在携带人类肿瘤的人源化小鼠模型中测试癌症免疫疗法
Summary
该协议概述了用于免疫肿瘤学研究的人免疫系统（HIS）小鼠的产生。介绍了使用该模型在植入该模型中的人类肿瘤上测试人类免疫疗法的说明和注意事项，重点是表征人类免疫系统对肿瘤的反应。
Abstract
逆转肿瘤微环境的免疫抑制性质对于用免疫治疗药物成功治疗癌症至关重要。鼠癌模型的多样性极其有限，并且无法转化为临床。为了作为免疫治疗研究的更生理学的临床前模型，已经开发了该协议来评估用人类免疫系统重组的小鼠中人类肿瘤的治疗。这种独特的方案展示了人类免疫系统（HIS，“人源化”）小鼠的发展，然后植入人类肿瘤，细胞系衍生异种移植（CDX）或患者来源的异种移植（PDX）。HIS小鼠是通过将从脐带血中分离的CD34 +人造血干细胞注射到新生儿BRGS（BALB / c Rag2-/- IL2RγC-/- NODSIRPα）高度免疫缺陷的小鼠中产生的，这些小鼠也能够接受异种肿瘤。强调了人体免疫系统发育和肿瘤植入的动力学和特征的重要性。最后，描述了使用流式细胞术对肿瘤微环境的深入评估。在使用该方案的众多研究中，发现单个肿瘤的肿瘤微环境在HIS-PDX小鼠中被概括;“热”肿瘤表现出大的免疫浸润，而“冷”肿瘤则没有。该模型是针对各种人类肿瘤的联合免疫疗法的试验场，也是寻求个性化医疗的重要工具。
Introduction
小鼠癌症模型对于建立肿瘤生长和免疫逃逸的基本机制很重要。然而，由于有限的同系模型和物种特异性差异，小鼠模型中的癌症治疗研究已经产生了有限的临床翻译1，2。免疫疗法作为控制肿瘤的主要方法的出现重申了对具有功能性人类免疫系统的体内模型的需求。在过去的十年中，人类免疫系统小鼠（HIS小鼠）的进步使得在各种癌症类型和免疫治疗剂中研究体内免疫肿瘤学成为可能3，4，5，6。人类肿瘤模型，包括细胞系衍生和患者来源的异种移植物（分别为CDX和PDX），在HIS小鼠中生长良好，并且在大多数情况下与它们在缺乏人类造血植入的免疫缺陷宿主中的生长几乎相同7，8。基于这一关键发现，研究人员一直在使用HIS小鼠模型来研究人类免疫疗法，包括旨在改变肿瘤微环境（TME）以减少免疫抑制从而增强免疫定向肿瘤杀伤的联合疗法。这些临床前模型有助于解决人类癌症的异质性问题，还可以预测治疗成功以及监测免疫相关药物毒性9，10。
通过引入人造血干细胞来生产具有人类免疫系统的小鼠模型需要不会排斥异种移植物的受体免疫缺陷小鼠。目前的HIS小鼠模型来源于30多年前报道的免疫缺陷小鼠品系。描述的第一个免疫缺陷小鼠品系是缺乏T细胞和B细胞的SCID小鼠11，其次是具有SIRPα多态性的杂交NOD-SCID，负责小鼠巨噬细胞对人类细胞的耐受性，这是由于NOD SIRPα等位基因与人CD47分子12，13的结合增加。在 2000 年代初期，由于基因缺失禁止宿主 NK 细胞发育，ALLB/c 和 NOD 免疫缺陷菌株上 IL-2 受体 （IL-2Rγc） 的共同 γ 链缺失改变了增强人类植入的游戏规则14，15，16，17。替代模型，例如BRG和NRG小鼠，通过删除Rag1或Rag2基因来实现T细胞和B细胞细胞缺乏，这是T细胞受体基因重排所必需的，因此淋巴细胞的成熟和存活18，19。本文使用的BRGS（BALB / c -Rag2 nullIl2RγCnullSirpα NOD）小鼠结合了IL-2Rγ链缺陷和Rag2-/-背景上的NOD SIRPα等位基因，导致没有T，B或NK细胞的高度免疫缺陷小鼠，但具有足够的活力和健康以允许长期植入超过30周13。
HIS小鼠可以通过多种方式产生，人PBMC注射是最直接的方法15，18，20。然而，这些小鼠具有活化的人T细胞的显着扩增，导致移植物抗宿主病（GVHD）在12周龄时，阻止了长期研究。或者，来自脐带血（CB）、骨髓和胎儿肝脏的人造血干细胞也可用于从头植入和生产人体免疫系统。在该系统中，造血干细胞产生多谱系人类免疫系统，与主要发育T细胞的PBMC小鼠相比，T，B和先天免疫细胞的产生对小鼠宿主具有重要的耐受性。因此，GVHD 不存在或大大延迟，研究可以扩展到 10 个月大的小鼠。CB提供了一种简单，可及且无创的CD34 +人造血干细胞来源，有助于植入具有遗传相同免疫系统的多只HIS小鼠17，18，20，21。在过去的几年中，HIS小鼠模型已被广泛用于研究免疫疗法和TME3，4，5，6。尽管这些小鼠发育了人类来源的免疫系统，但与对照免疫缺陷小鼠相比，人类异种移植肿瘤的生长速度相似，并且允许癌细胞和免疫细胞之间的复杂相互作用，这对于维持移植PDX的微环境很重要3，7，8.该协议已被用于进行50多项研究，测试具有PDX和CDX的HIS-BRGS小鼠的治疗。一个重要的结论是，HIS小鼠中的人类肿瘤保持其独特的TME，这是通过相对于初始患者样本和免疫浸润特征的肿瘤的分子评估所定义的3，22，23。我们小组专注于使用多参数流式细胞术深入评估免疫器官和肿瘤中的HIS。在此，我们描述了用于BRGS小鼠人源化，嵌合体评估，人类肿瘤植入，肿瘤生长测量，癌症治疗给药以及通过流式细胞术分析HIS细胞的方案。
