分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析
Summary
这里介绍的是胶质瘤浸润外周血单核细胞的分离和流式细胞术分析能够产生在进入早期脑肿瘤微环境的免疫细胞的数目和激活状态依赖于时间的定量数据一个直接的方法。
Abstract
我们的实验室已最近表明，天然杀伤（NK）细胞能够颅内植入后CNS-1恶性胶质瘤很快消除原位移植小鼠GL26和大鼠如果癌细胞在它们的β半乳糖苷结合凝集素的galectin表达呈现缺陷的-1（加仑-1）。更近期的工作现在显示的Gr-1 + / CD11b的+髓样细胞群是这种效果的关键。为了更好地了解由NK细胞和骨髓细胞协作以赋予加仑-1缺失的肿瘤排斥我们已经开发了全面的协议用于分离和胶质瘤浸润外周血单核细胞（PBMC）的分析的机制。该方法是在这里证明了外周血单个核细胞浸润比较成的肿瘤微环境GAL-1表达GL26胶质瘤与那些通过shRNA的敲除呈现GAL-1缺失。该协议开始的描述如何培养并准备GL26 CELLS接种入同系的C57BL / 6J小鼠的大脑。然后，它解释了涉及从早期脑肿瘤微环境胶质瘤浸润的PBMC的分离和流式细胞术分析的步骤。该方法是适应若干体内 ，其中免疫渗入脑颞数据是必需的实验设计。该方法灵敏度高，重现性好，为神经胶质瘤浸润外周血单个核细胞可以从颅内肿瘤一旦被隔离24小时后移植瘤与时间点匹配的肿瘤遍布独立的实验中观察到类似的细胞计数。单个实验者可以从大脑收获执行该方法流胶质瘤浸润的PBMC的流式细胞术分析中大约4-6小时取决于待分析的样品的数目。替代胶质瘤模型和/或细胞特异性的检测抗体，也可以使用在实验者自行决定，以评估其它几个与免疫的渗透Ë细胞类型，而不需要改变对整体过程的兴趣。
Introduction
胶质瘤是一类从转化胶质中枢神经系统（CNS）内所产生的神经上皮脑癌。在所有的胶质瘤，世界卫生组织（WHO）Ⅳ级神经胶质瘤，或胶质母细胞瘤（GBM），是最常见和致命的1。 GBM是高度耐火到当前标准的转交它由肿瘤切除术，以随后辐射加伴随和辅助化疗与替莫唑胺2尽可能。这些致命的癌症随身携带的只有15〜18个月的生存率从最初诊断时，只有5％的患者生存的疾病预后极差5 年 1之后。
的血脑屏障（BBB）的存在，缺乏专业的抗原呈递细胞的（的APC），和脑4内的以前未知的存在真正的淋巴结构导致基底膜免疫特权的概念。然而，大量的研究现在SHO瓦特这些脑癌确实产生外周免疫细胞主要是粒细胞中的起源，其中包括单核细胞，巨噬细胞和髓源抑制细胞（肌源性干细胞）5的募集。 GBM还影响脑驻留小胶质细胞的活性而成为亲致瘤6,7。淋巴样细胞例如CD8 + T细胞8和CD56 +自然杀伤细胞9也是在肿瘤微环境中存在的，但在少得多的数量，想到了一个事实是由于策动胶质瘤衍生的因子对肿瘤相关巨噬细胞的免疫抑制功能（噬细胞）10。 CD4 + T细胞也存在于GBM，但许多这类人群也表达CD25和FoxP3的，免疫调节性T（ 调节性T细胞）11机。 GBM的整体免疫抑制状态的高潮在促进免疫逃逸和肿瘤进展12。
<p class="“jove_content”">更好地了解GBM免疫抑制的机制是旨在刺激免疫系统对肿瘤免疫治疗的有效战略至关重要。在过去的15年来，我们的实验室一直在努力克服脑肿瘤immunosuppresson的机制，以便制定有效的新抗GBM免疫疗法13-19。这项工作的高潮已经导致了旨在评估组合的细胞毒性和免疫刺激治疗的患者初诊GBM（ClinicalTrials.gov标识符：NCT01811992）临床试验。我们最近的工作表明，小鼠GL26和大鼠CNS-1 GBM细胞通过产生大量的β半乳糖苷结合凝集素半乳糖凝集素1（GAL-1）20块抗肿瘤NK细胞的免疫监视。这证明通过抑制加仑-1在神经胶质瘤细胞使用shRNA介导的基因敲除的表达。 体外 experimenTS表明加仑-1缺失神经胶质瘤细胞中培养正常增殖，但颅内植入进同源的C57BL / 6J或RAG1之后不久进行快速排斥– / -小鼠 ，从而建立T-或B细胞的独立性对这种形式的肿瘤排斥。 NK细胞免疫耗竭具有抗唾液酸GM 1抗血清或导致颅内GAL-1缺失的脑胶质瘤生长完全恢复单克隆抗体NK1.1，建立NK细胞GAL-1缺失的神经胶质瘤抑制的作用。我们现在表明的Gr-1 + / CD11b的+髓样细胞的免疫耗竭足以防止加仑-1缺失胶质瘤排斥尽管NK细胞的存在，从而揭示在NK介导的伽援用于骨髓细胞一个不可或缺的辅助作用的-1缺陷型肿瘤溶解（未公布的数据）。这一出人意料的结果使我们制定一个全面的协议，用于外周血单核细胞的分离和分析（外周血单个核细胞），其渗透到脑肿瘤微环境很快颅内植入后，让我们可以更好地表征，上游GAL-1缺失的胶质瘤抑制免疫渗透活动。
该方法是这里演示用鼠标GL26胶质瘤细胞组成性表达mCitrine荧光蛋白，叫GL26-CIT，它通过荧光显微镜21个州允许直接肿瘤细胞的可视化。这些细胞被立体定位植入进同源C57BL / 6J小鼠的脑和被允许之前小鼠安乐死生长24，48，或72小时。胶质瘤浸润的PBMC，然后分离和免疫标记使用抗-CD45，-GR-1，-CD11b和-NK1.1细胞表面的抗体与细胞内的免疫标记为颗粒酶B（GZMB）。这种特异性抗体的组合允许肿瘤浸润的Gr-1 + / CD11b的+髓样细胞的鉴定和NK1.1 + NK细胞，细胞类型，我们有bEEN牵连GAL-1缺失的肿瘤排斥。的GAL-1缺失GL26-CIT胶质瘤免疫浸润轮廓，这里称为GL26-CIT-gal1i，然后比较该胶质瘤表达加仑-1称为GL26-CIT-NT的正常水平，包含一非易失的针对对照shRNA的发夹。该协议开始于上GL26-CIT胶质瘤如何来培养细胞的说明在体外 ，随后是关于如何原位嫁接这些细胞进同源C57BL / 6J小鼠的纹状体的解释。然后，它前进到枚举涉及分离和胶质瘤浸润的PBMC用于流式细胞术分析的免疫标记的步骤。协议结尾标准数据分析和图形表示的解释。
