使用类似人类单细胞的THP-1白血病细胞系，将巨噬细胞分化和极化成M2样表型
Summary
M2样肿瘤相关巨噬细胞（TAM）与肿瘤进展和癌症预后不佳有关。本协议作为在 14 天内可重复区分和分化 THP-1 单细胞状细胞成 M2 样巨噬细胞的详细指南。该模型是研究TAM在肿瘤微环境内的抗炎作用的基础。
Abstract
肿瘤相关巨噬细胞 （TAM） 可以根据外部刺激切换其表达和细胞因子特征。这种非凡的可塑性使 TAM 能够适应肿瘤微环境内的持续变化。巨噬细胞可以主要具有抗炎（M1样）或抗炎（M2样）属性，并且可以在这两个主要状态之间不断切换。肿瘤环境中的M2样巨噬细胞与多种癌症的癌症进展和预后不佳有关。诱导THP-1细胞分化和极化的许多不同方法用于研究细胞和细胞间机制以及TAM在肿瘤微环境下的影响。目前，没有使用THP-1细胞系的M2样巨噬细胞偏振的既定模型，由于某些体外刺激，巨噬细胞的表达和细胞因子特征的结果因研究而异。此协议作为详细的指导，将 THP-1 单细胞状细胞区分为 M0 巨噬细胞，并在 14 天内将细胞进一步分化为 M2 样表型。我们使用光显微镜演示了THP-1单细胞状细胞、分化巨噬细胞和偏振M2样巨噬细胞的形态变化。该模型是细胞系模型研究TAM的抗炎作用及其与肿瘤微环境其他细胞群相互作用的基础。
Introduction
肿瘤相关巨噬细胞（TAM）及其在慢性炎症、癌症发病和肿瘤发育中的作用是最近研究1、2的重要目标。被招募到发育中的肿瘤组织微环境的外周血单核细胞分化成巨噬细胞，可分化成巨噬细胞3的两个主要亚型。经典激活的巨噬体主要代表亲炎M1样表型，而替代激活的M2样亚型主要表现出抗炎特性4。巨噬细胞可以根据细胞代谢在这两种主要表型之间动态切换，中间亚型具有炎症和抗炎特性5。TAM 代表两种表型的异质种群。然而，不同类型癌症的肿瘤促进功能和预后差，尤其与M2样巨噬细胞6、7、8有关。
巨噬细胞的功能特征及其与肿瘤微环境中其他细胞的相互作用，在肿瘤持续发展的不断变化的环境中具有复杂性和挑战性。细胞系可以提供同质细胞群，在培养中具有稳定的生存能力，从而促进演示定义的细胞和细胞间机制的过程。单核细胞样THP-1细胞系是原发性人类单核细胞9的合法模型系统。这种自发不朽的细胞系是从一名患有急性单细胞白血病的一岁婴儿的外周血液中获得的。THP-1细胞的分化和极化已经通过几项研究进行了报告，并以多种不同的方式进行了11、12、13、14。激活，因此，巨噬细胞的两极分化成M1样表型后，一个补偿性的抗炎反弹机制，促进M2样表型通过细胞因子产生的炎症性巨噬细胞，如间脂6（IL-6）或异位15，16。这可能作为一个断裂机制，以减轻细胞激活17后过度的炎症反应。将单核细胞和THP-1单核细胞分化成抗炎M2样表型的过程本身也伴随着必须克服的亲炎刺激。炎症细胞因子反应可由机械应激18引起，如改变介质以重新喂养细胞，或添加化合物来分化THP-1细胞，如磷12-肌酸13-醋酸酯（PMA），并诱导肿瘤坏死因子α（TNF+）、白细胞介质1+（IL-1+）或IL-6 19的产生。这种改变的细胞因子表达配置文件作为对PMA的反应可以影响和防止随后的巨噬细胞偏振20。充足的休息时间，如PMA治疗后报告，允许这些炎症反应减少和促进细胞偏振成一个独特的M2样表型21。
该协议演示了一种在 14 天内将 THP-1 单细胞状细胞分化和偏振成 M2 样巨噬细胞表型的方法。
Protocol
注：此协议中描述的步骤概述见 图 1。购买了人类单细胞样白血病细胞系THP-1。进行了短串联重复分析，以验证 THP-1 细胞线。在无菌条件下执行所有步骤。THP-1单细胞系在悬浮中生长，不附着在细胞培养表面。通过机械应力或PMA的特定治疗，可以通过将单核细胞分化成大噬细胞来诱导粘性。 1. THP-1单细胞样细胞的培养和维护 设置 150s 的定?…
Representative Results
以M2样巨噬细胞为特征，使用分化标记簇（CD）CD14、CD11b、CD80（M1样标记）和CD206（M2样标记）的流细胞学验证M2极化。流细胞测量染色是根据制造商的说明进行的。巨噬细胞用PBS/5S清洗，并孵育与Fcé受体块，以避免未特定的结合。然后，细胞被HITCH结合的小鼠抗人类CD14和CD80抗体染色，与PE结合的小鼠抗人类CD11b抗体，并与PE-串联结合小鼠抗人类CD206抗体和同型匹配IgG（材料表）在4°C?…
Discussion
此关于在 14 天内区分和偏振 THP-1 单细胞状细胞的协议提供了一种方法，通过在步骤之间有足够休息时间的细胞进行长期治疗孵化，获得具有不同 M2 样表型的巨噬细胞。
某些步骤对此协议至关重要。THP-1单核细胞的加倍时间约为26小时。细胞可以以 9 x 105/mL的细胞密度进行分裂，每次分裂时应以 3 x 105/mL 的密度播种。分裂可以在不去除所有使用过的（旧）细胞?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
路易斯维尔大学价格外科研究所得到约翰·普莱斯和芭芭拉·普劳顿·阿特伍德价格信托基金的财政支持。资金来源在研究的设计和进行以及数据的收集、管理、分析和解释方面没有作用。
