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Intracaecal Orthotopic Colorectal Cancer Xenograft Mouse Model
脑内原位结直肠癌异种移植小鼠模型   

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Abstract

The host microenvironment plays a prominent role in tumor growth, angiogenesis, invasion, metastasis, and response to therapy. Orthotopic tumor model mimics the natural environment of tumor development and provides an effective approach to investigate tumor pathophysiology and develop therapeutic strategies. This protocol describes the technique involving injection of colorectal cancer cell suspension into the intestinal wall of mice to establish an orthotopic colorectal tumor model.

Keywords: Colorectal cancer(结直肠癌), Orthotopic model(原位模型), Cecum(盲肠), Ascending colon(升结肠), Nude mice(裸鼠)

Background

Various murine models have been developed to facilitate studies of human cancers and allow better understanding of mechanisms contributing to tumor growth. While heterotopic xenograft models involve implanting cancer cells into the flank of immunocompromised mouse subcutaneously, orthotopic tumor models more closely resemble the original tumor development due to the implantation of tumor cells directly into the organ of origin (Richmond and Su, 2008). Although orthotopic xenograft models are technically challenging and labor-intensive, orthotopic transplants are able to more accurately mimic human tumor and better predict a patient’s response to chemotherapy in comparison with heterotopic transplants because of the effects of tumor microenvironment (Talmadge et al., 2007). With increased knowledge regarding the important role of tumor-host cell interaction during tumor progression, genetically engineered mouse (GEM) models using immunocompetent mice extend our ability to model the complexity of human cancers (Gopinathan and Tuveson, 2008; Zitvogel et al., 2016). However, GEM models are more expensive, often require months to a year to develop tumors, and have the drawbacks regarding the heterogeneity of tumor frequency, latency and growth. By contrast, xenografts are less expensive, require less time to establish tumors, and have better reproducibility (Vandamme, 2014). In this protocol, we describe the procedure of generating orthotopic colorectal cancer by injecting human cancer cells into immunocompromised mice (Tseng et al., 2007; Liao et al., 2015).

Materials and Reagents

  1. 27 G x 5/8 syringes (BD, catalog number: 329412 )
  2. 0.5-μm membrane filter (EMD Millipore, catalog number: FHLC04700 )
  3. 6-8-week old female NU/J nude mice (THE JACKSON LABORATORY, catalog number: 007850 ) or CB17 severe combined immunodeficient (SCID) mice (Charles River, Quebec, Canada)
  4. Phosphate-buffered saline (PBS), sterile (Corning, catalog number: 21-040 )
  5. Buprenorphine SR-LAB (Zoopharm, Windsor, CO)
  6. Betadine
  7. 70% alcohol
  8. Ophthalmic ointment
  9. 0.9% sodium chloride irrigation (Hospira, catalog number: 0409-6138-22 )
  10. 2,2,2-Tribromoethanol (Sigma-Aldrich, catalog number: T48402 )
  11. 2-methyl-2-butanol (Sigma-Aldrich, catalog number: 152463 )
  12. Avertin working solution or ketamine/xylazine/acepromazine (see Recipes)

Equipment

  1. Forceps (sterilize before use) (Fine Science Tools, catalog number: 11006-12 )
  2. Surgical scissors (sterilize before use) (Fine Science Tools, catalog number: 91402-12 )
  3. Reflex wound closure clip applier (Fine Science Tools, catalog number: 12020-09 )
  4. Reflex wound closure clips (Fine Science Tools, catalog number: 12022-09 )
  5. Reflex wound clip remover (Fine Science Tools, catalog number: 12023-00 )
  6. Heat lamp (Morganville Scientific, catalog number: HL0100 )
  7. BETADINE® Solution Swab Aid® Antiseptic pads (Moore medical, catalog number: 90697 )
  8. Virkon® disinfectant cleaner (Sigma-Aldrich, catalog number: Z692158 )
  9. Trimmer/clipper (Wahl Clipper, catalog number: 8685 )

