合成肺腺癌细胞原位移植研究 pd-l1 表达
Summary
本文介绍了一种小鼠肺腺癌细胞微创全基因移植模型, 作为研究非小细胞肺癌的时间和成本降低模型。
Abstract
小鼠模型的应用是研究各种疾病的病理生理学不可缺少的。关于肺癌, 有几种模型, 包括基因工程模型以及移植模型。然而, 基因工程小鼠模型是耗时和昂贵的, 而一些原位移植模型是很难重现。本文介绍了一种非侵入性肺肿瘤细胞气管内给药方法, 作为一种替代的原位移植模型。使用小鼠肺腺癌细胞和合成形移植接受者可以在完全活跃的免疫系统存在下研究肿瘤的发生。此外, 移植前肿瘤细胞的基因操作使这一模型成为研究遗传因素在生理条件下对肿瘤生长和肿瘤细胞基因表达谱的影响的一个有吸引力的节省时间的方法。利用该模型, 我们表明, 肺腺癌细胞在自然环境中生长时, t 细胞抑制器编程死亡配体 1 (death-ligand) 的水平与体外培养相比有了提高。
Introduction
肺癌仍然是迄今为止男性和女性中最大的癌症相关杀手.事实上, 根据美国癌症协会的数据, 每年死于肺癌的人数超过死于乳腺癌、前列腺癌和结肠癌的人1 人。直到最近, 大多数患有非小细胞肺癌 (nsclc) 的患者, 是肺癌最丰富的亚型, 在一线环境下接受了铂基化疗, 主要是血管生成的补充抑制剂2。只有一组患者在表皮生长因子受体 (egfr)、再生淋巴瘤激酶 (alk) 或 ros1 中存在致癌突变, 可以用可用的靶向药物3,4进行治疗。随着免疫检查抑制剂的出现, 肺癌患者出现了新的希望, 尽管直到现在, 只有 20%-40% 的患者对免疫疗法做出反应5。因此, 需要进一步研究, 通过微调免疫检查点疗法和调查组合治疗方案来改善这一结果。
为了研究肺癌, 有大量的临床前模型, 包括由化学物质和致癌物质引发的自发模型和基因工程小鼠模型 (gemm), 在这些模型中, 有条件的激活后, 本土肿瘤就会出现。癌基因和肿瘤抑制基因6,7,8的失活。这些模型对研究肺肿瘤发展的基本过程特别有价值, 但也需要广泛的小鼠繁殖, 实验很耗时。因此, 许多评价潜在抑制剂的研究利用皮下 (患者衍生) 异种移植模型, 其中人肺癌细胞系皮下注射到免疫缺陷小鼠9。
在这些模型中, 肿瘤的微元没有相应的表现;因此, 研究人员还使用原位移植模型, 其中肿瘤细胞被静脉注射, 胸内注射, 或直接注入肺实质10,11, 12, 13, 14,15,16,17,18,19,20。其中一些方法在技术上具有挑战性, 难以重现, 需要对研究人员进行强化培训。21在这里, 我们在免疫能力强的小鼠身上采用了一种非侵入性原位原位气管内移植方法, 在这种方法中, 肿瘤在3-5周内发展, 与人类肿瘤有显著的相似性, 以诱导 t 细胞的表达抑制细胞上的程序化死亡配体 1 (death-ligand)。11,12,20使用从 gemm 模型和同源受者小鼠中提取的小鼠肿瘤细胞, 可以对包括免疫细胞在内的肿瘤微环境进行适当的研究。此外, 基因编辑工具,如 crispr/cas9 技术22可以在移植前的体外使用, 这有助于调查遗传因素对肺肿瘤发生的影响。
Protocol
下文概述的所有实验规程均遵循道德准则, 并得到奥地利联邦科学、研究和经济部的批准。 请注意:这里的方案描述了小鼠肺腺癌细胞成合合受者的原位移植模型。如果内部有细胞, 可从 kraslsl-g12d:p 53fl/fl (kp) 小鼠7、18的肿瘤肺中分离出细胞, 并移植到背景和性别相同的小鼠体内。如果细胞是由其他研究小…
Representative Results
我们通过气管内肿瘤细胞传递的原位移植模型来测试肿瘤微环境是否刺激 pd-l1 表达。因此, 我们从自主 kp 模型 (kp 细胞) 中分离出小鼠肺交流细胞, 10周后通过 cre-re这时-重组表达腺病毒 (adre) 分娩24。随后, 我们用绿色荧光蛋白 (gfp) 标记肺交流细胞, 表达慢病毒25 , 并通过气管内传递将其移植到免疫能力强的同种小鼠体内。为了验证…
Discussion
研究肺生理和病理事件, 广泛使用侵入性和非侵入性气管插管方法注入各种试剂26、27、28、29。 ,30,31,32。在癌症领域, 研究人员使用气管内 (和鼻内) 注入 cre-re卖给表达的病毒, 在肺上皮细胞中引入体细胞突变。在 kras-lsl-g12d…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢萨菲亚扎赫马帮助准备组织部分。
Materials
| mouse lung adenocarcinoma cell line | isolated in house | ||
| C57Bl/6 mice | F1 of the cross of the two backgrounds may be used (8-12 weeks) | ||
| 129S mice | |||
| RPMI 1640 Medium | Life Technologies | 11544446 | |
| Fetal Calf Serum | Life Technologies | 11573397 | |
| Penicillin/Streptomycin Solution | Life Technologies | 11548876 | |
| L-Glutamine | Life Technologies | 11539876 | |
| Trypsin, 0.25% (1X) with EDTA | Life Technologies | 11560626 | |
| UltraPure 0.5M EDTA, pH 8.0 | Thermo Fisher Scientific | 15575020 | |
| Ketasol (100 mg/ml Ketamine) | Ogris Pharma | 8-00173 | |
| Xylasol (20 mg/ml Xylazine) | Ogris Pharma | 8-00178 | |
| BD Insyste (22GA 1.00 IN) | BD | 381223 | |
| Blunt forceps | Roboz | RS8260 | |
| Leica CLS150 LED | Leica | 30250004 | Fibre Light Illuminator |
| Student Iris Scissors | Fine Science Tools | 91460-11 | |
| DNase I (RNase-Free) | New England Biolabs | M0303S | |
| Collagenase Type I | Life Technologies | 17100017 | |
| ACK Lysing Buffer | Lonza | 10-548E | |
| CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), PE-Cyanine7 | eBioscience | 25-5982-82 | |
| Rat IgG2a kappa Isotype Control, PE-Cyanine7 | eBioscience | 25-4321-82 |
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Citer Cet Article
Moll, H. P., Mohrherr, J., Breitenecker, K., Haber, M., Voronin, V., Casanova, E. Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression. J. Vis. Exp. (143), e58101, doi:10.3791/58101 (2019).