处理棉鼠的研究为溶瘤病毒的临床前评估
Summary
Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.
Abstract
Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.
Introduction
溶瘤病毒（OV）通过利用正常和肿瘤细胞1,2-之间的生化差异选择性地复制在肿瘤细胞中。有两种类型的OVS：那些不需要的突变来实现选择性溶瘤，称为天然存在的野生型病毒和那些必须被改造以实现选择性溶瘤。在给定的肿瘤类型突变的集合确定为一个OV 2的选择生长优势正常细胞的性质。使用OVS的安全和利益已被证明在临床试验中3-7。尽管在溶瘤病毒疗法的领域中的先进存在临床前和临床结果之间的间隙，这表明需要更好的模型来评价OVS的抗肿瘤功效。
牛疱疹病毒1型（BHV-1）是疱疹病毒家族的一个成员，并且Alphaherpesviridae亚科。 BHV-1 initi茨牛呼吸系统疾病综合症在牛，体现在各种各样的症状类似感冒8,9。 BHV-1结合使用的HSV-1的附件和条目受体如硫酸乙酰肝素和的Nectin-1 10。然而，它在Nectin-2的10的地方结合CD155。 BHV-1具有一个非常窄的宿主范围，使得其不能有效地进入并启动复制在正常和转化的鼠细胞3,4,10。这使得使用常规的鼠模型的有问题的。 BHV-1的溶瘤能力已被证明在体外 11,12。 BHV-1已经显示出从各种组织学起源，包括乳腺癌细胞和乳腺癌起始细胞11,12的启动在复制和杀死人肿瘤细胞。然而，BHV-1的抗肿瘤能力，必须在体内的免疫活性宿主的范围内进行评价。
人类腺病毒（广告），对于这有57种血清型鉴定，最常见的导致呼吸系统疾病的人。溶瘤腺病毒载体已被评估为他们的抗肿瘤功效与若干前进进入临床试验阶段13-15。尽管有前途的临床前数据，临床效果已经达到人们的期望。人肿瘤异种移植模型通常用于研究Ad载体的抗肿瘤功效，尽管它们表现出减弱的免疫应答的病毒16,17。此外，同基因小鼠模型是非允许对广告感染，使得使用这些模型不切实际17,18宿主的免疫应答的评估。
宿主免疫系统已被列为最具影响力的机制，OVS引起肿瘤细胞死亡19。耐受和非耐受的肿瘤相关抗原之间的抗肿瘤反应（TAA）的型号而异，并且可以极大地影响OV疗法的成功。在HSV-1 OV KM100（ICP0 N212VP16 在 1814年20）20,21引起肿瘤消退中的80％的荷瘤小鼠中的鼠多瘤中间T抗原乳腺癌模型22。然而，在HER-2 / neu的型号，KM100的抗肿瘤功效在同系小鼠的20％完全消退和肿瘤停滞之间在转基因变化，HER-2耐受小鼠。总之，这些数据突出的利用动物模型最能概括人类的免疫景观，充分了解哪些功能确定治疗的成功充分评估OVS的重要性。
棉鼠（Sigmodon hispidus），原产于北美和南美，是最常用的呼吸道合胞病毒感染模型（如5审查）。棉鼠中也使用抗BHV-1接种研究，他们概括与牛呼吸道疾病6,23相关联的病理。此外，BHV-1感染棉鼠是免疫原性，诱导持续粘膜和系统免疫应答6,23-25。细胞系已被来自自发纤维肉瘤和乳腺（LCRT）和骨（CCRT和VCRT），分别为26的骨肉瘤。棉鼠已被用来评估溶瘤腺病毒载体在体内的效力，因为它们很容易受到感染的广告，并表现出相似的病理学人类27-29。对于OVS的临床前评价使用免疫受损模型不仅少指示对治疗的临床反应，但它们不能顾及在溶瘤病毒疗法30,31免疫系统的作用。因此，同系和乳腺癌和骨肉瘤的肿瘤耐受棉鼠模型是相关的模型，其中，以评估OVS，如BHV-1和Ad的临床前功效不能使用常规的小鼠模型的研究。
Protocol
Representative Results
Discussion
Cotton rats are highly excitable and have a strong flight response. Therefore, special care should be taken to minimize any undue stress on the animal. The cage setup described will allow for safe and easy capture of the animals, with the placement of the enrichment tube being of the utmost importance. When setting up cages, ensure that the enrichment tubes meet the size and shape requirements, and are placed in proper orientation in the cage. It is also important to ensure that any technicians who might be aiding in ani…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Breanne Cuddington holds a fellowship from the Canadian Breast Cancer Foundation. This work was sponsored by operating grants from the Cancer Research Society and the Canadian Cancer Society Research Institute (formerly the Canadian Breast Cancer Research Alliance). We thank Ann Tollefson (Saint Louis University School of Medicine) for LCRT cells and Dr. Kathleen Delaney and Marion Corrick for technical assistance with cotton rat housing and sedation.