Protocol
所有动物工作均根据科罗拉多大学丹佛分校机构动物护理和使用委员会批准的动物协议（IACUC 协议 #00593 和 #00021）进行。所有动物工作均按照科罗拉多大学丹佛安舒茨医学院的美国实验动物护理协会认可的设施实验动物资源办公室 （OLAR） 进行。所有人类脐带血样本都是从去识别化的捐赠者那里获得的，因此无需人类研究伦理委员会的批准。 注意：协议中提到的所有介质和?…
Representative Results
按照侧腹肿瘤方案和实验时间表（图1），在两种不同的人结直肠癌（CRC）PDX中研究了肿瘤生长和对靶向酪氨酸激酶抑制剂（TKI）治疗和纳武利尤单抗联合治疗的免疫反应。TKI药物已在免疫缺陷宿主中进行了研究，以评估肿瘤生长仅29。该模型能够单独研究TKI免疫反应的变化，更重要的是，与抗PD-1联合使用。这项研究的重点是两个不同实验中的联合治疗队列…
Discussion
在过去的6年中，利用我们在免疫学和人源化小鼠方面的专业知识，我们的研究团队开发了一个急需的临床前模型来测试免疫疗法对各种人类肿瘤3，7，30，31。该协议强调考虑模型的可变性，特别关注以免疫治疗为中心的人类T细胞群。在该协议中，描述了HIS小鼠的产生，以及其淋巴器官和肿瘤的免疫?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢动物研究机构（OLAR）对我们小鼠的照顾，以及我们研究所癌症中心支持补助金（P30CA046934）支持的流式细胞术共享资源，感谢他们对我们所有工作的巨大帮助。我们还感谢Gail Eckhardt和Anna Capasso在我们的HIS-BRGS模型中研究人类PDX免疫疗法的首次合作。这项研究得到了美国国立卫生研究院P30CA06934癌症中心支持基金的部分支持，并使用PHISM（临床前人类免疫系统小鼠模型）共享资源，RRID：SCR_021990和流式细胞术共享资源，RRID：SCR_022035。这项研究得到了美国国立卫生研究院NIAID的部分支持，合同号为75N93020C00058。
Materials
| 1 mL syringe w/needles | McKesson | 1031815 | |
| 15 mL tubes | Grenier Bio-One | 188271 | |
| 2-mercaptoethanol | Sigma | M6250 | |
| 50 mL tubes | Grenier Bio-One | 227261 | |
| AutoMACS Pro Separator | Miltenyi | 130-092-545 | |
| BD Golgi Stop Protein Transport Inhibitor with monensin | BD Bioscience | BDB563792 | |
| BSA | Fisher Scientific | BP1600100 | |
| Cell Stim Cocktail | Life Technologies | 509305 | |
| Chill 15 Rack | Miltenyi | 130-092-952 | |
| Cotton-plugged glass pipettes | Fisher Scientific | 13-678-8B | |
| Cultrex Basement membrane extract | R&D Systems | 363200502 | |
| Cytek Aurora | Cytek | ||
| DNase | Sigma | 9003-98-9 | |
| eBioscience FoxP3/Transcription Factor Staining Buffer Set | Invitrogen | 00-5523-00 | |
| Embryonic Stemcell FCS | Gibco | 10439001 | |
| Eppendorf Tubes; 1.5 mL volume | Grenier Bio-One | 616201 | |
| Excel | Microsoft | ||
