Name: establishment of zebrafish patient-derived xenografts from pancreatic cancer for chemosensitivity testing
Uploaded: 2023-05-12T12:00:00
Description: Watch this Scientific Journal Video about Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing at JoVE.com

这项工作由比萨基金会（项目114/16）资助。作者要感谢Azienda Ospedaliera Pisana组织病理学部门的Raffaele Gaeta为患者样本选择和病理学支持。我们还要感谢Alessia Galante在实验中的技术支持。本文基于COST行动TRANSPAN的工作，CA21116，由COST（欧洲科学和技术合作）支持。

意大利公共卫生部根据关于动物使用和护理的指令2010/63 / EU批准了所有描述的动物实验。当地伦理委员会批准了这项研究，注册号为70213。从所有相关受试者获得知情同意。在开始之前，应准备好所有溶液和设备（第1节），并应将鱼穿过（第2节）。
1. 溶液和设备的准备
注：有关要制备的溶液和培养基，请参见 表1 。
<ol><li>琼?...

<ol>
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E., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2022.">Cancer statistics, 2022.</a> CA Cancer Journal for Clinicians. 72 (1), 7-33 (2022).</li><li>Trunk, 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=Emerging+treatment+strategies+in+pancreatic+cancer.">Emerging treatment strategies in pancreatic cancer.</a> Pancreas. 50 (6), 773-787 (2021).</li><li>Moffat, G. T., Epstein, A. S., O'Reilly, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer-A+disease+in+need:+Optimizing+and+integrating+supportive+care.">Pancreatic cancer-A disease in need: Optimizing and integrating supportive care.</a> Cancer. 125 (22), 3927-3935 (2019).</li><li>Sarantis, P., Koustas, E., Papadimitropoulou, A., Papavassiliou, A. G., Karamouzis, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+ductal+adenocarcinoma:+Treatment+hurdles,+tumor+microenvironment+and+immunotherapy.">Pancreatic ductal adenocarcinoma: Treatment hurdles, tumor microenvironment and immunotherapy.</a> World Journal of Gastrointestinal Oncology. 12 (2), 173-181 (2020).</li><li>Marshall, L. J., Triunfol, M., Seidle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+vs.+organoids:+a+preliminary+analysis+of+cancer+research+output,+funding+and+human+health+impact+in+2014-2019.">Patient-derived xenograft vs. organoids: a preliminary analysis of cancer research output, funding and human health impact in 2014-2019.</a> Animals. 10 (10), 1923 (2020).</li><li>Li, Y., Tang, P., Cai, S., Peng, J., Hua, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+based+personalized+medicine:+from+bench+to+bedside.">Organoid based personalized medicine: from bench to bedside.</a> Cell Regeneration. 9 (1), 21 (2020).</li><li>Jung, J., Seol, H. S., Chang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+and+application+of+patient-derived+xenograft+model+for+cancer+research.">The generation and application of patient-derived xenograft model for cancer research.</a> Cancer Research and Treatment. 50 (1), 1-10 (2018).</li><li>Rizzo, G., Bertotti, A., Leto, S. M., Vetrano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+tumor+models:+a+more+suitable+tool+for+pre-clinical+studies+in+colorectal+cancer.">Patient-derived tumor models: a more suitable tool for pre-clinical studies in colorectal cancer.</a> Journal of Experimental &amp; Clinical Cancer Research. 40 (1), 178 (2021).</li><li>Usai, 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=Zebrafish+patient-derived+xenografts+identify+chemo-response+in+pancreatic+ductal+adenocarcinoma+patients.">Zebrafish patient-derived xenografts identify chemo-response in pancreatic ductal adenocarcinoma patients.</a> Cancers. 13 (16), 4131 (2021).</li><li>Usai, 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=A+model+of+a+zebrafish+avatar+for+co-clinical+trials.">A model of a zebrafish avatar for co-clinical trials.</a> Cancers. 12 (3), 677 (2020).</li><li>Chen, X., Li, Y., Yao, T., Jia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benefits+of+zebrafish+xenograft+models+in+cancer+research.">Benefits of zebrafish xenograft models in cancer research.</a> Frontiers in Cell and Developmental Biology. 9, 616551 (2021).</li><li>Miserocchi, 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=Management+and+potentialities+of+primary+cancer+cultures+in+preclinical+and+translational+studies.">Management and potentialities of primary cancer cultures in preclinical and translational studies.</a> Journal of Translational Medicine. 15 (1), 229 (2017).</li><li>Baghban, R., 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=Tumor+microenvironment+complexity+and+therapeutic+implications+at+a+glance.">Tumor microenvironment complexity and therapeutic implications at a glance.</a> Cell Communication and Signaling. 18 (1), 59 (2020).</li><li>Albini, 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=Cancer+stem+cells+and+the+tumor+microenvironment:+interplay+in+tumor+heterogeneity.">Cancer stem cells and the tumor microenvironment: interplay in tumor heterogeneity.</a> Connective Tissue Research. 56 (5), 414-425 (2015).</li><li>Avdesh, 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> Journal of Visualized Experiments. (69), e4196 (2012).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nature Medicine. 19 (11), 1423-1437 (2013).</li><li>Tavares Barroso, M., 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=Establishment+of+pancreatobiliary+cancer+zebrafish+avatars+for+chemotherapy+screening.">Establishment of pancreatobiliary cancer zebrafish avatars for chemotherapy screening.</a> Cells. 10 (8), 2077 (2021).</li><li>Kopetz, S., Lemos, R., Powis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+patient-derived+xenografts:+the+best+laid+plans+of+mice+and+men.">The promise of patient-derived xenografts: the best laid plans of mice and men.</a> Clinical Cancer Research. 18 (19), 5160-5162 (2012).</li><li>Xing, F., Saidou, J., Watabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+associated+fibroblasts+(CAFs)+in+tumor+microenvironment.">Cancer associated fibroblasts (CAFs) in tumor microenvironment.</a> Frontiers in Bioscience. 15 (1), 166-179 (2010).</li><li>Str&#228;hle, U., 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=Zebrafish+embryos+as+an+alternative+to+animal+experiments-a+commentary+on+the+definition+of+the+onset+of+protected+life+stages+in+animal+welfare+regulations.">Zebrafish embryos as an alternative to animal experiments-a commentary on the definition of the onset of protected life stages in animal welfare regulations.</a> Reproductive Toxicology. 33 (2), 128-132 (2012).</li><li>Hidalgo, M., 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=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discovery. 4 (9), 998-1013 (2014).</li></ol>

