胰腺癌矫形小鼠模型
1Pancreatic Research Group, South Western Sydney Clinical School,University of New South Wales, 2Ingham Institute for Applied Medical Research, 3Centre for Circulating Tumour Cell Diagnostics and Research,Ingham Institute for Applied Medical Research, 4Surgical Innovations Unit,Westmead Hospital, 5Westmead Clinical School,University of Sydney
Summary
在临床背景下，局部胰腺癌患者将接受胰腺切除术，然后进行辅助治疗。这里报道的这个协议旨在建立一个安全和有效的方法，通过正交植入胰腺癌，然后进行分立胰腺切除术和切除术，在裸体小鼠中模拟这一临床场景。
Abstract
在考虑接受胰腺癌手术（PC）的患者中，缺乏令人满意的动物模型来研究辅助疗法和/或新辅助疗法。为了解决这一缺陷，我们描述了一个涉及 PC 正交植入的鼠标模型，然后是切除胰腺切除术和切除术。该模型已被证明是安全的和适当的灵活，用于研究各种治疗方法的辅助和新辅助设置。
在这个模型中，胰腺肿瘤首先通过将人类胰腺癌细胞（路西法酶标记的AsPC-1）和人类癌症相关胰腺板状细胞的混合物植入Balb/c水生裸鼠的离散胰腺而生成。三周后，癌症通过切除术、切除胰腺切除术和切除术进行切除。在这个模型中，生物发光成像可用于跟踪癌症发展的进展和切除/治疗的影响。切除后，可给予辅助治疗。或者，在切除前可以进行新辅助治疗。
介绍了来自45只小鼠的代表数据。所有小鼠都成功地进行了胰腺切除术/切除术，没有血吸虫病问题。在 43 （96%） 中实现了大于 5 mm 的宏观近胰腺边缘小 鼠。胰腺切除技术成功率为100%，早期死亡率和发病率为0%。在切除后的一周内，没有一只动物死亡。
总之，我们描述了一个强大和可重复的技术，用于模拟临床场景的小鼠胰腺癌手术切除模型。该模型可能有助于测试辅助剂和新辅助治疗。
Introduction
胰腺管腺癌（胰腺癌[PC]）与不良预后1有关。手术切除术仍然是PC唯一潜在的治疗方法，对于患有早期疾病的患者应予以考虑。不幸的是，即使R0切除（即切除边缘没有肿瘤），复发率（局部或从未被发现的转移性疾病）是高2，3。因此，全身辅助疗法在几乎所有接受切除4的患者中都显示。此外，虽然新辅助疗法现在只推荐给边缘可切除的癌症，其适应症正在扩大，使其常规使用是许多临床研究的重点5，6，7，8。为了开发涉及切除的 PC 的新疗法，这些方法需要首先在临床前模型中进行评估，以准确回顾临床设置。
矫型小鼠型号的PC已经经常用于测试药物治疗9，10。其中许多是由单独注射癌细胞到小鼠胰腺产生的，导致肿瘤缺乏PC特有的突出的频闪。最近，联合注射正交模型，如我们最初通过注射人类PC和人类胰腺恒星细胞的混合物（PSC，PC中胶原体频闪的主要生产者）而开发的模型，已经进入常规使用11，12。这种癌症和频闪细胞的联合注射产生的肿瘤既表现出癌症元素和PC的特有的频闪（去塑性）成分，（二）增强癌细胞增殖和转移11。因此，这个模型非常类似于人类PC。虽然一些矫形PC的切除模型被描述为13，14，15，16，但没有一个反映临床现实的胰腺切除在人类的准确如这个模型，因此一直不尽如人意的测试辅助剂或新辅助治疗。
演示的小鼠模型的目的是演示如何：（i） 成功植入正交胰腺癌，同时最大限度地减少无意的腹内传播和 （ii） 随后完全切除癌症。本文强调了该技术的技巧和潜在陷阱。
Protocol
所有程序均由新南威尔士大学动物护理和伦理委员会（17/109A）批准。雌性水性巴尔布/c裸鼠，年龄8-10周，体重16-19克，用于此协议。老鼠被安置在微型隔离笼中，并喂养市售的辣椒食品和水。 1. 矫形胰腺癌植入 为植入细胞做好准备。首先，计算手术所需的细胞数量（每只动物需要1 x10 6 个路西法酶标记的AsPC-1细胞和1 x10 6 个与癌症相关的人类胰?…
Representative Results
连续有59只老鼠接受了植入手术。总泄漏发生在八 （14%）小 鼠。注射时的泄漏程度估计如下协议部分所述。三周后，为了让这些植入的肿瘤生长，在切除前进行了预切除生物发光成像，排除了患有严重转移性疾病的小鼠。45 （76%）老鼠接受了手术切除手术。 所有 45 （100%）小鼠成功地进行了切除胰腺切除术/切除术，没有出血问题。在 43 （96%） 中实现了大于 5 mm 的宏观近胰?…
Discussion
胰腺癌的切除矫形小鼠模型很重要，因为它允许检测辅助剂和新辅助治疗。这在胰腺癌中尤为重要，因为手术仍然是最有效的治疗方法，但与复发的高风险有关。本文描述了一种可以可靠地产生胰腺癌的方法，这种方法通过切除可以治愈，复制了需要新辅助/辅助疗法的临床场景。
对现有方法的意义
尽管辅助疗法和新辅助疗法在胰腺癌中的重要性，但文献中很?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者得到了阿夫纳胰腺癌基金会的支持。
Materials
| Animals, Materials and Equipment for Implantation Procedure | |||
| AsPC-1 human pancreatic cancer cell line, luciferase tagged (luc+ gene from Promega PGL3 Basic plasmid) | American Type Culture Collection, Manassas, VA, USA | supplied by Professor Takashi Murakami, Saitama Medical University, Saitama, Japan | |
| Autoclip wound clips, 9 mm | Becton Dickson Pty Ltd, North Ryde, NSW, Australia | 500346 | |
| Basic Dressing Pack | Multigate Medical Products Pty Ltd, Villawood, NSW, Australia | ||
| Cancer associated human pancreatic stellate cells | Pancreatic Research Group cell bank | In house cell bank | |