| FBS | Benchmark | 100-106 500mL | |
| Ficoll Hypaque | GE Healthcare | 45001752 | |
| FlowJo Software | BD Biosciences | ||
| Forceps – fine | Roboz Surgical | RS5045 | |
| Forceps normal | Dumont | RS4919 | |
| Formaldehyde | Fisher | F75P1GAL | |
| Frosted Glass Slides | Corning | 1255310 | |
| Gentlemacs C-Tubes | Miltenyi | 130-096-334 | |
| GentleMACS Dissociator | Miltenyi | 130-093-235 | |
| glass pipettes | DWK Life Sciences | 63A53 | |
| Glutamax | Gibco | 11140050 | |
| HBSS w/ Ca & Mg | Sigma | 55037C | |
| HEPES | Corning | MT25060CI | |
| IgG standard | Sigma | I2511 | |
| IgM standard | Sigma | 401108 | |
| IMDM | Gibco | 12440053 | |
| Liberase DL | Roche | 5466202001 | |
| LIVE/DEAD Fixable Blue | Thermo | L23105 | |
| MDA-MB-231 | ATCC | HTB-26 | |
| MEM | Gibco | 1140050 | |
| mouse anti-human IgG-AP | Southern Biotech | JDC-10 | |
| mouse anti-human IgG-unabeled | Southern Biotech | H2 | |
| mouse anti-human IgM-AP | Southern Biotech | UHB | |
| mouse anti-human IgM-unlabeled | Southern Biotech | SA-DA4 | |
| MultiRad 350 | Precision X-Ray | ||
| PBS | Corning | 45000-446 | |
| Pen Strep | Gibco | 15140122 | |
| Petri Dishes | Fisher Scientific | FB0875713A | |
| p-nitrophenyl substrate | Thermo | 34045 | |
| PRISM | Graphpad | ||
| Rec Hu FLT3L | R&D systems | 308-FK-005/CF | |
| Rec Hu IL6 | R&D systems | 206-IL-010/CF | |
| Rec Hu SCF | R&D systems | 255SC010 | |
| RPMI 1640 | Corning | 45000-39 | |
| Saponin | Sigma | 8047-15-2 | |
| Scissors | McKesson | 862945 | |
| Serological pipettes 25 mL | Fisher Scientific | 1367811 | |
| Sterile filter | Nalgene | 567-0020 | |
| Sterile molecular water | Sigma | 7732-18-5 | |
| Yeti Cell Analyzer | Bio-Rad | 12004279 | |
| Zombie Green | biolegend | 423112 |
Referências
- Chulpanova, D. S., Kitaeva, K. V., Rutland, C. S., Rizvanov, A. A., Solovyeva, V. V. Mouse tumor models for advanced cancer immunotherapy. International Journal of Molecular Sciences. 21 (11), 4118 (2020).