Procedure

  1. Autoclave forceps, surgical scissors, wound closure applier and clips prior to procedure.
  2. The entire surgical procedure is to be carried out under a sterile hood.
  3. Clean surgical equipment with Virkon® disinfectant cleaner between mice.
  4. Trypsinize 80% confluent colorectal cancer cells, e.g., HT-29 and HCT116, and wash cells with PBS, centrifuge at 200 x g for 5 min at 4 °C. Discard supernatant and repeat the wash step three times.
  5. Keep cells (5 x 106-1 x 107 cells in 50 μl PBS) on ice and check cell viability before injection. Example: For 10 injections, prepare at least 12 x 5 x 106 to 12 x 1 x 107cells in 12 x 50 μl PBS.
  6. Administer buprenorphine pre-operatively (0.1 mg/kg, subcutaneously) 10 min before surgery.
  7. Inject mice with Avertin solution (0.5 ml/25 mg mouse) or ketamine/xylazine/acepromazine (0.2 ml/20 mg mouse) intraperitoneally to anesthetize the mouse before surgery.
  8. Check hind limb reflex by pinching foot to make sure that mice are asleep. Induction time is around 1-2 min. Surgical anesthesia lasts approximately for 15-45 min with a sleep time of 60-120 min if Avertin is used.
    Note: Surgical anesthesia lasts approximately for 30-40 min with a sleep time of 60-180 min if ketamine/xylazine/acepromazine is used.
  9. Preparation of animals: 1) Remove hair (if SCID mice are used) from the surgical area by using a trimmer/clipper; 2) apply betadine in a circular fashion starting at the surgical incision and rotating outward; 3) alternate betadine with 70% alcohol; 4) repeat three times, discard cotton pad after each use; 5) Before surgery, apply ophthalmic ointment to both eyes to prevent desiccation; 6) before starting, cover the rodent with sterile drape to avoid contamination of the incision; 7) during surgery, use thermal pads to maintain normal body temperature.
  10. Place the animal in ventral recumbency. Make a 0.5-inch long midline skin incision along the lower abdomen around 1 cm anterior to the genital by a pair of forceps and surgical scissors. The body wall is then incised in the same length. Gently pull out cecum and ascending colon from the incision. Keep the exposed cecum and ascending colon moist with 0.9% sodium chloride during the entire procedure.
  11. Inject 5 x 106-1 x 107 colon cancer cells in 50 μl PBS into the cecum wall (Figure 1). (Tseng et al., 2007)
    Note: Make sure that bubbles are removed from the cell suspension.


    Figure 1. Schematic diagram of mouse gastrointestinal tract. Cecum and the injection site are indicated in the diagram.

  12. Place cecum and ascending colon back into the abdominal cavity.
    Note: Be careful not to squeeze the injection area.
  13. Suture abdominal muscle layer, and then close the skin by wound clips.
  14. Keep mice warm under heat lamp until the mice recover from anesthesia. The lamp should be kept 12-14 inches away from the mouse and attentive monitoring should be performed to prevent overheating. Surgical heating pads or a temperature-controlled cage/incubator can be used as alternatives. Mice should be monitored until maintaining upright posture and walking normally before returning to the animal housing room.
  15. Administer buprenorphine post-operatively (0.1 mg/kg, subcutaneously in one 72-h injection) as needed.
  16. Check mice every day following surgery for infection or signs of pain. Antibiotics may be used if infections occur.
  17. Remove clips by wound clip remover after 7-10 days once the incision has healed.

Data analysis

One month after surgery, tumors can be harvested to measure tumor weights and tumor volumes. However, the tumor growth rate and tumor volumes depend on the cell lines that are injected. Tumor diameters are measured by a digital caliper, and the tumor volume (mm3) is calculated by the formula: Volume = (width)2 x length/2. Tumors can be further confirmed by immunohistochemical staining for Ki67/haematoxylin after resection. In addition, depends on availability of equipment and reagents, small animal imaging instrumentations, including x-ray computed tomography (CT), magnetic resonance imaging (MRI) or bioluminescence imaging, are useful tools to non-invasively measure burden of xenografted tumors. Lymph node, liver, and lung metastases can be further examined by bioluminescence imaging (if cells contain luciferase) or by microscopic analysis after Ki67/haematoxylin staining within 16 weeks after injection. At least five mice are recommended for each group. A P value < 0.05 is considered as significant using Student’s t-test. However, the number of mice to be used depends on the hypothesis tested and on the statistical test. A power analysis should be done prior to the start of the experiment.