Materials
| 0.4% trypan blue | VWR, Radnor, USA | 152-5061 | |
| 1.5 mL microcentrifuge tube | USA Scientific, Ocala, USA | 1615-5510 | |
| 10 mL serological pipet | VWR, Radnor, USA | 89130-898 | |
| 1000 μL TipOne pipet tips | USA Scientific, Ocala, USA | 1111-2821 | |
| 15 mL Centrifuge tube | VWR, Radnor, USA | 89039-664 | |
| 20 μL TipOne pipet tips | USA Scientific, Ocala, USA | 1120-1810 | |
| 200 μL TipOne pipet tips | USA Scientific, Ocala, USA | 1120-8810 | |
| 25 mL serological pipet | VWR, Radnor, USA | 89130-900 | |
| 5 mL serological pipet | VWR, Radnor, USA | 89130-896 | |
| 50 mL Centrifuge tube | VWR, Radnor, USA | 89039-662 | |
| Accutase solution 500 mL | Sigma, St. Louis, USA | A6964 | |
| Antibiotic Antimycotic Solution (100x), stabilized | Sigma, St. Louis, USA | A5955-100 mL | with 10,000 units penicillin, 10 mg of streptomycin and 25 μg of amphotericin B per mL, sterile-filtered, BioReagent, suitable for cell culture |
| Binder CO2 Incubator | VWR, Radnor, USA | C170-ULE3 | |
| CytoOne T-75cm flask with filter cap | USA Scientific, Ocala, USA | CC7682-4875 | |
| Dulbecco’s Phosphate Buffered Saline (PBS) | Sigma, St. Louis, USA | D8537-500 mL | PBS without calcium chloride and magnesium chloride should be used, since both can alter macrophage polarization |
| Eppendorf Centrifuge 5804 R (refrigerated) | Eppendorf, Enfield, USA | – | |
| Ethyl alcohol (70%) | – | – | |
| FACSCalibur flow cytometer | BD Biosciences, San Diego, USA | – | The flow cytometer operates with CellQuest software (BD Biosciences) |
| Falcon 24-well plate | VWR, Radnor, USA | 353504 | |
| Fetal Bovine Serum (FBS) | ATCC, Manassas, USA | 30-2020 | |
| FITC Mouse Anti-Human CD14 | BD Biosciences, San Diego, USA | 555397 | Flow cytometry, myeloid cell marker (100 tests) |
| FITC Mouse Anti-Human CD80 | BD Pharmingen, San Diego, USA | 557226 | Flow cytometry, M1 marker (100 tests) |
| FITC Mouse IgG1 κ Isotype Control | BD Pharmingen, San Diego, USA | 555748 | Flow cytometry, isotype control for CD80 (100 tests) |
| FITC Mouse IgG2a, κ Isotype Control | BD Biosciences, San Diego, USA | 553456 | Flow cytometry, isotype control for CD14 (100 tests) |
