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Protocol
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Cancer Research

研究癌症相关成纤维细胞在肿瘤生长中的作用的小鼠模型

Published: December 22, 2020
doi:
10.3791/61883
, , , , , , , , , , , , , , , , ,
1Department of Molecular, Cell and Developmental Biology,University of California, Los Angeles, 2Department of Biological Chemistry, David Geffen School of Medicine,University of California, Los Angeles, 3Department of Molecular Biology,Princeton University, 4Molecular Biology Institute,University of California, Los Angeles

Summary

提供了共同注射癌细胞和成纤维细胞并随着时间的推移监测肿瘤生长的方案。该协议可用于了解成纤维细胞作为肿瘤生长调节因子的作用的分子基础。

Abstract

癌症相关成纤维细胞（CAF）可以通过创造促进肿瘤的微环境在肿瘤生长中发挥重要作用。研究CAF在肿瘤微环境中作用的模型有助于理解成纤维细胞，来自不同组织的成纤维细胞以及成纤维细胞中特定遗传因子的功能重要性。小鼠模型对于了解体内肿瘤生长和进展的贡献因素至关重要。在这里，提供了一种方案，其中癌细胞与成纤维细胞混合并引入小鼠以发展肿瘤。确定肿瘤大小随时间的变化和最终的肿瘤权重并在组间进行比较。所描述的方案可以更深入地了解CAF在肿瘤生长和进展中的功能作用。

Introduction

在肿瘤微环境中，最突出的细胞类型之一是癌症相关成纤维细胞（CAF）1。这些癌相关成纤维细胞可发挥肿瘤抑制作用2，3。例如，表达S100A的成纤维细胞分泌胶原蛋白，可以包封致癌物并防止癌的形成4。此外，胰腺癌中α平滑肌肌动蛋白（SMA）阳性肌成纤维细胞的耗竭会导致免疫抑制并加速胰腺癌的进展2。CAF还可以与癌细胞共同进化并促进肿瘤进展5，6，7，8。成纤维细胞可以合成和分泌细胞外基质蛋白，从而产生促进肿瘤的环境8。这些细胞外基质蛋白可引起组织的机械僵硬，这与肿瘤进展有关9，10。沉积的细胞外基质可以作为抑制免疫浸润的物理屏障11。CAF的基质沉积也与肿瘤浸润有关，因为CAF产生的纤连蛋白已被证明可以促进肿瘤浸润12。CAF通过分泌转化生长因子-β（TGF-β），血管内皮生长因子（VEGF），白细胞介素-6（IL-6）和CXC-趋化因子配体12（CXCL12）13，14，15，促进血管生成并将免疫抑制细胞募集到肿瘤微环境中。由于其在促进肿瘤生长中的核心作用，癌症相关成纤维细胞是抗癌治疗的新兴靶标6，16，17，18。

下面的协议描述了一种在成熟且广泛使用的肿瘤生长小鼠模型中测试成纤维细胞如何影响肿瘤生长的方法。为了了解成纤维细胞在肿瘤微环境中的重要性，修改了将癌细胞引入小鼠以监测其生长的标准方案，以包括癌细胞引入的成纤维细胞。癌细胞可以皮下或皮内引入。皮内引入会导致由皮肤本身引起的肿瘤。将癌细胞和成纤维细胞共同注射到小鼠体内的异种移植物代表了剖析成纤维细胞，成纤维细胞亚群和蛋白质因子在促进癌症生长能力中的作用的重要方法工具19，20，21。提供了将癌细胞和成纤维细胞共同注射到小鼠中的详细方案。该方法可用于比较成纤维细胞的存在与否，比较来自不同来源的成纤维细胞20，或比较具有和没有特定蛋白质表达的成纤维细胞19。引入癌细胞和成纤维细胞后，可以随着时间的推移监测肿瘤大小。在实验结束时，可以解剖和称重肿瘤。通过监测肿瘤随时间的生长，可以剖析不同因素的重要性。