Notes

  1. Complete the injection as soon as possible after the cells are trypsinized. Ideally, cells should be injected within one hour after trypsinization.
  2. Colon cancer cells aggregate easily. Ensure cells are completely separated when calculating the cell numbers for injection in order to reduce the variations between different mice.
  3. Yellowing of the Avertin solution indicates toxic degradation. Discard the solution when it turns yellow.
  4. NIH and European guidelines discourage the use of Avertin. The use of Avertin must be justified and approved by The Institutional Animal Care and Use Committee (IACUC). Alternatively, ketamine/xylazine/acepromazine cocktail is another commonly used injectable anesthetic agents.

Recipes

  1. Avertin working solution
    1. Dissolve 2.5 g tribromoethanol in 5 ml 2-methyl-2-butanol. Heat to approximately 40 °C and stir vigorously in the dark
    2. Add distilled water to a final volume of 200 ml, stirring continuously in the dark until the solid is dissolved
    3. Filter the solution through a 0.5-µm membrane filter
    4. Store filtered solution in the dark at 4 °C. Discard the unused solution after 2 weeks
  2. Ketamine/xylazine/acepromazine cocktail
    1. Mix 1 ml Ketamine (100 mg/ml), 0.5 ml xylazine (20 mg/ml) and 0.3 ml acepromazine (10 mg/ml) and then add 8.2 ml normal saline (0.9%) or PBS
    2. Keep the sterile solution in a sterile container
    Notes:
    1. Make fresh mixture and use the ketamine/xylazine/acepromazine cocktail within one week, as the is mixture are not stable and may lose potency.
    2. Ketamine/xylazine in combination with NSAID prior to surgery or addition of acepromazine will augment the anesthetic effect of ketamine/xylazine.

Acknowledgments

This work was supported by NIH (CCSG CA16672); Cancer Prevention and Research Institute of Texas (RP150245); The University of Texas MD Anderson-China Medical University and Hospital Sister Institution Fund; Ministry of Science and Technology, International Research-intensive Centers of Excellence in Taiwan (I-RiCE; MOST 105-2911-I002-302); Ministry of Health and Welfare, China Medical University Hospital Cancer Research Center of Excellence (MOHW106-TDU-B-2 12-144003). This protocol was adapted from previous work published in the Journal of Clinical Investigation (Liao et al., 2015).

References

  1. Gopinathan, A. and Tuveson, D. A. (2008). The use of GEM models for experimental cancer therapeutics. Dis Model Mech 1(2-3): 83-86.
  2. Liao, H. W., Hsu, J. M., Xia, W., Wang, H. L., Wang, Y. N., Chang, W. C., Arold, S. T., Chou, C. K., Tsou, P. H., Yamaguchi, H., Fang, Y. F., Lee, H. J., Lee, H. H., Tai, S. K., Yang, M. H., Morelli, M. P., Sen, M., Ladbury, J. E., Chen, C. H., Grandis, J. R., Kopetz, S. and Hung, M. C. (2015). PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response. J Clin Invest 125(12): 4529-4543.
  3. Richmond, A. and Su, Y. (2008). Mouse xenograft models vs GEM models for human cancer therapeutics. Dis Model Mech 1(2-3): 78-82.
  4. Talmadge, J. E., Singh, R. K., Fidler, I. J. and Raz, A. (2007). Murine models to evaluate novel and conventional therapeutic strategies for cancer. Am J Pathol 170: 793-804.
  5. Tseng, W., Leong, X. and Engleman, E. (2007). Orthotopic mouse model of colorectal cancer. J Vis Exp 10: 484.
  6. Vandamme, T. F. (2014). Use of rodents as models of human diseases. J Pharm Bioallied Sci. 6(1): 2-9.
  7. Zitvogel, L., Pitt, J. M., Daillère, R. and Smyth, M. J. (2016). Mouse models in oncoimmunology. Nat Rev Cancer 16:759-773.