- Olson, B., Li, Y., Lin, Y., Liu, E. T., Patnaik, A. Mouse models for cancer immunotherapy research. Cancer Discovery. 8 (11), 1358-1365 (2018).
- Marin-Jimenez, J. A., et al. Testing cancer immunotherapy in a human immune system mouse model: correlating treatment responses to human chimerism, therapeutic variables and immune cell phenotypes. Frontiers in Immunology. 12, 607282 (2021).
- Yin, L., Wang, X. J., Chen, D. X., Liu, X. N., Wang, X. J. Humanized mouse model: a review on preclinical applications for cancer immunotherapy. American Journal of Cancer Research. 10 (12), 4568-4584 (2020).
- Cogels, M. M., et al. Humanized mice as a valuable pre-clinical model for cancer immunotherapy research. Frontiers in Oncology. 11, 784947 (2021).
- Jin, K. T., et al. Development of humanized mouse with patient-derived xenografts for cancer immunotherapy studies: A comprehensive review. Cancer Science. 112 (7), 2592-2606 (2021).
- Capasso, A., et al. Characterization of immune responses to anti-PD-1 mono and combination immunotherapy in hematopoietic humanized mice implanted with tumor xenografts. Journal for Immunotherapy of Cancer. 7 (1), 37 (2019).
- Wang, M., et al. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy. The FASEB Journal. 32 (3), 1537-1549 (2018).
- Yong, K. S. M., et al. Humanized mouse as a tool to predict immunotoxicity of human biologics. Frontiers in Immunology. 11, 553362 (2020).
- Shen, H. W., Jiang, X. L., Gonzalez, F. J., Yu, A. M. Humanized transgenic mouse models for drug metabolism and pharmacokinetic research. Current Drug Metabolism. 12 (10), 997-1006 (2011).
- Bosma, G. C., Custer, R. P., Bosma, M. J. A severe combined immunodeficiency mutation in the mouse. Nature. 301 (5900), 527-530 (1983).
- Shultz, L. D., et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. The Journal of Immunology. 154 (1), 180-191 (1995).
- Legrand, N., et al. Functional CD47/signal regulatory protein alpha (SIRP(alpha)) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo. Proceedings of the National Academy of Sciences. 108 (32), 13224-13229 (2011).
- Ishikawa, F., et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood. 106 (5), 1565-1573 (2005).
- Ito, M., et al. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood. 100 (9), 3175-3182 (2002).
- Shultz, L. D., et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. The Journal of Immunology. 174 (10), 6477-6489 (2005).
- Traggiai, E., et al. Development of a human adaptive immune system in cord blood cell-transplanted mice. Science. 304 (5667), 104-107 (2004).
- Theocharides, A. P., Rongvaux, A., Fritsch, K., Flavell, R. A., Manz, M. G. Humanized hemato-lymphoid system mice. Haematologica. 101 (1), 5-19 (2016).
- Goldman, J. P., et al. Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor gamma chain. British Journal of Haematology. 103 (2), 335-342 (1998).
- Stripecke, R., et al. Innovations, challenges, and minimal information for standardization of humanized mice. EMBO Molecular Medicine. 12 (7), (2020).
- Allen, T. M., et al. Humanized immune system mouse models: progress, challenges and opportunities. Nature Immunology. 20 (7), 770-774 (2019).
- Gammelgaard, O. L., Terp, M. G., Preiss, B., Ditzel, H. J. Human cancer evolution in the context of a human immune system in mice. Molecular Oncology. 12 (10), 1797-1810 (2018).
- Rios-Doria, J., Stevens, C., Maddage, C., Lasky, K., Koblish, H. K. Characterization of human cancer xenografts in humanized mice. Journal for Immunotherapy of Cancer. 8 (1), 000416 (2020).