癌症研究中的体内模型为了解癌症生物学和预测癌症治疗反应提供了宝贵的工具。目前，可以使用不同的体内模型，例如转基因动物（转基因和敲除小鼠）或来自人类原代细胞的患者来源的异种移植物。尽管有许多最佳功能，但每个功能都有各种限制。特别是，上述模型缺乏一种可靠的方法来模拟患者的肿瘤组织微环境。
有人提出，肿瘤微环境可能在药物反应?...

临床癌症研究的一个问题是，癌症不是一种单一的疾病，而是可以随着时间的推移而演变的各种不同的疾病，需要根据肿瘤本身和患者的特点进行特定的治疗1。因此，挑战在于转向以患者为导向的癌症研究，以便为癌症治疗结果的早期预测确定新的个性化策略2。这与胰腺导管腺癌（PDAC）尤其相关，因为它被认为是一种难以治疗的癌症，5年生存率为11%3。
诊断较晚、进展迅速、缺乏有效治疗仍然是PDAC最紧迫的临床问题。因此，主要的挑战是对患者进行建模并确定可在临床中应用的生物标志物，以选择符合个性化医疗的最有效疗法4，5，6。随着时间的推移，已经提出了新的方法来模拟癌症疾病：患者来源的类器官（PDO）和小鼠患者来源的异种移植物（mPDX）起源于人类肿瘤组织的来源。它们已被用于复制疾病以研究对治疗的反应和抵抗力，以及疾病复发7，8，9。
同样，对基于斑马鱼的患者来源异种移植（zPDX）模型的兴趣也有所增加，这要归功于其独特且有前途的特性10，代表了癌症研究的快速和低成本工具11，12。zPDX模型只需要很小的肿瘤样本量，这使得化疗的高通量筛选成为可能13。用于zPDX模型的最常见技术是基于原代细胞群的完整样品消化和植入，其部分复制肿瘤，但具有缺乏肿瘤微环境和恶性细胞与健康细胞之间串扰的缺点14。
这项工作展示了如何将zPDXs用作临床前模型来识别胰腺癌患者的化学敏感性特征。有价值的策略促进了异种移植过程，因为不需要细胞扩增，从而加速了化疗筛选。该模型的优势在于，所有微环境成分都保持原样，因为它们在患者癌症组织中，因为众所周知，肿瘤的行为取决于它们的相互作用15，16。这比文献中的替代方法非常有利，因为它可以保持肿瘤异质性并有助于以患者特异性的方式提高治疗结果和复发的可预测性，从而使zPDX模型能够用于联合临床试验。这份手稿描述了制作zPDX模型所涉及的步骤，从一段患者肿瘤切除开始，然后对其进行治疗以分析对化疗的反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-fluorouracil</td>
			<td>Teva Pharma AG</td>
			<td>SMP 1532755</td>
			<td></td>
		</tr>
		<tr>
			<td>48 multiwell plate</td>
			<td>Sarstedt</td>
			<td>83 3923</td>
			<td></td>
		</tr>
		<tr>
			<td>96 multiwell plate</td>
			<td>Sarstedt</td>
			<td>82.1581.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Merck</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose powder&#160;</td>
			<td>Merck</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nuclei Antibody, clone 235-1</td>
			<td>Merck</td>
			<td>MAB1281&#160;</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Aquarium net QN6</td>
			<td>Penn-plax</td>
			<td>0-30172-23006-6</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Merck</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34567</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker CM-DiI&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>C7001</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Deep Red&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34565</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>9661S</td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)&#160;</td>
			<td>PanReac AppliChem ITW Reagents</td>
			<td>A3672,0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid -&#160; Lederfolin</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries, 3.5&#34;</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>Outer diameter = 1.14 mm. Inner diameter = 0.53 mm.&#160;</td>
		</tr>
		<tr>
