隔离和中性粒细胞的表征与抗肿瘤性质
Summary
Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.
Abstract
嗜中性粒细胞，最丰富的全白血细胞在人体循环的，起到对入侵微生物的宿主防御中起重要作用。此外，嗜中性粒细胞在肿瘤细胞的免疫监视中心作用。他们必须认识到肿瘤细胞，并诱导肿瘤细胞死亡或者通过涉及过氧化氢，或通过抗体依赖性细胞介导的细胞毒性（ADCC）的细胞接触依赖性机制的能力。嗜中性粒细胞具有抗肿瘤活性可以从癌症患者和荷瘤小鼠的外周血中分离出来。这些嗜中性粒细胞被称为肿瘤夹带的嗜中性粒细胞（TEN）从健康受试者或天真的小鼠表明，没有显著的肿瘤细胞毒活性的嗜中性粒细胞区分开来。与其他的白血细胞相比，嗜中性粒细胞显示出不同的浮力使得当经受密度梯度它可行，得到> 98％纯的嗜中性白细胞群体。然而，除了到正常的高密度嗜中性白细胞群体脱氮（HDN），在癌症患者中，在肿瘤的小鼠，以及下慢性炎性病症，不同的低密度中性粒细胞种群（LDN）出现在循环中。 LDN共纯化的单核组分，并且可以从使用正或负选择策略的单核细胞中分离出来。一旦分离嗜中性粒细胞的纯度通过流式细胞术确定的，它们可以被用于在体外和体内功能性测定法。我们描述的技术用于监测中性粒细胞的抗肿瘤活性，它们的迁移，并产生活性氧，以及监测其吞噬能力的离体的能力。我们进一步描述的技术来标记的嗜中性粒细胞在体内跟踪，并确定其在体内的抗转移能力。所有这些技术是必不可少的理解如何获得和具有抗肿瘤表征嗜中性粒细胞功能。
Introduction
嗜中性粒细胞被初步定性为它作为对抗入侵微生物的第一道防线的先天免疫细胞。目前已知有中性粒细胞更为深远的功能，被卷入安装适应性免疫反应对抗外来抗原1,2，调节造血3，4血管新生和伤口愈合5。此外，嗜中性粒细胞可影响肿瘤的生长和转移进程凭借自己亲和抗肿瘤活性6,7。嗜中性粒细胞的特征是多态分段核（因此称为多形核（PMN）白细胞）和含有颗粒的至少三个不同的亚类，以及分泌囊泡8（ 图1A-C）。
嗜中性粒细胞具有较高的吞噬能力和高NADPH氧化酶的活性微生物消灭批判，并分泌一系列趋化因子在重要的牵引的附加 中性粒细胞和其它免疫细胞到炎症8,9的部位。嗜中性粒细胞的特征是大量的表面受体，包括Toll样受体（TLR）的表达，C型凝集素受体 （的CLR），补体受体3（细胞CD11b / CD18）和其它粘附分子（ 例如，L-选择LFA-1，VLA-4和癌胚抗原相关细胞粘附分子3（CEACAM3 / CD66b）），趋化因子受体（ 例如，CXCR1，CXCR2，CCR1，CCR2），趋化因子受体（ 例如，PAFR，LTB 4 R和C5aR的） ，细胞因子受体（ 例如，G-CSFR，IL-1R，IL-4R，IL-12R，IL-18R，TNFR），甲酰基肽受体（ 例如，FPR1-3），和Fc受体（ 例如，CD16（FcγRIII ），CD32（FcγRII），CD64和（FcγRI）10。在小鼠中，中性粒细胞通常被认定为细胞CD11b + Ly6G +，而人类嗜中性粒细胞使用的CD11b，CD15，CD16和CD66b白细胞识别标志。它也被普遍接受染色的颗粒蛋白髓过氧化物酶（MPO）和中性粒细胞弹性蛋白酶（NE）检测组织中嗜中性粒细胞。