该演示可以发现，GR-1 + / CD11b的+骨髓细胞和NK1.1 + NK细胞优先积累在48中的GAL-1缺失的脑肿瘤微环境中小时肿瘤植入，因此这有助于解释为什么这些肿瘤迅速进行完整的肿瘤溶解大约一周后的肿瘤植入20。该方法是很容易适应一些，其中对免疫渗入脑颞数据，需要在体内不同的实验设计。单个实验者可以从大脑收获执行协议流胶质瘤浸润的PBMC的流式细胞术分析在约4-6小时取决于待分析的样品的数目。该方法还可以用实验的目的是表征循环的PBMC在荷瘤小鼠对与那些渗入脑如此识别由肿瘤微环境特异性诱导的免疫抑制的表型比较的信息相结合。这和类似的方法应用应有助于更好地理解所涉及的外周免疫细胞贩运到大脑肿瘤微环境的因素。
Protocol
Representative Results
Discussion
这个协议描述一种可再生的方法的PBMC的分离和流式细胞仪分析已渗入早期鼠脑肿瘤微环境。在由被立体定位植入到小鼠脑的纹状体的实验者指定的浓度产生神经胶质瘤细胞悬浮液。小鼠然后安乐死在由实验设计指定的预定的时间点和他们的大脑收获和加工以分离这些免疫标记与荧光标记的初级抗体的组合胶质瘤浸润的PBMC;免疫标记细胞，然后通过流式细胞术进行定量。虽然这里给出的示范着重于?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是支持的美国国立卫生研究院神经疾病和中风（NIH / NINDS）授予R01-NS074387，R01-NS057711和R21-NS091555到的MGC /研究所; NIH / NINDS授予R01-NS061107，R01-NS076991，R01-NS082311和R21-NS084275到PRL;从利亚的快乐心助学金，密歇根大学综合癌症中心颁发给MGC和PRL大学;神经外科，医学密歇根大学的大学部;密歇根州的临床规范研究所与健康研究，由美国国立卫生研究院资助2UL1-TR000433支持;由NIH / NCI（国家癌症研究所）资助T32-CA009676支持的密歇根大学癌症生物学培训资助;密歇根培训的大学临床和基础神经科学由美国国立卫生研究院/ NINDS支持授予T32-NS007222;和密歇根大学医学院科学家培训项目的大学由美国国立卫生研究院/ NIGMS（通用医学科学研究所）的支持给予T32-GM007863。作者一再次感谢来自卡琳Muraszko博士和神经外科接收的学术领导和支持;到M.达尔格伦，D TomFord的，和S. Napolitan高超的行政支持;以M. Dzaman优秀的技术援助;和菲尔楼詹金斯向购买蔡司3D扫描电子显微镜的慷慨支持。我们也承认Kuchroo实验室在哈佛医学院从脑单个核细胞分离的密度离心媒体介导的战略的修改后的版本被吸引。
Materials
| Dulbecco’s Modification of Eagles Medium | Gibco | 12430-054 | with 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red |
| Dulbecco’s Phosphate-buffered Saline | Gibco | 14190-144 | without calcium chloride or Magnesium chloride |
| Fetal bovine serum | Omega Scientific Inc. | FB-11 | |
| Penicillin Streptomycin | Gibco | 15140-122 | 10,000U/ml Penicillin; 10,000μg/ml Streptomycin |
| L-glutamine | Gibco | 25030-081 | 200mM |
| G418 sulfate | Omega Scientific Inc. | GN-04 | |
| Puromycin dihydrochloride | Sigma-Aldrich | P8833 | from Streptomyces alboniger |
| HyClone HyQTase non-mammalian trypsin alternative | Thermo Scientific | SV30030.01 | in DPBS with EDTA |
| Phase contrast hemocytometer | Sigma-Aldrich | Z359629 | 0.1mm deep |
| Trypan blue stain | Gibco | 15250-061 | |
| Sterile 0.9% NaCl injection, USP | Hospira | NDC: 0409-4888 | 10ml vials |
| 0.6ml conical polypropylenemi microtubes | Genesee Scientific | 22-272 | |
| Atipamezole hydrochloride injection | Orion Pharma | NDC: 52483-6298 | 5mg/ml solution |
| Ketamine hydrochloride injection | Fort Dodge | NDC: 0409-2051 | 100mg/ml solution |
| Dexmedetomidine hydrochloride injection | Zoetis | NDC: 54771-2805 | 0.5mg/ml solution |
| Xylazine hydrochloride injection | Lloyd | NDC: 61311-481 | 100mg/ml solution |
| Carprofen injection | Pfizer | NDC: 61106-8501 | 50mg/ml solution |
