识别骨髓增生异常综合征和急性髓系白血病中的骨髓微环境群体
1Wilmot Cancer Institute,University of Rochester Medical Center, 2Department of Biomedical Engineering,University of Rochester, 3Department of Biomedical Genetics,University of Rochester Medical Center, 4Genomics Research Center,University of Rochester Medical Center, 5Division of Endocrinology and Metabolism, Department of Medicine,University of Rochester Medical Center
Summary
本文介绍了从骨髓增生异常综合征和急性髓系白血病的小鼠模型中分离和表征骨髓微环境群体的详细方案。该技术可识别非造血骨髓生态位（包括内皮细胞和间充质基质细胞）随疾病进展的变化。
Abstract
骨髓微环境由不同的细胞群组成，例如间充质基质细胞、内皮细胞、骨系细胞和成纤维细胞，它们为造血干细胞 （HSC） 提供支持。除了支持正常的造血干细胞外，骨髓微环境还在造血干细胞疾病的发展中发挥作用，例如骨髓增生异常综合征 （MDS） 和急性髓系白血病 （AML）。造血干细胞中 MDS 相关突变导致分化受阻和进行性骨髓衰竭，尤其是在老年人中。MDS通常可进展为难治性AML，这是一种以未成熟髓母细胞快速积累为特征的疾病。已知这些骨髓肿瘤患者的骨髓微环境会发生变化。在这里，描述了从骨髓增生异常综合征和急性髓系白血病的小鼠模型中分离和表征骨髓微环境细胞的综合方案。分离和表征骨髓生态位群体的变化有助于确定它们在疾病发生和进展中的作用，并可能导致开发针对骨髓基质群体中促癌改变的新疗法。
Introduction
骨髓微环境由造血细胞、非造血基质细胞和细胞外基质组成 1,2。这种微环境可以促进造血干细胞自我更新，调节谱系分化，并为骨组织提供结构和机械支持1,2,3,4,5。基质生态位包括骨系细胞、成纤维细胞、神经细胞和内皮细胞6，而造血生态位由淋巴和髓系群体组成 1,2,3。除了支持正常的造血干细胞外，骨髓微环境还可以在造血干细胞疾病的发展中发挥作用，例如MDS和AML 7,8,9,10,11。骨系细胞的突变已被证明可促进 MDS、AML 和其他骨髓增生性肿瘤的发展 10,12,13,14。
骨髓增生异常综合征是一组由造血干细胞突变引起的白血病前期疾病。MDS 通常与 HSC 分化受阻和发育不良细胞的产生有关，这通常会导致骨髓衰竭。MDS是美国最常诊断的髓系肿瘤，3年生存率为35%-45%15。MDS通常与转化为急性髓系白血病的高风险有关。这可能是一种致命的并发症，因为MDS衍生的AML对大多数疗法都有抵抗力，并且可能复发。由于造血干细胞和祖细胞的易位或突变而新发的 AML 也通常对标准化疗产生耐药性 16,17。由于MDS和AML主要是老年人的疾病，大多数患者在60岁以上被诊断出来，因此大多数患者不符合根治性骨髓移植的条件。因此，迫切需要确定疾病进展的新调节因子。由于骨髓微环境可以为恶性细胞提供支持14，因此定义随着疾病进展的骨髓生态位的变化可能导致识别旨在抑制肿瘤生态位重塑的新疗法。因此，非常需要确定疾病进展的新调节因子。为此，识别和表征可能为恶性细胞提供支持的骨髓基质细胞群的变化至关重要。
已经生成了几种AML和MDS的小鼠模型，可用于研究疾病开始和进展期间骨髓微环境的变化6,1,19,20,21,22。在这里，描述了使用逆转录病毒诱导的 AML 6,20 的小鼠模型以及市售的高危 MDS 到AML 转化 19 的 Nup98-HoxD13 （NHD13） 模型来识别骨髓基质细胞群变化的方案。用从头移植 AML 细胞的小鼠在 20-30 天内死于该疾病6.NHD13小鼠在15-20周左右出现血细胞减少和骨髓发育不良，最终转化为AML，近75%的小鼠在32周左右死于这种疾病。为了分析小鼠模型骨髓微环境群体，收获骨骼，使用酶消化消化骨髓和骨针状体，然后通过磁性分选富集细胞以用于CD45-/Ter119-非造血群体。虽然之前已经描述了类似的分析11,13,22,23,24,25，但它们通常集中在骨髓或骨骼上，并且在分析中没有包括来自这两种来源的细胞。这些群体的综合表征与基因表达分析相结合，可以全面了解细胞造血微环境如何为疾病的发生和进展提供支持（图1）。虽然下面描述的方案侧重于逆转录病毒诱导的 AML 模型和遗传 MDS 模型，但这些策略可以很容易地用于研究任何感兴趣的小鼠模型的骨髓生态位的变化。
Protocol
所有动物实验均按照罗切斯特大学动物资源委员会批准的方案进行。小鼠在罗切斯特大学的动物护理设施中饲养和饲养。为了模拟高危 MDS，采用了市售的 NHD13 小鼠模型19。在该模型中，在疾病发作前 8 周龄的雌性 NHD13 小鼠中分析骨髓基质细胞。De novoAML的生成如前所述6,11,20。用于诱导 AML 的癌基因，例…
Representative Results
本文介绍了一种基于流式细胞术的方法，用于分析来自MDS和白血病小鼠模型的骨髓微环境群体，例如内皮细胞和间充质基质细胞（图1）。 图 2 描述了用于检测目标群体的门控策略，从通过前向和侧面散射曲线选择消化和 CD45/Ter119 耗尽部分中的细胞 （P1） 开始。白血病样品中细胞的门控示例如图1所示（图2A）。选择单峰，从该分…