Materials
| Name of Material/Equipment | Company | Catalog # | Comments/Description |
| Dulbecco’s modified Eagle’s medium | Gibco | 11965-092 | May use any brand |
| 1X Phosphate Buffered Saline | Can prepare in lab, filter to sterilize | ||
| 200 mM L-glutamine | Gibco | 25030164 | May use any brand |
| 100x Antibiotic-Antimycotic | Gibco | 15240-062 | May use any brand |
| Fetal bovine serum | Quality Biological Inc. | 110-001-101HI | May use any brand |
| T-150cm2 tissue culture flask | Fisher Scientific | 14-826-80 | May use any brand |
| 1X TypLE Express | Life Technologies | 12604-013 | |
| 12-well cell culture plate, flat bottom | Fisher Scientific | 08-772-29 | May use any brand, must be tissue culture treated |
| alamarBlue | Life Technologies | DAL1025 | May use an alternative reagent for determination of cell viability |
| 8640 Teklad 22/5 Rodent diet | Harlan | 8640 | |
| 1/8” corncob rodent bedding | Harlan | 7092 | |
| Nestlets | Ancare | – | Made of pulped virgin cotton fiber, dust-free and autoclavable |
| 50 mL Conical tubes | Fisher Scientific | 14-432-22 | May use any brand, must be sterile |
| Isoflurane USP, 99.9 %, inhalation anesthetic | Pharmaceutical Partners of Canada Inc. | M60302 | |
| 70% Ethanol | Can prepare in lab | ||
| 10 % Neutral Buffered Formalin | Sigma-Aldrich | HT501128 | May use any brand |
| Name of Material/Equipment | Company | Catalog # | Comments/Description |
| NAPCO NapFlow 1200 Class II A/B3 Biosafety Microbiological Safety Cabinet (cell culture hood) | NAPCO | Model used not currently available | May use any brand |
| Thermo Fisher Scientific Precision Heated Water Bath | Fisher Scientific | Model used not currently available | May use any brand |
| Reichert Bright-line Hemacytometer | Sigma-Aldrich | Z359629 | May use any brand |
| Typhoon Trio BioAnalyzer | GE Healthcare Life Sciences | Model used not currently available | May use any fluorescence plate reader |
| Tecan Safire2 Multi-detection Microplate Reader | Tecan | Model used not currently available | May use any fluorescence plate reader |
| Allegra 6R benchtop centrifuge | Beckman Coulter | 366816 | May use any brand |
| Table Top Anaesthesia machine | VetEquip | Model used not currently available | May use any brand, must be portable |
| Wahl Peanut Mini Clippers | Wahl | May use any brand of small clippers | |
| Insulin syringes 29 G x 1/2', 0.3 mL | BD | 329464 | May use any brand. Insulin syringes are recommended as they make injections easier through the rat’s tough skin. |
| Cotton swabs | MedPro | 018-425 | May use any brand |
| Sharp-Pointed Dissecting Scissors | Fisher Scientific | 8940 | May use any brand |
| Dissecting Tissue Forceps | Fisher Scientific | 13-812-41 | May use any brand |
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