目前还不清楚是否嗜中性粒细胞的不同功能由同一小区或由不同的细胞亚群介导的。累积的数据表明了异种嗜中性白细胞群体表现出可塑性受促炎性刺激物和微环境11,12的高度的存在。 Fridlender 等人 13已经严重分割在癌症中的嗜中性粒细胞分成两个主要的亚群称为N1具有抗肿瘤性质和N2与亲肿瘤性质。在癌症中，以及在慢性炎症中，有一个附加的子种群粒髓源抑制细胞（G-肌源性干细胞），该抑制T细胞反应14组成。 G-肌源性干细胞被认为是其特征在于由一个细胞CD11b +的Ly6C未成熟骨髓细胞低 Ly6G 喜表型的小鼠15，而具有CD15 + / CD16 低人体16的表型。 G-肌源性干细胞表达更高水平的精氨酸酶和髓过氧化物酶，细胞因子和趋化因子比正常循环中性粒细胞，而较低的水平。他们不太吞噬和游走性，但产生较高水平的ROS 15,17,18的。在本文中，我们将介绍一些基本的方法进行隔离中性粒细胞具有抗肿瘤特性和表征。
而中性粒细胞构成的所有白血细胞在人体循环的人口最多（45 – 70％; 1800 – 6000 /微升），在小鼠体内，正常情况下，他们是相当稀疏（10 – 15％; 300 – 500 /微升）。嗜中性粒细胞计数增加时炎症稳步偶尔在癌症，它代表的慢性炎症7的状态。嗜中性粒细胞多能，从普通的骨髓前体（CMP）CE开发LLS在骨髓，通过分化过程通过原粒细胞的阶段（MB），早幼粒细胞（PM），髓细胞（MC），晚幼（MM）和带细胞（BC）8。成熟，有丝分裂后的中性粒细胞可以保持骨髓4内- 7日他们被释放到循环8之前。嗜中性粒细胞周转在血液通常是迅速，平均半衰期为6 – 12小时，这可能是炎症条件下延长。未刺激的嗜中性粒细胞已通过暴露于佛波醇酯佛波12不限抗肿瘤发生活性，可以通过使幼稚中性粒细胞的趋化因子IL-8（CXCL2），CCL2，CCL5和CXCL5 6,19或人工地获取的特征， -myristate 13-乙酸酯（PMA）6。
短半衰期血中性粒细胞与低中性粒细胞数（约3 – 5×10 5）取血1ml的天真6实现- 8周旧鼠标，使人们艰难的探索循环中性粒细胞小鼠体外的功能。为了克服这个困难，其他来源的已被使用。例如，大量的中性粒细胞可以从骨髓20或以下无菌炎症的诱导腹膜得到的（ 例如 ，腹膜内注射硫乙醇酸盐肉汤或酵母聚糖A的之后）。应当指出的是，从腹腔得到嗜中性粒细胞不施加任何抗肿瘤发生活性（未发表的观察）。
。Granot酒店等 6观察到BALB / c小鼠接种原位用小鼠4T1乳腺癌细胞系开发嗜中性这恶化了与肿瘤进展6（ 图2A），以使得20 – 40万血嗜中性粒细胞可以容易地从1毫升血液中分离3 – 第4周后，肿瘤接种。这些嗜中性粒细胞已获得抗肿瘤活性，并因此被瑰奈德肿瘤夹带中性粒细胞（TEN），以便从幼稚中性粒细胞6（ 图2B）相区别。而高密度的中性粒细胞（HDN， 图1A）是高度抗肿瘤发生，在癌症的情况下产生的低密度中性粒细胞（LDN， 图1B）不是21。另外，来自肿瘤携带小鼠的骨髓及脾脏高密度的中性粒细胞具有抗肿瘤活性（未发表数据）。应当指出的是，与肿瘤进展脾脏逐渐放大（脾肿大），以增加嗜中性粒细胞的数量。
应当指出的是，也被在癌症包括自发（MMTV-PyMT和MMTV-WNT1乳腺肿瘤和K-ras基因驱动的肺肿瘤）其他模型生成并注射（AT-3（MMTV-PyMT）和E0771乳腺癌TEN细胞，LLC Lewis肺癌细胞和B16-F10黑素瘤细胞）。然而，在这些TUM中性粒细胞动员的程度或模型远比4T1-接种小鼠的少，达到5 – 10×10 6中性粒细胞在1ml血后3周。
Protocol
Representative Results
Discussion
嗜中性粒细胞是最丰富的全白血细胞，并且是在感染和炎症的情况下，第一反应者。因此，它们是外部线索高度敏感，并且容易激活。此外，嗜中性粒细胞具有非常短的半衰期和快速周转。总之，这些特点提高一些困难与中性粒细胞，这种独特的实验策略是必需的工作。例如，有几个中性粒细胞纯化策略，每一个都有自己的优点和缺点。
在中性粒细胞工作的一个关键步骤是?…
Declarações
The authors have nothing to disclose.