| Human BD Fc Block | BD Biosciences, San Diego, USA | 564220 | Flow cytometry, Fc block (0.25 mg) |
| Human interleukin 13 (IL-13) | R&D, Minneapolis, USA | IL-771-10 μg | |
| Human interleukin 4 (IL-4) | R&D, Minneapolis, USA | SRP3093-20 μg | |
| Labconco Biosafety Cabinet (Delta Series 36212/36213) | Labconco, Kansas City, USA | – | |
| L-Glutamine Solution, 200 mM | ATCC, Manassas, USA | 30-2214 | |
| Lipopolysaccharide (LPS) from E. coli 0111:B4 | Sigma, St. Louis, USA | L2630-100 mg | |
| Mini Cell Scrapers | Biotium, Fremont, USA | 22003 | |
| Neubauer hemocytometer | Fisher Scientific, Waltham, USA | 02-671-5 | |
| Nikon Eclipse inverted microscope TS100 | Nikon, Melville, USA | – | |
| Nuclease-free water | Invitrogen, Carlsbad, USA | AM9937 | |
| Olympus Light Microscope RH-2 | Microscope Central, Feasterville, USA | 40888 | |
| P10 variable pipet- Gilson | VWR, Radnor, USA | 76180-014 | |
| P1000 variable pipet-Gilson | VWR, Radnor, USA | 76177-990 | |
| P200 variable pipet- Gilson | VWR, Radnor, USA | 76177-988 | |
| PE Mouse Anti-Human CD11b | BD Biosciences, San Diego, USA | 555388 | Flow cytometry, myeloid cell marker (100 tests) |
| PE Mouse IgG1, κ Isotype Control | BD Biosciences, San Diego, USA | 555749 | Flow cytometry, isotype control for CD11b (100 tests) |
| PE-Cy 5 Mouse Anti-Human CD206 | BD Pharmingen, San Diego, USA | 551136 | Flow cytometry, M2 marker (100 tests) |
| PE-Cy 5 Mouse IgG1 κ Isotype Control | BD Pharmingen, San Diego, USA | 555750 | Flow cytometry, isotype control for CD206 (100 tests) |
| Phorbol 12-myristate 13-acetate (PMA) | Sigma, St. Louis, USA | P8139 | |
| Powerpette Plus pipettor | VWR, Radnor, USA | 75856-448 | |
| Precision Water bath (model 183) | Precision Scientific, Chicago, USA | 66551 | |
| RPMI-1640 Medium | ATCC, Manassas, USA | 30-2001 | |
| THP-1 cell line, American Type Culture Collection (ATCC) | ATCC, Manassas, USA | TIB-202 |
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Citazione di questo articolo
Scheurlen, K. M., Snook, D. L., Gardner, S. A., Eichenberger, M. R., Galandiuk, S. Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line. J. Vis. Exp. (174), e62652, doi:10.3791/62652 (2021).