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
Summary
该协议描述了基于病毒的双荧光标记肿瘤异种移植模型中的优化过程,使用幼虫斑马鱼作为宿主。这种异质异种异种移植模型模仿胰腺癌在体内微环境的组织组成,并作为在个性化zPDX(斑马鱼患者衍生异种移植)模型中评估药物反应的更精确的工具。
Abstract
患者衍生的肿瘤异种移植(PDX)和细胞衍生肿瘤异种移植(CDX)是临床前评估、药物指导和基础癌症研究的重要技术。传统宿主小鼠的一代 PDX 模型非常耗时,只适用于一小部分样本。近年来,斑马鱼PDX(zPDX)已成为一种独特的宿主系统,具有体积小、效率高的特点。在这里,我们描述了一种优化的方法,用于在zPDX模型中生成双荧光标记肿瘤异种移植模型,用于比较化疗评估。肿瘤细胞和成纤维细胞在不同的培养条件下从新鲜收获或冷冻的胰腺癌组织中丰富。两个细胞组都标有表达绿色或红色荧光蛋白的慢病毒,以及抗凋亡基因BCL2L1。转染细胞被预混合,并共同注入2dpf幼虫斑马鱼,然后在32°C的改性E3培养中繁殖。用化疗药物和/或BCL2L1抑制剂治疗异种移植模型,同时研究肿瘤细胞和成纤维细胞的毒性。总之,该协议允许研究人员快速生成具有异构肿瘤微环境的大量zPDX模型,并提供更长的观察窗口和更精确的定量评估药物候选者的效率。
Introduction
精准肿瘤学旨在为个别患者找到最有益的治疗策略。目前,提出了许多临床前模型,如体外原发培养、体外有机体培养2和小鼠中患者衍生的异种移植(PDX),在有机体培养前后被建议用于诊断和筛选/评估潜力治疗选择3.PDX模型由人类原发性癌细胞注入免疫功能受损的小鼠,是临床肿瘤学3、4中最有前途的个性化药物筛选工具之一。与体外培养的细胞系不同,PDX模型通常保持体内肿瘤环境的完整性和异质性,更好地模仿不同肿瘤患者的多样性和特异性,因此,可以预测患者的潜在医疗结果4。然而,在小鼠中生成PDX模型需要高质量的患者样本和数月时间来收集足够的细胞和模型进行多组实验,异种移植物的细胞/遗传成分可能与原来的细胞/遗传成分漂移病人的活检小鼠PDX模型的建立成功率也较低,难以在临床实践中得到广泛应用。对于携带胰腺癌等快速进展癌症的患者,他们可能无法及时从PDX实验中获得有价值的信息。
在过去几年中,斑马鱼被报道为不仅CDX(细胞衍生肿瘤异种移植)模型的潜在宿主,而且PDX模型5,6,7,8,9,10.作为脊椎动物的模型动物,斑马鱼在遗传学和生理学上都与哺乳动物有着足够的相似性,具有两个显著的优势:透明度和小尺寸11。斑马鱼的繁殖性也很高,几天之内就可以从一对12岁的成年人身上获得数百只近亲繁殖的幼虫。几项研究已经利用斑马鱼生成癌症疾病的转基因和异种移植模型13,14。与小鼠异种移植相比,斑马鱼异种移植允许在单细胞分辨率下进行跟踪。一定数量的人体组织能够产生数百种斑马鱼PDX模型(zPDX),而可能只能产生一对小鼠PDX模型15,16。此外,斑马鱼幼虫在2-5 dpf已经发展了完整的循环系统和代谢器官,如肝脏和肾脏,但不是免疫系统17,而剩余的蛋黄囊是一个天然的3D介质,理想的药物筛选,药物抗药性试验和肿瘤迁移观察6,18,19,20,21。
随着最终尝试使用zPDX作为临床使用的筛选/测试平台,在这里,我们描述了胰腺癌zPDX模型的优化方案,它允许在短时间内使用更少的细胞以更低的成本进行体内候选药物评估。与之前关于zPDX6、9、10的参考文献相比,我们引入了几种优化,使系统在临床个性化诊断中更加可行和可靠:1)对不同细胞进行预排序在原发性肿瘤组织中组并稳定原发细胞一周,然后再进行进一步的实验;2) 通过基于慢病毒的遗传修饰标记人体细胞,增强异种移植物的细胞活力;3) 优化营养补充剂(葡萄糖和谷氨酰胺)和温度的斑马鱼养殖条件;4) 以比较方式量化不同细胞类型的药物反应。我们还通过添加几种补充材料对注射液进行了更改。总之,这些改进提供了在斑马鱼宿主中快速产生一种更类似患者的异种移植物的可能性,这种异种移植可用于评估候选药物的反应。
Protocol
Representative Results
Discussion
PDX和CDX模型都是肿瘤生物学领域的重要平台,成功的物种间移植的关键步骤是提高异种移植的存活率。 最近,一些研究表明,BCL2L1(BCL-XL)或BCL2的瞬态表达可以显著提高小鼠宿主中人类胚胎干细胞的生存能力,而不会影响细胞特性和命运23,24,25.在我们的手稿中,我们标记了含有慢病毒的细胞,这些?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金81402582、上海自然科学基金12DZ2295100、14YF1400600和18ZR1404500的支持。
Materials
| DMEM | GIBCO | C11995500BT | |
| FBS | Hyclone | sv30087.03 | |
| Y-27632 | Cliniscience | Y0503 | Rho kinase inhibitor |
| Primocin | invivogen | ant-pm-1 | an antibiotic for primary cell cultures |
| Putrescine dihydrochloride | Sigma | P5780 | |
| Nicotinamide | Sigma | N3376 | |
| penicillin streptomycin | GIBCO | 15140122.00 | |
| phosphate buffer (PBS) | GIBCO | C10010500CP | |
| HBSS | GIBCO | 14170112.00 | |
| collagenase type IV | GIBCO | 17104019.00 | |
| hyaluronidase | Sigma | H3884 | |
| DnaseⅠ | Sigma | D5025 | |
| insulin | Sigma | I9278 | |
| b-FGF | GIBCO | PHG0264 | |
| EGF | GIBCO | PHG0314 | |
| pancreatic cancer fibroblasts inhibitor | CHI Scientific | FibrOUT | |
| 0.45 μm sterile filter | Millipore | SLHV033RB | |
| concentration column | Millipore | Millipore UFC910008 | Concentrate the virus |
| polybrene | Sigma | H9268 | |
| Hyaluronic Acid Sodium Salt | Sigma | H7630 | |
| L-glutamine | GIBCO | 21051024.00 | |
| gemcitabine | Gemzan | ||
| methylcellulose | Sigma | M0262 | |
| Navitoclax(ABT-263) | Selleck | S1001 | Bcl-xL inhibitor |
| Equipment | |||
| Microinjector | NARISHIGE | ||
| stereomicroscope | OLYMPUS | MVX10 | |
| Confocal Microscope | LEICA | SP8 | 0.00 |
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