Discussion
小鼠白血病模型已被广泛用于识别促进侵袭性髓系白血病进展的细胞内在和生态位驱动信号6,19,21。本文介绍了一种基于流式细胞术的综合方案，用于定义MDS和AML小鼠模型中骨髓微环境的细胞组成。
在从实验样品中获取流式细胞术数据之前，仔细补偿荧光重叠非常重要。包括所有适当的染色和门控也很重要…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢 URMC 流式细胞术核心。这项工作得到了美国血液学会学者奖、白血病研究基金会奖和美国国立卫生研究院R01DK133131资助和R01CA266617授予 JB 的支持。
Materials
| 1 mL pipette Tips | Genesee Scientific | 24-165RL | |
| 1.7 mL Microcentrifuge Tubes | AVANT | L211511-CS | |
| 10 µL pipette Tips | Genesee Scientific | 24-140RL | |
| 10 mL Individually Wrapped Sterile Serological Pipettes | Globe scientific | 1760 | |
| 1000 mL Vacuum Filtration Flask | NEST | 344021 | |
| 15 mL Centrifuge Tube | VWR | 10026-076 | |
| 2 mL Aspirating Pipette | NEST | 325011 | |
| 200 µL pipette Tips | Genesee Scientific | 24-150-RL | |
| 25 mL Individually Wrapped Sterile Serological Pipettes | Globe scientific | 1780 | |
| 5 mL Individually Wrapped Sterile Serological Pipettes | Globe scientific | 1740 | |
| 5 mL Polystyrene Round-Bottom Tube 12 x 75 mm style | Falcon | 352054 | |
| 5 mL Polystyrene Round-Bottom Tube with Cell Strainer Cap 12 x 75 mm style | Falcon | 352235 | |
| 50 mL Centrifuge Tube | NEST | 602052 | |
| 6 Well, Flat Bottom with Low Evaporation Lid | Falcon | 353046 | |
| Absorbent Underpads with Waterproof Moisture Barrier | VWR | 56616-031 | |
| APC MicroBeads | Miltenyi | 130-090-855 | |
| autoMACS Pro Separator | Miltenyi Biotec GmBH | 4425745 | |
| BD Pharmingen Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) | BD Biosciences | 553141 | 0.5 mg/mL |
| Bovine Serum Albumin | Sigma-Aldrich | A7906 | 66.000 g/mol |
| Brilliant Violet 421 anti-mouse Ly-6A/E (Sca-1) Antibody (D7) | Invitrogen | 404-5981 | 0.2 mg/mL |
| C57BL/6J Mice | Jackson Laboratory | 664 | |
| Carbon Dioxide Gas Tank | Airgas | CD50 | |
| CD31 (PECAM-1) Monoclonal Antibody (390), PE-Cyanine7 | Invitrogen | 25-0311-82 | 0.2 mg/mL |
| CD45 Monoclonal Antibody (30-F11), APC | Invitrogen | 17-0451-82 | 0.2 mg/mL |
| Cell Strainer 70 µm Nylon | Falcon | 352350 | |
| Cole-Parmer Essentials Mortar and Pestle; Agate, 125 mL | Cole-Parmer | EW-63100-62 | |
| Collagenase from Clostridium histolyticum | Sigma-Aldrich | C5138-500MG | |
| Collagenase Type I | STEMCELL | 7415 | |