| Buprenorphine hydrochloride injection | Reckitt Benckiser | NDC: 12496-0757 | 0.3mg/ml ampuls |
| 1ml tuberculin syringes | Covidien | 8881501400 | |
| 26G x ½ (0.45mm x 13mm) syringe needles | BD | 305111 | |
| Surgical clippers | 3M | 9661 | |
| Providone-iodine solution | Aplicare | 82-217 | NDC: 52380-1905 |
| Sterile petrolatum ophthalmic ointment | Dechra | NDC: 17033-211 | |
| 70% isopropyl alcohol prep pads | Kendall | 6818 | |
| Sterile gauze | Covidien | 8044 | non-woven, 4" x 4", 4-ply |
| 1.7ml conical polypropylene microtubes | Genesee Scientific | 22-281 | |
| Mouse stereotactic frame | Stoelting | 51730 | |
| Surgical lamp | Philips Burton | CS316W | Coolspot II Variable Spotlight |
| Curved dissecting forceps | Ted Pella Inc. | 5431 | |
| Colibri retractors | FST | 17000-03 | |
| Cordless precision power drill | Dremel | 1100-01 | Stylus model; 7.2-Volt Lithium-Ion with Docking Station |
| Engraving Cutter | Dremel | 105 | 1/32" or 0.8 mm bit diameter |
| Microliter syringe | Hamilton | 75 | Hamilton Microliter® Syringes 700 series; 5μl volume |
| Microliter syringe needles | Hamilton | 7762-06 | 33G, small hub RN NDL, 1.5 in, point style 3, 6/PK |
| Ethilon 3-0 black monofilament nylon sutures | Ethicon | 1663 | |
| Magnetic stir bar | VWR | 74950-296 | 7.9mm diameter x 50mm length (5/16" diameter x 2" length) |
| 1L glass screw-cap storage bottle | Corning | 1395 | Type 1, Class A borosilicate glass |
| Heparin sodium | Sagent | NDC: 25021-400 | 1,000U/ml solution |
| Peristaltic pump | Cole Parmer Instruments Group | 77200-60 | Master Flex Easy Load II console drive |
| compressed carbogen (95% O2 / 5% CO2) | available from local vendor | N/A | A-type, large cylinder |
| 20G x 1 ½" aluminum hub blunt needles | Kendall | 8881202363 | 0.9mm x 38.1mm |
| Polyurethane ice bucket | Fisherbrand | 02-591-45 | Capacity: 0.152 oz. (4.5L) |
| Collagenase (Type I-S) | Sigma Aldrich | C1639 | from Clostridium histolyticum |
| Deoxyribonuclease I | Worthington Biochemical Corp. | LS002007 | >2,000 Kunitz units per mg dry weight |
| Antistatic polystyrene hexagonal weighing dishes | Ted Pella Inc. | 20157-3 | top I.D. 115mm, base I.D. 85mm, 203ml volume |
| Stainless steel single edged razor blades | Garvey | 40475 | |
| Bone rongeurs | FST | 16001-15 | |
| Hemostat | FST | 13014-14 | |
| Large dissection scissors | Ted Pella Inc. | 1316 | |
| Small dissection scissors | FST | 14094-11 | |
| Blunt end forceps | Ted Pella Inc. | 13250 | |
| 7ml glass Dounce tissue grinder | Kontes | KT885300-0007 | |