- Gombash Lampe, S. E., Kaspar, B. K., Foust, K. D. Intravenous injections in neonatal mice. Journal of Visualized Experiments. (93), e52037 (2014).
- Lang, J., Weiss, N., Freed, B. M., Torres, R. M., Pelanda, R. Generation of hematopoietic humanized mice in the newborn BALB/c-Rag2null Il2rγnull mouse model: a multivariable optimization approach. Clinical Immunology. 140 (1), 102-116 (2011).
- Laskowski, T. J., Hazen, A. L., Collazo, R. S., Haviland, D. Rigor and reproducibility of cytometry practices for immuno-oncology: a multifaceted challenge. Cytometry Part A. 97 (2), 116-125 (2020).
- Bagby, S., et al. Development and maintenance of a preclinical patient derived tumor xenograft model for the investigation of novel anti-cancer therapies. Journal of Visualized Experiments. (115), e54393 (2016).
- Laajala, T. D., et al. Optimized design and analysis of preclinical intervention studies in vivo. Scientific Reports. 6, 30723 (2016).
- Na, Y. S., et al. Establishment of patient-derived xenografts from patients with gastrointestinal stromal tumors: analysis of clinicopathological characteristics related to engraftment success. Scientific Reports. 10 (1), 7996 (2020).
- Tentler, J. J., et al. RX-5902, a novel beta-catenin modulator, potentiates the efficacy of immune checkpoint inhibitors in preclinical models of triple-negative breast cancer. BMC Cancer. 20 (1), 1063 (2020).
- Lang, J., et al. Development of an adrenocortical cancer humanized mouse model to characterize anti-PD1 effects on tumor microenvironment. The Journal of Clinical Endocrinology & Metabolism. 105 (1), 26-42 (2020).
- Lang, J., et al. Studies of lymphocyte reconstitution in a humanized mouse model reveal a requirement of T cells for human B cell maturation. The Journal of Immunology. 190 (5), 2090-2101 (2013).
- Katano, I., et al. NOD-Rag2null IL-2Rγnull mice: an alternative to NOG mice for generation of humanized mice. Experimental Animalas. 63 (3), 321-330 (2014).
- Brehm, M. A., et al. Parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rγ(null) mutation. Clinical Immunology. 135 (1), 84-98 (2010).
- Hasgur, S., Aryee, K. E., Shultz, L. D., Greiner, D. L., Brehm, M. A. Generation of immunodeficient mice bearing human immune systems by the engraftment of hematopoietic stem cells. Methods in Molecular Biology. 1438, 67-78 (2016).
- Andre, M. C., et al. Long-term human CD34+ stem cell-engrafted nonobese diabetic/SCID/IL-2Rγnull mice show impaired CD8+ T cell maintenance and a functional arrest of immature NK cells. The Journal of Immunology. 185 (5), 2710-2720 (2010).
- Wunderlich, M., et al. Improved multilineage human hematopoietic reconstitution and function in NSGS mice. PLoS One. 13 (12), 0209034 (2018).
- Lee, J., Brehm, M. A., Greiner, D., Shultz, L. D., Kornfeld, H. Engrafted human cells generate adaptive immune responses to Mycobacterium bovis BCG infection in humanized mice. BMC Immunology. 14, 53 (2013).
- Masse-Ranson, G., et al. Accelerated thymopoiesis and improved T-cell responses in HLA-A2/-DR2 transgenic BRGS-based human immune system mice. European Journal of Immunology. 49 (6), 954-965 (2019).
- Oswald, E., et al. Immune cell infiltration pattern in non-small cell lung cancer PDX models is a model immanent feature and correlates with a distinct molecular and phenotypic make-up. Journal for Immunotherapy of Cancer. 10 (4), 004412 (2022).
Citar este artigo
Lanis, J. M., Lewis, M. S., Strassburger, H., Larsen, K., Bagby, S. M., Dominguez, A. T. A., Marín-Jiménez, J. A., Pelanda, R., Pitts, T. M., Lang, J. Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors. J. Vis. Exp. (190), e64606, doi:10.3791/64606 (2022).