下肿瘤通常附着在皮肤侧。打开伤口，用钳子轻轻握住肿瘤轴承侧的皮肤，并反转肿瘤，使肿瘤在外可见。用一把剪刀，从肿瘤胶囊的一半切开，从肿瘤底部最接近开口开始。对于50%的脱骨手术，切过肿瘤的中间。使用弯曲钳子挖出要切除的肿瘤部分。从德布尔克地区挖出任何残余物。对于 75% 的脱骨，首先执行 50% 的肿瘤脱布，然后切成一半的剩余的 50% 的肿瘤。使用弯曲钳子挖出25%的肿瘤。要关闭手术地点，请将剩余的肿瘤放回皮肤下方，并使用钳子，将皮肤皮瓣拉在一起，沿着伤口将皮肤拉上线。将皮肤从伤口边缘握在一起五毫米，并使用手术夹关闭伤口从侧面开始，最接近钳子。根据需要应用尽可能多的夹子，以确保没有底层组织暴露。通常，应用三到四个夹子，夹子之间有两毫米的间隙。将小鼠放入加热室，让小鼠恢复。将鼠标的笼子放在热垫上。监测加热室中的小鼠，直到它们从麻醉剂中恢复过来。然后，把老鼠放回笼子里，把笼子放在热垫上再呆10分钟，直到老鼠变得更活跃。给老鼠湿和软的食物。手术后一小时监测小鼠恢复。在一天结束和次日早晨再次监测小鼠，并确保剪辑保持到位。在任何给定时间，以出现或新辅助疗法治疗小鼠，具体取决于感兴趣的治疗。例如，在接种后的第15天，用一剂100微克的抗CTLA-4内皮治疗小鼠。或者，在接种日、15日、17日、19日，用三剂200微克的抗PD-1内皮治疗小鼠。50平方毫米肿瘤的不完全手术切除，这里用虚线表示，在没有辅助免疫治疗的情况下，肿瘤的再生率为100%。因此，肿瘤生长到50平方毫米的大小是部分脱发的理想大小。然后，该模型用于测试辅助免疫疗法，使用抗体对检查点模块，细胞毒性T淋巴细胞相关蛋白四和编程死亡受体1。对抗CTLA-4小鼠的治疗，治愈率为80%，而抗PD-1小鼠的治疗治愈率为25%，抗PD-1的抗反应为测试其新颖组合提供了机会，进一步提高了反应率。重要的是，剩余的肿瘤保持附着在皮肤上，否则血液供应将被切断，肿瘤将成为坏死。按照这个程序，肿瘤可以通过各种方法进行分析，如流细胞学，以评估手术如何影响肿瘤渗透细胞。这个小鼠模型使我们能够探索伤口愈合如何影响癌症免疫疗法。

Research

JoVE Journal

Medicine

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

科研

医学

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

临床试验和脊髓去除在肌萎缩性侧索硬化症的小鼠模型（ALS）

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

外科技术治疗脊髓交付：在哥廷根小型猪演示程序

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The In ovo CAM-assay as a Xenograft Model for Sarcoma

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

下腔静脉间置移植物在小鼠模型中植入

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

评价疗效<em&gt;小时。幽门螺杆菌</em&gt;蛋白HP-NAP为膀胱癌的治疗在原位小鼠模型的治疗工具

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A Mouse Model for Laser-induced Choroidal Neovascularization

小鼠模型的激光诱导脉络膜新生血管

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

骨科手术的小鼠模型研究术后认知功能障碍与组织再生

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...