| Cryogenic tubes, 1.0 mL | Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia | 366656 | |
| Disposable stainless-steel scalpel blade with handle, size 15 | Livingstone International, Mascot, NSW, | SCP15 | |
| Foetal bovine serum (FBS) | Life Technologies Corporation, Tullamarine, VIC, Australia | 16000044 | |
| Gilles fine tooth forceps 12 cm | Generic stainless steel microsurgical instrument set | ||
| Heated mats to maintain body temperature during surgery and postoperative recovery | Generic | ||
| Homozygous athymic nude mice: Strain BALB/c-Fox1nu/Ausb, female | Australian Bioresources, Moss Vale, NSW, Australia | ||
| Iscove's modified Dulbecco's medium (IMDM) with 4mM L-glutamine and no phenol red | Life Technologies Corporation, Tullamarine, VIC, Australia | 21056023 | |
| Jewellers forceps 11.5 cm | Generic stainless steel microsurgical instrument set | ||
| Micro needle holder (round handle) 15 cm straight | Generic stainless steel microsurgical instrument set | ||
| Micro scissors (round handle) 15 cm straight | Generic stainless steel microsurgical instrument set | ||
| Penicillin 10,000 U/mL, streptomycin 10,000 μg/mL | Life Technologies Corporation, Tullamarine, VIC, Australia | 15140122 | |
| Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle | Braun Australia Pty Ltd, Bella Vista, NSW, Australia | C1049407 | |
| Portable weighing scale | Precision balances, Bradford, MA, USA | ||
| Reflex clip applier and clip remover | World Precision Instruments, Sarasota, FL, USA | 500345 | |
| Roswell Park Memorial Institute (RPMI) 1640 with phenol red and 300 mg/L Lglutamine | Life Technologies Corporation, Tullamarine, VIC, Australia | 11875085 | |
| Round bodied vessel dilator 15 cm, 0.1 mm tip | Generic stainless steel microsurgical instrument set | ||
| Trypsin 0.05%, EDTA 0.02% | Life Technologies Corporation, Tullamarine, VIC, Australia | 25300054 | For pancreatic stellate cells |
| Trypsin 0.25%, EDTA 0.02% | Life Technologies Corporation, Tullamarine, VIC, Australia | 25200056 | For ASPC-1 cells |
| U-100 insulin syringes, 0.5 mL with 29 G (0.33 mm) × 13 mm needle | Terumo Medical Corporation, Elkton, MD, USA | ||
| Equipment for Resection Procedure | |||
| Alm self-retaining retractor | Generic stainless steel microsurgical instrument set | ||
| Autoclip wound clips 9 mm | Becton Dickson Pty Ltd, North Ryde, NSW | 500346 | |
| Basic Dressing Pack | Multigate Medical Products Pty Ltd, Villawood, NSW, Australia | 08-559NP | |
| Disposable stainless-steel scalpel blade with handle, size 15 | Livingstone International, Mascot, NSW, | SCP15 | |
| Gilles fine tooth forceps 12 cm | Generic stainless steel microsurgical instrument set | ||
| Hand-held high temperature fine tip cautery | Bovie Medical Corporation, Melville, NY, USA | AA01 | |
| Heated mats to maintain body temperature during surgery and postoperative recovery | Generic | ||