有可能的替代方法来研究成纤维细胞在肿瘤生长中的作用。例如，有基于Cre-loxed的模型，这些模型提供了组织特异性基因敲除，其驱动因素优先在成纤维细胞中表达。这些方法也为研究成纤维细胞中特定基因和途径对肿瘤进展的作用提供了机会。与基于Cre-lox的方法相比，所提供的方案将代表一种明显更快速的方法来监测成纤维细胞的作用，因为肿瘤生长将在短短几周内被监测。所提供的方法也便宜得多，因为它不需要产生和容纳基因工程小鼠的菌落。所提供的协议可用于使用shRNA快速测试不同基因的敲低效果，而无需开发小鼠集落。所提供的方法也更加灵活，因为它允许比较不同数量的成纤维细胞，不同比例的癌细胞和成纤维细胞，敲低不同的基因，甚至比较来自不同组织部位或物种的成纤维细胞。Cre-lox方法的优点是成纤维细胞在更生理的背景下存在于小鼠体内。

这里报道的方案对于寻求快速且经济高效地监测成纤维细胞对肿瘤生长影响的科学家来说很有价值。该协议对于研究来自不同来源的成纤维细胞或成纤维细胞的不同亚群对肿瘤生长的肿瘤生长特别有价值。如果肿瘤起始发生在生理背景下很重要，那么应考虑基因工程小鼠模型。

有几种可能的方法可以执行这些实验。免疫能力小鼠可用作宿主，这将允许研究成纤维细胞 – 免疫细胞相互作用。对于免疫能力小鼠模型，必须注射小鼠癌细胞和小鼠胚胎成纤维细胞（MEF）。MEF的使用还允许研究人员利用广泛的敲除小鼠品系来测试目的基因的存在与否。或者，免疫缺陷小鼠可用于测试人成纤维细胞在促进源自人类癌细胞的小鼠肿瘤生长中的作用。癌细胞的引入可以皮下或正位进行。对于黑色素瘤，如下所述，可以皮内注射肿瘤-成纤维细胞混合物以进行原位注射，从而更密切地模拟皮肤内黑色素瘤发展的位置。

Protocol

所有描述的实验都得到了加州大学洛杉矶分校动物护理委员会的批准。 注意：选择与宿主小鼠匹配的癌细胞和成纤维细胞进行小鼠品系。选择与宿主小鼠性别相匹配的癌细胞和成纤维细胞。从繁殖群体获得小鼠或从信誉良好的供应商处购买。将肿瘤引入~8-10周龄的小鼠体内。有毛的小鼠将处于毛囊周期的休止期或休息阶段。计划将成纤维细胞与癌细胞的比例定为 0.5-3。 <p c…

Representative Results

A2058人黑色素瘤细胞和原代人真皮成纤维细胞在无菌条件下培养。收集细胞并用PBS洗涤三次。免疫缺陷小鼠（NU / J – Foxn1裸体菌株）在一侧皮下注射，仅用25万个A2058黑色素瘤细胞。在另一侧，小鼠注射25万个A2058黑色素瘤细胞和75万个成纤维细胞的混合物。将细胞注射到12只免疫缺陷小鼠中。向左右侧注射是随机的。在注射后第12、14、16、19和21天监测肿瘤体积（<strong class="xf…