Acknowledgements
ZG支持从以色列科学基金会（批准号：41/11）的I-CORE项目赠款，在Abisch-福仁基金会，Rosetrees信托，以色列癌症研究基金会（ICRF – 研究职业发展奖）和令人关注的基础。 ZGF支持从以色列癌症研究基金会（ICRF – 研究职业发展奖）赠款，以色列卫生部和以色列肺脏协会的首席科学家。
Materials
| CELL LINES | |||
| Mouse 4T1 breast carcinoma cells | ADCC | CRL-2539 | Growth medium: DMEM + 10 % heat-inactivated FBS |
| PLASTIC WARES AND EQUIPMENTS | |||
| 24-well Tissue Culture Plate | Falcon | 353047 | Sterile |
| 100 mm Tissue Culture Plate | Corning | 430167 | Sterile |
| 25 cm2 Tissue Culture Flask | Nunc | 156340 | Sterile |
| 90 mm Bacterial Grade Culture Dish | Miniplast, Ein Shemer, Israel | 20090-01-017 | Sterile |
| 15 ml Sterile Conical Centrifuge Tube | Miniplast, Ein Shemer, Israel | 835015-40-111 | Sterile |
| 50 ml Sterile Conical Centrifuge Tube | Miniplast, Ein Shemer, Israel | 835050-21-111 | Sterile |
| Falcon 12×75 mm Round-Bottom Polystyrene Tube | Becton Dickinson | 352058 | Sterile |
| Millicell 24 Migration Plate with a pore size of 5μm | Merck Millipore | PSMT010R1 | Sterile |
| White 96-Flat-Bottom Well Plate | Costar | 3917 | Sterile |
| Cell Strainer (40 mm) | BD Falcon | 352340 | Sterile |
| 20G 1.5" Needle | BD Microlance 3 | 301300 | Sterile |
| 23G 1" Needle | BD Microlance 4 | 300800 | Sterile |
| 25Gx5/8" Needle | BD Microlance 5 | 300600 | Sterile |
| 0.3 ml Syringe with a 30Gx8mm Needle | BD Micro-Fine Plus Demi | 320829 | Sterile |
| 9 mm Clips | BD, AutoClip | 427631 | Sterile |
| EasySep Magnet | STEMCELL Technologies | 18000 | |
| MACS LS Separation Column | Miltenyi Biotech | 130-042-201 | Sterile |
| MidiMACS Separator Magnet | Miltenyi Biotech | 130-042-302 | |
| MACS MultiStand | Miltenyi Biotech | 130-042-303 | |
| Microscope Glass Slide | Menzel-Gläser Superfrost Plus Thermo | J1800AMNZ | |
| Orbital Shaker | Sky line, ELMI | S-3.02.10L | |
| Plate Reader | TECAN | InfiniteF200Pro | |
| POWDER | |||
| Bovine serum albumin (BSA), fraction V | Sigma | A7906 | |
| Bromodeoxyuridine (BrdU) | BD Pharmingen | 550891 | Sterile |
| CFSE (5-(and 6-)-Carboxyfluorescein diacetate, succinimidyl ester) | Molecular Probes | C1157 | |
| Dextran T500 | Sigma | 31392 | |
| Heparin sodium salt from porcine intestinal mucosa | Sigma | H3149 | |
| Sodium azide (NaN3) | Sigma | S8032 | Highly toxic, handle with care |
| Thioglycollate powder | Difco | 225650 | |
| Zymosan A | Sigma | Z4250 | |
| MEDIA AND SUPPLEMENTS | |||
| Dulbecco's modified Eagle medium (DMEM) | Sigma | D5796 | Sterile |
| Opti-MEM® I reduced serum medium | Life Technologies | 31985062 | Sterile |
| Roswell Park Memorial Institute (RPMI)-1640 medium | Sigma | R8758 | Sterile |
| Foetal bovine serum (FBS), heat-inactivated | Sigma | F9665 | Sterile |
| L-Glutamine | Biological Industries, Beth HaEmek, Israel | 03-020-1A | Sterile |
| Sodium pyruvate | Biological Industries, Beth HaEmek, Israel | 03-042-1B | Sterile |
| Penicillin Streptomycin x1000 solution | Biological Industries, Beth HaEmek, Israel | 03-031-5 | Sterile |
| Phosphate buffered saline (PBS) without Mg2+ and Ca2+ | Biological Industries, Beth HaEmek, Israel | 02-023-1 | Sterile |
| PBSx10 without Ca2+ and Mg2+ | Biological Industries, Beth HaEmek, Israel | 02-023-5A | Sterile |
| HPLC grade water | J.T. Baker | 4218-03 | Autoclave |
| SOLUTIONS | |||
| ACK – Ammonium-Chloride-Potassium | Life Technologies | A10492-01 | |
| Bromodeoxyuridine (BrdU) solution (10 mg/ml) in PBS | Dissolve 10 mg of BrdU in 1 ml PBS and sterile filter. | ||
| CFSE, 5 mM in DMSO | Dissolve 2.8 mg of CFSE in 1 ml DMSO. Divide into 10 ml aliquots in sterile 200 ml tubes and store in the dark at -20oC. | ||