| Percoll Density Centrifugation Media | GE Healthcare | GE17-0891-01 | |
| 10 mL serological pipettes | Genesee Scientific | 12-104 | single use; sterile |
| 70μm sterile nylon mesh cell strainers | Fisherbrand | 22363548 | |
| Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11) | Biolegend | 103128 | |
| APC-conjugated mouse anti-mouse NK1.1 (clone: PK136) | eBiosciences | 17-5941-82 | |
| PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5) | BD Pharmigen | 553128 | |
| PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70) | Biolegend | 101228 | |
| Alexa Fluor 700-conjugated rat IgG2b, κ (clone: RTK4530) isotype control | Biolegend | 400628 | |
| APC-conjugated mouse IgG2a, κ (clone: eBM2a) isotype control | eBioscience | 17-4724 | |
| PE-conjugated rat IgG2b, κ (clone: eB149/10H5) isotype control | eBioscience | 12-4031-82 | |
| PerCP/Cy5.5-conjugated rat IgG2b, κ (clone: RTK4530) isotype control | Biolegend | 400632 | |
| Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11) | Biolegend | 515403 | |
| Pacific Blue-conjugated mouse IgG1, κ (Clone: MOPC-21) isotype control | Biolegend | 400151 | |
| Cytofix/Cytoperm | BD | 554714 | |
| 15ml polypropylene centrifuge tubes | Genesee Scientific | 21-103 | conical bottom |
| 50ml polypropylene centrifuge tubes | Genesee Scientific | 21-108 | conical bottom |
| 12x75mm round-bottom polypropylene FACS tubes | Fisherbrand | 14-956-1B | |
| FACSAria Special Order Research Product flow cytometer/cell sorter | BD | 650033 | |
| Class II Biological Safety Cabinet | The Baker Company | SG603 | model: SterilGARD III Advance |
Riferimenti
- Bleeker, F. E., Molenaar, R. J., & Leenstra, S. Recent advances in the molecular understanding of glioblastoma. J Neurooncol. 108, 11-27 (2012).
- Stupp, R. et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 352, 987-996 (2005).
- Ostrom, Q. T. et al. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007-2011. Neuro Oncol. 16 Suppl 4, iv1-63 (2014).
- Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels. 523, 337-341 Nature. (2015).
- Kennedy, B. C. et al. Tumor-associated macrophages in glioma: friend or foe? J Oncol. 2013, 486912 (2013).
- Markovic, D. S., Glass, R., Synowitz, M., Rooijen, N., & Kettenmann, H. Microglia stimulate the invasiveness of glioma cells by increasing the activity of metalloprotease-2. J Neuropathol Exp Neurol. 64, 754-762 (2005).
- Zhai, H., Heppner, F. L., & Tsirka, S. E. Microglia/macrophages promote glioma progression. Glia. 59, 472-485 (2011).
