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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 5-fluorouracil | Teva Pharma AG | SMP 1532755 | |
| 48 multiwell plate | Sarstedt | 83 3923 | |
| 96 multiwell plate | Sarstedt | 82.1581.001 | |
| Acetone | Merck | 179124 | |
| Agarose powder | Merck | A9539 | |
| Amphotericin | Thermo Fisher Scientific | 15290018 | |
| Anti-Nuclei Antibody, clone 235-1 | Merck | MAB1281 | 1:200 dilution |
| Aquarium net QN6 | Penn-plax | 0-30172-23006-6 | |
| BSA | Merck | A9418 | |
| CellTrace | Thermo Fisher Scientific | C34567 | |
| CellTracker CM-DiI | Thermo Fisher Scientific | C7001 | |
| CellTracker Deep Red | Thermo Fisher Scientific | C34565 | |
| Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb | Cell Signaling Technology | 9661S | 1:250 dilution |
| Dimethyl sulfoxide (DMSO) | PanReac AppliChem ITW Reagents | A3672,0250 | |
| Dumont #5 forceps | World Precision Instruments | 501985 | |
| Folinic acid - Lederfolin | Pfizer | ||
| Glass capillaries, 3.5" | Drummond Scientific Company | 3-000-203-G/X | Outer diameter = 1.14 mm. Inner diameter = 0.53 mm. |
| Glass vials | VWR International | WHEAW224581 | |
| Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Thermo Fisher Scientific | A-21244 | 1:500 dilution |
| Goat serum | Thermo Fisher Scientific | 31872 | |
| Hoechst 33342 | Thermo Fisher Scientific | H3570 | |
| Irinotecan | Hospira | ||
| Low Temperature Freezer Vials | VWR International | 479-1220 | |
| McIlwain Tissue Chopper | World Precision Instruments | ||
| Microplate Mixer | SCILOGEX | 822000049999 | |
| Oxaliplatin | Teva | ||
| Paraformaldehyde | Merck | P6148-500G | |
| PBS | Thermo Fisher Scientific | 14190094 | |
| Penicillin-streptomycin | Thermo Fisher Scientific | 15140122 | |
| Petri dish 100 mm | Sarstedt | 83 3902500 | |
| Petri dish 60 mm | Sarstedt | 83 3901 | |
| Plastic Pasteur pipette | Sarstedt | 86.1171.010 | |
| Poly-Mount | Tebu-bio | 18606-5 | |
| Propidium iodide | Merck | P4170 | |
| RPMI-1640 medium | Thermo Fisher Scientific | 11875093 | |
| Scalpel blade No 10 Sterile Stainless Steel | VWR International | SWAN3001 | |
| Scalpel handle #3 | World Precision Instruments | 500236 | |
| Tricaine | Merck | E10521 | |
| Triton X-100 | Merck | T8787 | |
| Tween 20 | Merck | P9416 | |
| Vertical Micropipette Puller | Shutter instrument | P-30 |
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