			<td>Glass vials&#160;</td>
			<td>VWR International</td>
			<td>WHEAW224581</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit&#160;IgG&#160;(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21244&#160;&#160;</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>31872</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Irinotecan</td>
			<td>Hospira</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Temperature Freezer Vials</td>
			<td>VWR International</td>
			<td>479-1220</td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Mixer</td>
			<td>SCILOGEX</td>
			<td>822000049999</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxaliplatin</td>
			<td>Teva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck</td>
			<td>P6148-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 100 mm</td>
			<td>Sarstedt</td>
			<td>83 3902500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60 mm</td>
			<td>Sarstedt</td>
			<td>83 3901</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette</td>
			<td>Sarstedt</td>
			<td>86.1171.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Mount</td>
			<td>Tebu-bio</td>
			<td>18606-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Merck</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade No 10 Sterile Stainless Steel</td>
			<td>VWR International</td>
			<td>SWAN3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle #3</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Merck</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Merck</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Micropipette Puller</td>
			<td>Shutter instrument</td>
			<td>P-30&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of zebrafish patient-derived xenografts from pancreatic cancer for chemosensitivity testing

癌症是全世界死亡的主要原因之一，许多类型癌症的发病率继续增加。在筛查、预防和治疗方面取得了很大进展;然而，仍然缺乏预测癌症患者化学敏感性特征的临床前模型。为了填补这一空白，开发并验证了一种 体内 患者衍生的异种移植模型。该模型基于受精后2天的斑马鱼 （Danio rerio） 胚胎，这些胚胎被用作从患者手术标本中提取的肿瘤组织的异种移植片段的接受者。 
还值得注意的是，生物样本没有被消化或分解，以维持肿瘤微环境，这对于分析肿瘤行为和对治疗的反应至关重要。该协议详细介绍了一种从原发性实体瘤手术切除中建立基于斑马鱼的患者来源异种移植物（zPDX）的方法。经解剖病理学家筛选后，使用手术刀刀片解剖标本。去除坏死组织、血管或脂肪组织，然后切成 0.3 mm x 0.3 mm x 0.3 mm 的碎片。 
然后将这些碎片进行荧光标记并异种移植到斑马鱼胚胎的周围空间中。可以低成本处理大量胚胎，从而能够对zPDX对多种抗癌药物的化学敏感性进行高通量 体内分析 。常规采集共聚焦图像以检测和量化化疗诱导的凋亡水平与对照组相比。异种移植程序具有显着的时间优势，因为它可以在一天内完成，为进行联合临床试验的治疗筛选提供了合理的时间窗口。

该协议描述了从原发性人胰腺腺癌中建立zPDX的实验方法。收集肿瘤样品，切碎并使用荧光染料染色，如方案第4节所述。然后通过将一块肿瘤植入 2 个 dpf 斑马鱼胚胎的围膜空间成功建立 zPDX，如协议第 5 节所述。如方案第6节所述，进一步筛选zPDX以确定患者来源癌细胞的化疗敏感性谱。例如，测试了化疗组合 FOLFOXIRI（5-氟尿嘧啶、亚叶酸、奥沙利铂和伊立替康），因为它被用作晚期胰腺导管腺癌?...