- Kmiecik, J. et al. Elevated CD3+ and CD8+ tumor-infiltrating immune cells correlate with prolonged survival in glioblastoma patients despite integrated immunosuppressive mechanisms in the tumor microenvironment and at the systemic level. J Neuroimmunol. 264, 71-83 (2013).
- Yang, I., Han, S. J., Sughrue, M. E., Tihan, T., & Parsa, A. T. Immune cell infiltrate differences in pilocytic astrocytoma and glioblastoma: evidence of distinct immunological microenvironments that reflect tumor biology. J Neurosurg. 115, 505-511 (2011).
- Wagner, S. et al. Microglial/macrophage expression of interleukin 10 in human glioblastomas. Int J Cancer. 82, 12-16 (1999).
- El Andaloussi, A., & Lesniak, M. S. An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme. Neuro Oncol. 8, 234-243 (2006).
- Kostianovsky, A. M., Maier, L. M., Anderson, R. C., Bruce, J. N., & Anderson, D. E. Astrocytic regulation of human monocytic/microglial activation. J Immunol. 181, 5425-5432 (2008).
- Curtin, J. F. et al. HMGB1 mediates endogenous TLR2 activation and brain tumor regression. PLoS Med. 6, e10 (2009).
- Curtin, J. F. et al. Fms-like tyrosine kinase 3 ligand recruits plasmacytoid dendritic cells to the brain. J Immunol. 176, 3566-3577 (2006).
- Mineharu, Y. et al. Engineering the brain tumor microenvironment enhances the efficacy of dendritic cell vaccination: implications for clinical trial design. Clin Cancer Res. 17, 4705-4718 (2011).
- Larocque, D. et al. Exogenous fms-like tyrosine kinase 3 ligand overrides brain immune privilege and facilitates recognition of a neo-antigen without causing autoimmune neuropathology. Proc Natl Acad Sci U S A. 107, 14443-14448 (2010).
- Curtin, J. F. et al. Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials. PLoS One. 3, e1983 (2008).
- Ali, S. et al. Combined immunostimulation and conditional cytotoxic gene therapy provide long-term survival in a large glioma model. Cancer Res. 65, 7194-7204 (2005).
- Dewey, R. A. et al. Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials. Nat Med. 5, 1256-1263 (1999).
- Baker, G. J. et al. Natural killer cells eradicate galectin-1-deficient glioma in the absence of adaptive immunity. Cancer Res. 74, 5079-5090 (2014).
- Baker, G. J. et al. Mechanisms of glioma formation: iterative perivascular glioma growth and invasion leads to tumor progression, VEGF-independent vascularization, and resistance to antiangiogenic therapy. Neoplasia. 16, 543-561 (2014).
- Ormerod, M. G., & Novo, D. Flow cytometry : a basic introduction. (2008).
- Stirling, D. P., & Yong, V. W. Dynamics of the inflammatory response after murine spinal cord injury revealed by flow cytometry. J Neurosci Res. 86, 1944-1958 (2008).
- Hirai, T. et al. The prevalence and phenotype of activated microglia/macrophages within the spinal cord of the hyperostotic mouse (twy/twy) changes in response to chronic progressive spinal cord compression: implications for human cervical compressive myelopathy. PLoS One. 8, e64528 (2013).
- Fleming, T. J., Fleming, M. L., & Malek, T. R. Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family. J Immunol. 151, 2399-2408 (1993).
- Wiesner, S. M. et al. De novo induction of genetically engineered brain tumors in mice using plasmid DNA. Cancer Res. 69, 431-439 (2009).
- Holland, E. C. et al. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat Genet. 25, 55-57 (2000).
- Lynes, J. et al. Lentiviral-induced high-grade gliomas in rats: the effects of PDGFB, HRAS-G12V, AKT, and IDH1-R132H. Neurotherapeutics. 11, 623-635 (2014).
- de Vries, N. A. et al. Rapid and robust transgenic high-grade glioma mouse models for therapy intervention studies. Clin Cancer Res. 16, 3431-3441 (2010).
- Uhrbom, L. et al. Ink4a-Arf loss cooperates with KRas activation in astrocytes and neural progenitors to generate glioblastomas of various morphologies depending on activated Akt. Cancer Res. 62, 5551-5558 (2002).
- Marumoto, T. et al. Development of a novel mouse glioma model using lentiviral vectors. Nat Med. 15, 110-116 (2009).
- Assanah, M. et al. Glial progenitors in adult white matter are driven to form malignant gliomas by platelet-derived growth factor-expressing retroviruses. J Neurosci. 26, 6781-6790 (2006).