Discussion

在图1的实验中，与引入人A2058黑色素瘤细胞相比，与引入黑色素瘤细胞而不共同注射成纤维细胞时，导致更大的肿瘤。这种差异可以很容易地根据肿瘤体积和肿瘤重量检测到。结果与癌症相关成纤维细胞可以促进肿瘤生长的多个报道一致5，6，7，8。除了这里讨论的终点，如肿瘤…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者要感谢Coller实验室的所有成员的有益意见。H.A.C.是丽塔·艾伦基金会的米尔顿·E·卡塞尔学者。我们感谢NIH / NCI 1 R01 CA221296-01A1，NIH 1 R01 AR070245-01A1，黑色素瘤研究联盟团队科学奖，癌症研究所临床实验室整合计划奖，Iris Cantor妇女健康中心/ UCLA CTSI NIH Grant UL1TR000124，加州大学癌症研究协调委员会，大卫格芬医学院代谢主题奖，临床转化科学研究所和Jonsson综合癌症中心， 布罗德干细胞研究中心（Rose Hills和Ha Gaba）的创新奖，加州大学洛杉矶分校前列腺癌孢子奖（美国国立卫生研究院国家癌症研究所，奖项编号P50CA092131），布罗德干细胞中心的创新奖，加州大学洛杉矶分校Eli和Edythe Broad再生医学和干细胞研究中心， 肿瘤细胞生物学培训计划（USHHS Ruth L. Kirschstein 机构国家研究服务奖 # T32 CA009056）、加州大学洛杉矶分校 AR071307 的皮肤病学 T32 计划以及加州大学洛杉矶分校肌肉细胞生物学、病理生理学和治疗学 T32 培训计划 5 T32 AF 65972。

Materials

26G Needles Fisher Scientific 14-826-10
Alcohol swabs Fisher Scientific 326895
Animal clipper miniARCO with surgical blade #40 WAHL Professional 8787-450A
Athymic nude mice (NU/J) The Jackson labs 002019 These mice are immunocompromised and can be used for experiments in which human cells are introduced. Immunocompetent mice can also be used if mouse cancer cells and fibroblasts will be introduced.
Cancer cells ATCC ATCC® CRL-11147™ This is the catalog number for a primary human melanoma cell line. Other cancer cell types can also be used.
Cell Culture Multi Flasks Fisher Scientific 14-826-95
Centrifuge for conical tubes capable of reaching 180 x g Fisher Scientific 14-432-22
Countess Cell Counting Chamber Fisher Scientific C10228
Dulbecco's Modified Eagle Medium Fisher Scientific 11965-118
Fetal bovine serum Fisher Scientific MT35010CV
Fibroblasts  ATCC PCS-201-012 We isolate fibroblasts from skin in our lab. This is a catalog number for an adult primary human dermal fibroblast cell line. MEFs and fibroblasts derived from other sites can also be used.
Isoflurane Henry Schein Animal Health NDC 11695-6776-2
PBS USP grade for injection into mice Fisher Scientific 50-751-7476
Sterile 10 ml serological pipet Celltreat 667210B
Sterile 5 ml serological pipet Celltreat 229005B
Sterile 50 ml centrifuge tubes Genesee Scientific 28-108
Sterile Syringe Filters pore size 0.2 microns Fisher Scientific 09-740-61A
Sterile tissue culture-grade Trypsin-EDTA Fisher Scientific 15400054
Sterile tissue-culture grade PBS Fisher Scientific 50-751-7476
Sterle 25 ml serological pipet Celltreat 667225B
TC treated 100 x 20 mm dishes Genesee Scientific 25-202
TC treated 150 x 20 mm dishes Genesee Scientific 25-203
TC treated 60 x 15 mm dishes Genesee Scientific 25-260
Trypan blue Fisher Scientific C10228
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Tags

Keywords: Cancer-associated FibroblastsTumor GrowthMelanomaMouse ModelCell CultureTrypsin-EDTAHemocytometerCell Counting
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Jelinek, D., Zhang, E. R., Ambrus, A., Haley, E., Guinn, E., Vo, A., Le, P., Kesaf, A. E., Nguyen, J., Guo, L., Frederick, D., Sun, Z., Guo, N., Sevier, P., Bilotta, E., Atai, K., Voisin, L., Coller, H. A. A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth. J. Vis. Exp. (166), e61883, doi:10.3791/61883 (2020).
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