| IVIS Lumina II Bioluminescent Imaging Device | Caliper Life Sciences, Hopkinton, MA, USA | ||
| Jewellers forceps 11.5 cm | Generic stainless steel microsurgical instrument set | ||
| Micro needle holder (round handle) 15 cm straight | Generic stainless steel microsurgical instrument set | ||
| Micro scissors (round handle) 15 cm straight | Generic stainless steel microsurgical instrument set | ||
| Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle | Braun Australia Pty Ltd, Bella Vista, NSW, Australia | C1049407 | |
| Portable weighing scale | Precision balances, Bradford, MA, USA | ||
| Reflex wound clip applier and clip remover | World Precision Instruments, Sarasota, FL, USA | 500345 | |
| Round bodied vessel dilator 15 cm, 0.1 mm tip | Generic stainless steel microsurgical instrument set | ||
| Titanium “Weck style” Ligaclip, small | HZMIM, Hangzhou, China | ||
| Titanium Ligaclip applier for open surgery, small | HZMIM, Hangzhou, China | ||
| Volatile anaesthetic machine, including vapouriser and induction chamber | Generic | Generic vapouriser and induction chamber | |
| Drugs for Procedures | |||
| 70% w/w ethanol solution | Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia | Applied topically as surgical skin preparation | |
| Buprenorphine 0.3 mg/mL | Troy Laboratories Pty Ltd, Glendenning, NSW, Australia | Dose: 0.05 mg/kg s.c. | |
| D-Luciferin (1 U/g) | PerkinElmer, Inc., Waltham, MA, USA | 122799 | diluted in PBS to 15 mg/mL. Dose: 150 mg/kg i.p |
| Enrofloxacin 50 mg/mL | Troy Laboratories Pty Ltd, Glendenning, NSW, Australia | Dose: 5 mg/kg s.c. | |
| Flunixin 50 mg/mL | Norbrook Laboratories Australia, Tullamarine, VIC, Australia | Dose: 2.5 mg/kg s.c. | |
| Isoflurane | Zoetis Australia Pty Ltd., Rhodes, NSW, Australia | Dose (vapourised with oxygen): 4% induction, 3% maintenance | |
| Ketamine 100 mg/mL | Maylab, Slacks Creek, QLD, Australia | Dose: 80 mg/kg i.p. | |
| Povidone-Iodine 10% w/v solution | Perrigo Australia, Balcatta, WA, Australia | RIO00802F | Applied topically to the anterior abdomen as surgical skin preparation |
| Refresh eye ointment (liquid paraffin 42.5% w/w, soft white paraffin 57.3% w/w) | Allergan Australia Pty Ltd, Gordon, NSW, Australia | Applied to both eyes | |
| Sodium chloride 0.9% w/v | Braun Australia Pty Ltd, Bella Vista, NSW, Australia | 9481P | Dose: 900 μL s.c. |
| Water for injections BP | Pfizer Australia, Sydney, NSW, Australia | For dilution of drugs | |
| Xylazine 20 mg/mL | Troy Laboratories Pty Ltd, Glendenning, NSW, Australia | Dose: 10 mg/kg i.p. |
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Cite This Article
Pang, T. C. Y., Xu, Z., Mekapogu, A. R., Pothula, S., Becker, T. M., Goldstein, D., Pirola, R. C., Wilson, J. S., Apte, M. V. An Orthotopic Resectional Mouse Model of Pancreatic Cancer. J. Vis. Exp. (163), e61726, doi:10.3791/61726 (2020).