| Eosin Y solution | Sigma | HT110-2-32 | |
| Hanks' balanced salt solution | Biological Industries, Beth HaEmek, Israel | 02-016-1A | Sterile |
| Heparin, 20 mg/ml in PBS | Dissolve 100 mg Heparin in 5 ml sterile PBS, and sterile filter through a 0.2 mm filter. | ||
| Histopaque-1119 | Sigma | 11191 | Sterile filter through a 0.2 mm filter. |
| Histopaque-1077 | Sigma | 10771 | Sterile filter through a 0.2 mm filter. |
| Luciferase cell culture lysis buffer x5 | Promega | E153A | Dilute 1:5 in sterile water just before use. |
| Luciferase assay solution | Promega | E1501 | Contains luciferase assay substrate powder (E151A) and luciferase assay buffer (E152A) |
| Mayer's Hematoxylin solution | Sigma | MHS-32 | |
| PBS+0.5% BSA | Dissolve 2.5g BSA in 500 ml PBS, and sterile filter through a 0.2 mm filter. | ||
| PBS+1% BSA | Dissolve 1g BSA in 100 ml PBS, and sterile filter through a 0.2 mm filter. | ||
| 5x PBS with 2.5% BSA | Dissolve 12.5g BSA in a mixture of 250 ml sterile HPLC-grade water and 250 ml PBSx10, and sterile filter through a 0.2 mm filter. | ||
| PBS containing 0.5% BSA and 2 mM EDTA | Dissolve 250 mg BSA in 50 ml sterile PBS and add 200 ml of 0.5M EDTA pH 8.0, sterile filter through a 0.2 mm filter. | ||
| FACS buffer (PBS containing 0.5% BSA, 2 mM EDTA and 0.02% NaN3) | Dissolve 250 mg BSA in 50 ml sterile PBS and add 200 ml of 0.5M EDTA pH 8.0 and 500 ml of 2% NaN3, sterile filter through a 0.2 mm filter. | ||
| Saline (0.9% NaCl) | Dissolve 9 g NaCl in 1000 ml ddw, autoclave | ||
| 0.2% NaCl solution | Dissolve 2 g NaCl in 1000 ml ddw, autoclave | ||
| 1.6% NaCl solution | Dissolve 16 g NaCl in 1000 ml ddw, autoclave | ||
| 2 % Sodium azide | Dissolve 1g sodium azide in 50 ml sterile ddw, keep at 4oC. Highly toxic. | ||
| 3% Thioglycollate solution | Dissolve 3 g of thioglycollate powder in 100 ml ddw. Boil until solution becomes yellow and autoclave. | ||
| Trypan blue solution (0.4%) | Sigma | T8154 | Dilute 1:10 in PBS to get a 0.04% solution. |
| Trypsin solution B | Biological Industries, Beth HaEmek, Israel | 03-046-1 | Sterile |
| 1 mg/ml Zymosan A | Resuspend 1 mg Zymosan A in 1 ml sterile PBS in an Eppendorf tube. Vortex vigorously and incubate the tube at 37 oC for 30 min. Do not autoclave. Prepare the solution freshly before use. | ||
| KITS | |||
| EasySep PE selection kit | STEMCELL Technologies | 18557 | |
| EasySep PE selection cocktail | STEMCELL Technologies | 18151 | |
| the EasySep magnetic nanoparticles | STEMCELL Technologies | 18150 | |
| Anti-Ly6G mouse MicroBead Kit | Miltenyi Biotec | 130-092-332 | |
| EasySep Mouse Neutrophil Enrichment Kit | STEMCELL Technologies | 19762 | |
| EasySep Human Neutrophil Enrichment Kit | STEMCELL Technologies | 19257 | |
| FITC BrdU flow kit | BD Pharmingen | 559619 | |
| MACS Neutrophil isolation kit | Miltenyi Biotec | 130-097-658 | |
| Phagocytosis Assay Kit | Cayman Chemical Company | 500290 | |
| ANTIBODIES | |||
| FcR blocking antibody | Biolegend | 101302 | |
| Purified rat anti-Ly6G antibody | BD Pharmingen | 551459 | Clone 1A8 |
| PE-conjugated rat anti-mouse Ly6G antibody | Biolegend | 127608 | Clone 1A8 |
| FITC-conjugated rat anti-mouse Ly6G | BD Pharmingen | 551460 | Clone 1A8 |
| PerCP-Cy5.5 rat anti-mouse Ly6G | TONBO Biosciences | 65-1276 | Clone 1A8 |
| violetFluor 450-conjugated rat anti-mouse Ly6G | TONBO Biosciences | 75-1276 | Clone 1A8 |
| FITC-conjugated rat anti-mouse CD11b | BD Pharmingen | 553310 | Clone M1/70 |