简介

宿主微环境在肿瘤生长,血管生成,侵袭,转移和治疗反应中起着重要作用。原位肿瘤模型模拟肿瘤发展的自然环境,并提供了一种有效的方法来研究肿瘤病理生理学和开发治疗策略。该方案描述了将结肠直肠癌细胞悬液注射到小鼠肠壁中以建立原位结肠直肠肿瘤模型的技术。

背景 已经开发了各种鼠模型来促进对人类癌症的研究,并且更好地了解有助于肿瘤生长的机制。虽然异位异种移植模型包括将癌细胞植入免疫受损小鼠的侧腹皮下,原位肿瘤模型由于将肿瘤细胞直接植入原发器官而更接近于原始肿瘤发展(Richmond和Su,2008)。虽然原位异种移植模型在技术上是具有挑战性和劳动密集型的,但由于肿瘤微环境的影响,原位移植物能够更准确地模拟人类肿瘤并更好地预测患者对化疗的反应与异位移植相比(Talmadge等, / em>。,2007)。随着关于肿瘤进展期间肿瘤宿主细胞相互作用的重要作用的知识的增加,使用免疫活性小鼠的遗传工程小鼠(GEM)模型扩展了我们模拟人类癌症复杂性的能力(Gopinathan和Tuveson,2008; Zitvogel等人。,2016)。然而,GEM模型更昂贵,通常需要数月至一年才能开发肿瘤,并且具有关于肿瘤频率,潜伏期和生长异质性的缺点。相比之下,异种移植物较便宜,需要更少的时间建立肿瘤,并具有更好的再现性(Vandamme,2014)。在本方案中,我们描述了通过将人癌细胞注射到免疫受损小鼠中产生原位结肠直肠癌的程序(Tseng等人,2007; Liao等人,2015) 。

关键字:结直肠癌, 原位模型, 盲肠, 升结肠, 裸鼠

材料和试剂

  1. 27 G x 5/8注射器(BD,目录号:329412)
  2. 0.5μm膜过滤器(EMD Millipore,目录号:FHLC04700)
  3. 6-8周龄的雌性NU / J裸鼠(THE JACKSON LABORATORY,目录号:007850)或CB17严重联合免疫缺陷(SCID)小鼠(Charles River,Quebec,Canada)
  4. 磷酸盐缓冲盐水(PBS),无菌(Corning,目录号:21-040)
  5. 丁丙诺啡SR-LAB(Zoopharm,Windsor,CO)
  6. Betadine
  7. 70%酒精
  8. 眼科软膏
  9. 0.9%氯化钠灌洗(Hospira,目录号:0409-6138-22)
  10. 2,2,2-三溴乙醇(Sigma-Aldrich,目录号:T48402)
  11. 2-甲基-2-丁醇(Sigma-Aldrich,目录号:152463)
  12. Avertin工作溶液或氯胺酮/赛拉嗪/ acepromazine(参见食谱)

设备

  1. 镊子(使用前灭菌)(精细科学工具,目录号:11006-12)
  2. 手术剪刀(使用前灭菌)(精细科学工具,目录号:91402-12)
  3. 反射伤口闭合夹子(精细科学工具,目录号:12020-09)
  4. 反射伤口闭合夹(精细科学工具,目录号:12022-09)
  5. 反射伤口夹去除器(精细科学工具,目录号:12023-00)
  6. 加热灯(Morganville Scientific,目录号:HL0100)
  7. BETADINE ® Solution Swab Aid ®防腐垫(Moore medical,目录号:90697)
  8. 消毒剂清洁剂(Sigma-Aldrich,目录号:Z692158)
  9. 修剪器/剪刀(Wahl Clipper,目录号:8685)