Watch this Scientific Journal Video about Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing at JoVE.com

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

临床前模型旨在提高癌症生物学知识并预测治疗效果。本文描述了基于斑马鱼的患者来源的带有肿瘤组织碎片的异种移植物（zPDX）的产生。zPDXs用化疗治疗，根据移植组织的细胞凋亡评估其治疗效果。

建立胰腺癌斑马鱼患者来源的异种移植物进行化学敏感性测试

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

斑马鱼患者来源的异种移植物是一种强大的临床前模型，用于预测癌症患者体内的化学敏感性特征。该协议描述了从患者手术标本开始的模型生成。通过异种移植小组织碎片而不是将细胞分离到斑马鱼胚胎中，可以维持肿瘤微环境，这是肿瘤进展和化疗反应的关键步骤。该模型的最大优势在于它有可能在短短一周的临床相关时间范围内预测患者的预后，作为个性化医疗的工具。通过用五毫升新鲜肿瘤培养基洗涤从患者收集的整个肿瘤组织开始样品处理。使用塑料巴斯德移液器上下移液10次。在重复洗涤三次之前，小心吸出并丢弃洗涤介质。将样品浸入一到两毫升新鲜肿瘤培养基中。使用手术刀刀片将肿瘤样本切成一到两毫米的立方体碎片，然后将它们放入装有肿瘤培养基的无菌五毫升塑料管中。将McIlwain组织切碎器设置为100微米厚度后，将肿瘤碎片放在切碎器的圆形塑料台上并切碎。将桌子旋转 90 度并重复切碎。将切碎的肿瘤碎片收集在含有肿瘤培养基的15毫升塑料管中。将切碎的肿瘤碎片以300G离心三分钟，然后吸出并丢弃上清液。将片段与细胞痕量溶液或替代荧光细胞追踪剂（如CM-DiI或深红）在37摄氏度的水浴中孵育30分钟。在孵育过程中，每10分钟轻轻重悬一次片段。在染色结束时，通过添加补充至少1%蛋白质的肿瘤培养基来去除任何游离染料。体积将是染色体积的五倍。将碎片以300G离心三分钟，弃去上清液。加入一毫升Dulbecco的磷酸盐缓冲盐水或DPBS，并重复此洗涤步骤三次。完成后，将碎片悬浮在60毫米培养皿中的5毫升DPBS中。为了建立基于斑马鱼的患者来源的异种移植物或zPDX，麻醉受精后两天的胚胎。在具有三个琼脂糖圆柱体的培养皿中，将斑马鱼胚胎放在露出一侧的圆柱体上。去除多余的溶液，将胚胎保持在薄膜中。使用无菌镊子，将染色的肿瘤组织碎片从60毫米培养皿转移到放置胚胎的1%琼脂糖载体中。然后拿起组织并将其放在胚胎蛋黄上，然后使用热拉玻璃微针将其推入阴道空间。在胚胎中加入一些E3 1%青霉素链霉素，使其重新进入液体中。在所有胚胎中重复植入肿瘤组织后，从培养皿中取出琼脂糖载体并在35摄氏度下孵育胚胎。植入后两小时，使用荧光体视显微镜检查胚胎是否有正确的染色阳性异种移植物。丢弃死胚胎和没有肿瘤碎片的胚胎完全在围膜空间内。完成后，将胚胎随机分布在六个多孔板中，根据实验计划平均分成几组。通过将药物混合到E3 1%笔链球菌中来稀释药物，以制备FOLFOXIRI鸡尾酒。植入两个小时后，从每个孔中取出培养基并加入药物鸡尾酒。将胚胎放入35摄氏度的培养箱中三天，每天更换培养基以更新药物。对于成像，在共聚焦显微镜的40X物镜下捕获图像。使用 1024 x 512 像素的分辨率，Z 间距为 5 微米。为分析氰基中检测到的细胞凋亡而采集的zPDX的整个安装图像显示，与对照组相比，联合治疗增加了细胞凋亡。该案例研究报告称，与对照组相比，FOLFOXIRI治疗组的植入异种移植物中凋亡细胞的比例在统计学上显着增加。该协议显示了使用胰腺癌组织的一个例子，但斑马鱼患者来源的异种移植物可以从不同类型的癌症中建立。此外，由于该模型的伦理影响较小，因此很容易用于新药的体内筛选。该方法在技术上具有挑战性，需要专业操作员。斑马鱼胚胎操作和显微注射方面的先前专业知识将加快该技术的培训。

Research

JoVE Journal

Cancer Research

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

科研

癌症研究

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

患者衍生的胰腺癌异质异种移植模型,使用斑马鱼幼虫作为比较药物评估的宿主

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

患者衍生胰腺腺腺腺腺癌有机体模型的分离和特征

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

在全融合微流体平台中增强患者衍生异种移植物的外体 3D 水凝胶培养物的生存能力

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...