| FITC-conjugated rat anti-mouse Ly-6G and Ly-6C (GR-1) | BD Pharmingen | 553127 | Clone RB6-8C5 |
| PE-conjugated rat anti-mouse CD45 | BD Pharmingen | 553081 | Clone 30-F11 |
| FITC-conjugated rat anti-mouse F4/80 | Abcam | ab60343 | Clone BM8 |
| FITC-conjugated mouse anti-human CD66b | Biolegend | 305103 | Clone G10F5 |
| Purified rat isotype control antibody (IgG2a, k) | BD Pharmingen | 553927 | Clone R35-95 |
| LEAF purified Armenian hamster anti-mouse CD3e antibody | BioLegend | 100314 | Clone 145-2C11 |
Referências
- Tangye, S. G., Brink, R. A helping hand from neutrophils in T cell-independent antibody responses. Nat Immunol. 13, 111-113 (2012).
- Mantovani, A., Cassatella, M. A., Costantini, C., Jaillon, S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol. 11, 519-531 (2011).
- Pruijt, J. F., et al. Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice. Proc Natl Acad Sci U S A. 99, 6228-6233 (2002).
- Tecchio, C., Cassatella, M. A. Neutrophil-derived cytokines involved in physiological and pathological angiogenesis. Chem Immunol Allergy. 99, 123-137 (2014).
- Liu, M., et al. Formylpeptide receptors mediate rapid neutrophil mobilization to accelerate wound healing. PloS one. 9, e90613 (2014).
- Granot, Z., et al. Tumor entrained neutrophils inhibit seeding in the premetastatic lung. Cancer Cell. 20, 300-314 (2011).
- Sionov, R. V., Fridlender, Z. G., Granot, Z. The Multifaceted Roles Neutrophils Play in the Tumor Microenvironment. Cancer Microenviron. , (2014).
- Borregaard, N. Neutrophils, from marrow to microbes. Immunity. 33, 657-670 (2010).
- Yamashiro, S., et al. Phenotypic and functional change of cytokine-activated neutrophils: inflammatory neutrophils are heterogeneous and enhance adaptive immune responses. J Leukoc Biol. 69, 698-704 (2001).
- Futosi, K., Fodor, S., Mocsai, A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol. 17, 638-650 (2013).
- Scapini, P., Cassatella, M. A. Social networking of human neutrophils within the immune system. Blood. 124, 710-719 (2014).
- Fridlender, Z. G., et al. Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils. PloS one. 7, e31524 (2012).
- Fridlender, Z. G., et al. Polarization of tumor-associated neutrophil phenotype by TGF-beta: ‘N1’ versus ‘N2. TAN. Cancer Cell. 16, 183-194 (2009).
- Talmadge, J. E., Gabrilovich, D. I. History of myeloid-derived suppressor cells. Nat Rev Cancer. 13, 739-752 (2013).
- Youn, J. I., Nagaraj, S., Collazo, M., Gabrilovich, D. I. Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol. 181, 5791-5802 (2008).
- Choi, J., et al. CD15+/CD16low human granulocytes from terminal cancer patients: granulocytic myeloid-derived suppressor cells that have suppressive function. Tumour Biol. 33, 121-129 (2012).
- Brandau, S., et al. Myeloid-derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties. J Leukoc Biol. 89, 311-317 (2011).
- Pillay, J., Tak, T., Kamp, V. M., Koenderman, L. Immune suppression by neutrophils and granulocytic myeloid-derived suppressor cells: similarities and differences. Cell Mol Life Sci. 70, 3813-3827 (2013).
- Lopez-Lago, M. A., et al. Neutrophil chemokines secreted by tumor cells mount a lung antimetastatic response during renal cell carcinoma progression. Oncogene. 32, 1752-1760 (2013).
- Boxio, R., Bossenmeyer-Pourie, C., Steinckwich, N., Dournon, C., Nusse, O. Mouse bone marrow contains large numbers of functionally competent neutrophils. J Leukoc Biol. 75, 604-611 (2004).
- Sagiv, J., et al. Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer. Cell Reports. , (2015).