| Corning Mini Centrifuge | CORNING | 6770 | |
| Corning Stripettor Ultra Pipet Controller | Corning | 4099 | |
| Deoxyribonuclease I from bovine pancreas | Sigma-Aldrich | D4513 | |
| Dispase II, powder | Gibco | 117105041 | |
| DPBS 10x | gibco | 14200-075 | |
| eBioscience 1x RBC Lysis Buffer | Invitrogen | 00-4333-57 | |
| Ethanol absolute, KOPTEC, meets analytical specification of BP, Ph. Eur., USP (200 Proof) | VWR | 89125-174 | |
| Fine scissors – sharp | Fine Science Tools | 14061-10 | |
| Foundation B Fetal Bovine Serum | GeminiBio | 900-208 | |
| Gilson PIPETMAN L Pipette Starter Kits | FisherScientific | F167370G | |
| Graefe Forceps | Fine Science Tools | 11051-10 | |
| Hank's Balanced Salt Solution (HBSS) 10x | gibco | 14185-052 | |
| Hemocytometer | Fisher | 02-671-10 | |
| Incubator | BINDER | C150-UL | |
| Kimwipes | KIMTECH | K222101 | |
| LABGARD Class II, Type A2 Biological Safety Cabinet | Nuaire | NU-425-400 | |
| LD Columns | Miltenyi Biotec GmBH | 130-042-901 | |
| LSE Vortex Mixer | CORNING | 6775 | |
| LSRII/Fortessa/Symphony A1 | Becton, Dickinson and Company | 647800L6 | |
| MACS MULTI STAND | Miltenyi Biotec GmBH | 130-042-303 | |
| MACsmix Tube Rotator | Miltenyi Biotec GmBH | 130-090-753 | |
| mIgG | Millipore-Sigma | 18765-10mg | 2 mg/mL |
| Nup98-HoxD13 (NHD13) Mice | Jackson Laboratory | 010505 | |
| PE anti-mouse CD51 Antibody (RMV-7) | Biolegend | 104106 | 0.2 mg/mL |
| PE/Cyanine5 anti-mouse CD140a Antibody (RUO) | Biolegend | 135920 | 0.2 mg/mL |
| Penicillin-Streptomycin | Gibco | 15140122 | 10,000 U/mL |
| Plastipak 3 mL Syringe | Becton, Dickinson and Company | 309657 | |
| Propidium Iodide – 1.0 mg/mL Solution in Water | ThermoFisher Scientific | P3566 | |
| QuadroMACS Separator | Miltenyi Biotec GmBH | 130-090-976 | |
| Sorvall X Pro / ST Plus Series Centrifuge | Thermo Scientific | 75009521 | |
| TER-119 Monoclonal Antibody (TER-119), APC | Invitrogen | 17-5921-82 | 0.2 mg/mL |
| Trypan Blue Solution 0.4% | Gibco | 15-250-061 | |
| Ultrapure 0.5 M EDTA, pH 8.0 | Invitrogen | 15575-038 |
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Kaszuba, C. M., Rodems, B. J., Sharma, S., Franco, E. I., Ashton, J. M., Calvi, L. M., Bajaj, J. Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia. J. Vis. Exp. (201), e66093, doi:10.3791/66093 (2023).