程序

  1. 高压灭菌镊子,手术剪刀,伤口闭合敷贴器和手术前的夹子。
  2. 整个外科手术应在无菌罩下进行
  3. 在小鼠之间使用Virkon ®消毒剂清洁剂清洁手术设备。
  4. 胰蛋白酶消化80%汇合的结肠直肠癌细胞,例如HT-29和HCT116,并用PBS洗涤细胞,在4℃下以200×g离心5分钟。弃上清,重复洗涤步骤三次。
  5. 在冰上保存细胞(5×10 6 -1 x 10 7 细胞),并在注射前检查细胞活力。实施例:对于10次注射,在12×50μlPBS中制备至少12×5×10 6个至12×1×10 7个细胞。
  6. 术前10分钟施用丁丙诺啡(0.1 mg / kg,皮下注射)。
  7. 用Avertin溶液(0.5ml / 25mg小鼠)或氯胺酮/甲苯噻嗪/乙酰丙嗪(0.2ml / 20mg小鼠)腹膜内注射小鼠,在手术前麻醉小鼠。
  8. 通过捏脚检查后肢反射,以确保小鼠睡着了。感应时间约1-2分钟。如果使用Avertin,手术麻醉持续约15-45分钟,睡眠时间为60-120分钟。
    注意:如果使用氯胺酮/甲苯噻嗪/乙酰丙嗪,手术麻醉持续约30-40分钟,睡眠时间为60-180分钟。
  9. 动物的制备:1)通过使用修剪器/剪刀从手术区域去除头发(如果使用SCID小鼠) 2)从外科切口开始以旋转方式施用betadine,向外旋转; 3)替代甜菜碱与70%酒精; 4)重复三次,每次使用后丢弃棉垫; 5)手术前应用双眼眼药膏以防止干涩; 6)开始前,用无菌悬垂覆盖啮齿动物,避免切口污染; 7)在手术过程中,使用热垫保持体温正常。
  10. 将动物置于腹侧卧位。通过一对镊子和手术剪刀,沿着生殖器前方约1厘米的下腹部做一个0.5英寸长的中线皮肤切口。然后将体壁切开相同的长度。从切口轻轻拉出盲肠和升肠。在整个手术过程中,保持暴露的盲肠和上升的结肠潮湿0.9%氯化钠
  11. 将50μlPBS中的5×10 6个结肠癌细胞注入盲肠壁(图1)。 (Tseng等人,2007)
    注意:确保从细胞悬浮液中除去气泡。


    图1.小鼠胃肠道示意图盲肠和注射部位如图所示。

  12. 将盲肠和上结肠放回腹腔。
    注意:注意不要挤压注射区域。
  13. 缝合腹肌层,然后用伤口夹紧肌肤。
  14. 保持老鼠在加热灯下温暖,直到小鼠从麻醉中恢复。灯泡应保持距离鼠标12-14英寸,应进行细心监控以防止过热。手术加热垫或温控笼/培养箱可作为替代品使用。监护小鼠,直至保持直立姿势,并在返回动物房间前正常行走。
  15. 根据需要,术后给予丁丙诺啡(0.1 mg / kg,一次72 h注射后皮下注射)。
  16. 手术后每天检查小鼠感染或疼痛迹象。如果发生感染,可以使用抗生素。
  17. 7-10天后,一旦切口愈合,请用伤口夹去除器取下夹子。

数据分析

手术后一个月,可以收获肿瘤以测量肿瘤重量和肿瘤体积。然而,肿瘤生长速度和肿瘤体积取决于注射的细胞系。肿瘤直径用数字卡尺测量,肿瘤体积(mm 3 )通过下列公式计算:体积=(宽度) 2 x长度/ 2。切除后可通过Ki67 /苏木精的免疫组织化学染色进一步证实肿瘤。另外,取决于设备和试剂的可用性,小动物成像仪器,包括X射线计算机断层扫描(CT),磁共振成像(MRI)或生物发光成像,是非侵入性测量异种移植肿瘤负担的有用工具。可以通过生物发光成像(如果细胞含有荧光素酶)或通过在注射后16周内Ki67 /苏木精染色后的显微镜分析进一步检查淋巴结,肝和肺转移。每组至少要推荐5只小鼠。 A 值< 0.05被认为是显着的使用学生的测试。然而,使用的小鼠数量取决于所测试的假设和统计测试。功率分析应在实验开始之前进行。

笔记

  1. 在细胞进行胰蛋白酶消化后,尽快完成注射。理想情况下,细胞应在胰蛋白酶消化后1小时内注射。
  2. 结肠癌细胞容易聚集。在计算注射细胞数时,确保细胞完全分离,以减少不同老鼠之间的差异
  3. Avertin溶液黄化表明毒性降解。当黄色变为黄色时,放弃解决方案。
  4. NIH和欧洲指南不鼓励使用Avertin。 Avertin的使用必须由机构动物护理和使用委员会(IACUC)证明和批准。或者,氯胺酮/赛拉嗪/ acepromazine鸡尾酒是另一种常用的可注射麻醉剂。

食谱

  1. Avertin工作解决方案
    1. 将2.5g三溴乙醇溶于5ml 2-甲基-2-丁醇中。加热至约40°C,并在黑暗中剧烈搅拌
    2. 加入蒸馏水至终体积200毫升,在黑暗中连续搅拌直至固体溶解
    3. 通过0.5μm膜过滤器过滤溶液
    4. 将过滤的溶液在4℃的黑暗中储存。 2周后丢弃未使用的溶液
  2. 氯胺酮/赛拉嗪/ acepromazine鸡尾酒
    1. 混合1ml氯胺酮(100mg / ml),0.5ml甲苯噻嗪(20mg / ml)和0.3ml乙酰丙嗪(10mg / ml),然后加入8.2ml生理盐水(0.9%)或PBS
    2. 将无菌溶液保存在无菌容器中
    注意:
















    1. 在手术或加入乙酰丙嗪之前,氯胺酮/赛拉嗪与NSAID联合将增加氯胺酮/赛拉嗪的麻醉效果。

致谢

这项工作得到NIH(CCSG CA16672)的支持。德州癌症防治研究所(RP150245);德克萨斯大学MD安德森中国医科大学和医院姐妹机构基金会;科技部,台湾国际研究密集型中心(I-RiCE; MOST 105-2911-I002-302);中国医科大学附属医院癌症研究中心生福利部(MOHW106-TDU-B-2 12-144003)。该协议改编自 Journal of Clinical Investigation (Liao等人,2015)之前的工作。

参考

  1. Gopinathan,A.和Tuveson,DA(2008)。&nbsp; 使用GEM模型进行实验性癌症治疗。 Dis Model Mech 1(2-3):83-86。
  2. 摘要:目的:研究iao,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Lee,HH,Tai,SK,Yang,MH,Morelli,MP,Sen,M.,Ladbury,JE,Chen,CH,Grandis,JR,Kopetz,S.and Hung,MC(2015) =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/26571401”target =“_ blank”> EGF受体的PRMT1介导的甲基化调节信号传导和西妥昔单抗应答。 > J Clin Invest 125(12):4529-4543。
  3. Richmond,A.and Su,Y.(2008)。小鼠异种移植模型与用于人类癌症治疗的GEM模型。模型机械 1(2-3):78-82。
  4. Talmadge,JE,Singh,RK,Fidler,IJ and Raz,A.(2007)。&nbsp; 评估癌症的新型和常规治疗策略的小鼠模型。 Am J Pathol 170:793-804。
  5. Tseng,W.,Leong,X.和Engleman,E。(2007)。结直肠癌的原位小鼠模型 J Vis Exp 10:484.
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引用:Liao, H. and Hung, M. (2017). Intracaecal Orthotopic Colorectal Cancer Xenograft Mouse Model. Bio-protocol 7(11): e2311. DOI: 10.21769/BioProtoc.2311.
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