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Macrophage Polarization by Tumor-induced MDSCs Assay
利用肿瘤诱导的骨髓源抑制细胞(MDSCs)进行的巨噬细胞极化实验   

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Abstract

Myeloid derived suppressor cells (MDSCs) are a subset of granulocytes (immature myeloid cells) that exploit a variety of mechanism to modulate the innate and adaptive immune system. MDSCs are present normally in the body, but their numbers increase during inflammation and in cancer, promoting an immunosuppressive microenvironment. In addition to MDSCs, macrophages also play an important role during cancer development. There are two subsets of tumor associated macrophages (TAMs): M1 and M2. M1 are “anti-tumor” macrophages that are activated by interferon gamma (IFN-γ) and/or Lipopolysaccharide (LPS) and secrete high amount of interleukin 12 (IL-12) thereby inducing a Th1 anti-tumor immune response. M2 or “pro-tumorigenic” macrophages are activated by interleukin 4 (IL-4) and interleukin 10 (IL-10) and secrete large amounts of IL-10, which promotes tumor progression (Gabrilovich et al., 2012).

Interaction between MDSCs and macrophages in the tumor microenvironment was shown to enhance immune suppression mediated by these subsets. MDSCs influence TAMs by producing IL-10 that, in turn, induces a down-regulation of IL-12 and polarizes M1 into M2 macrophages. In our study, we use the following protocol to evaluate the ability of tumor induced MDSCs to polarize LPS activated M1 into M2 macrophages (Vences-Catalan et al., 2015). This protocol was adapted from a previous study (Sinha et al., 2007).

Keywords: MDSCs(肌源性干细胞), Macrophage(巨噬细胞), Tumor(肿瘤), Macrophage polarization(巨噬细胞极化), Cytokines(细胞因子)

Materials and Reagents

  1. 70 μm cell strainer (Corning, Falcon®, catalog number: 352350 )
  2. 1 and 10 ml syringe (BD, catalog number: 309659 and 309604 )
  3. 15 and 50 ml polypropylene conical tubes (Corning, Falcon®, catalog number: 352096 and 352070 )
  4. 18 gauze needle (BD, catalog number: 305196 )
  5. 24-well plates (Corning, Falcon®, catalog number: 351147 )
  6. Mice
    Note: We use 6-8 weeks old female Balb/c mice, but any strain of mice can be used as long as both macrophages and MDSCs are from the same genetic background.
  7. Cells
    Note: We use 4T1 breast cancer cell line syngeneic to Balb/c; this tumor model is known to induce a strong accumulation of MDSCs in blood, spleen and tumor.
  8. Thioglycolate (BD, catalog number: 211716 )
    Note: A 3% solution in water and sterilized in autoclave at 121 °C for 15 min has been used.
  9. PBS (Corning, Cellgro, catalog number: 21-031-CV )
  10. RPMI 1640 (Corning, Cellgro, catalog number: 10-040-CV
  11. DMEM media (Corning, Cellgro, catalog number: 10-017-CV )
  12. Penicillin-Streptomycin (Pen-Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-22 )
    Note: 5 ml of this solution was used in 500 ml of RPMI 1640 media.
  13. Fetal calf serum (FCS) (GE Life Sciences, HyCloneTM, FetalClone®III, catalog number: SH30109.03 )
  14. ACK buffer (Quality Biological, catalog number: 118-156-101 )
  15. IL-12p70 and IL-10 ELISA kit (Biolegend, Legend MaxTM, catalog number: 431417 and 433607 )
  16. CD11b PE (clone: M1/70) (BD, catalog number: 553311 )
  17. F4/80 APC (clone: BM8) (eBioscience, catalog number: 17-4801-82 )
  18. Anti Ly6G and Ly6c (Gr1) APC (clone: RB6-8C5) (BD, catalog number: 553129 )
  19. LPS 1 mg/ml (Sigma-Aldrich, catalog number: L3012-5MG )
    Note: If using a different tumor model where MDSCs in blood or spleen represents a small percentage of total Peripheral Blood Mononuclear Cells or splenocytes, respectively, purify MDSCs with myeloid-derived suppressor cell isolation kit and follow instructions according to manufacturers protocol (Miltenyi Biotec, catalog number: 130-094-538 ).

Equipment

  1. Centrifuge (Eppendorf, model: 5810 R )

Procedure

Note: Isolation of peritoneal macrophages as well as MDSCs should be performed under sterile conditions.

  1. Macrophage isolation
    1. Inject intraperitoneally (i.p.) 3-4 naïve Balb/c mice with 1 ml of sterile 3% thioglycolate 4-5 days before macrophage isolation.
    2. 4-5 days later, euthanize mice by cervical dislocation (AVMA Guidelines for the Euthanasia of Animals: 2013 Edition). Carefully make a small incision on the skin to expose the peritoneal wall, avoid cutting the peritoneal wall to prevent leakage of the PBS. Harvest peritoneal cells by injection of 5-10 ml of sterile cold PBS into the peritoneal cavity; give a small massage to shed the peritoneal cells (use 18 gauze needle for the extraction, wash several times with PBS). Collect PBS each time you wash the peritoneum (Lu, 2013). 
    3. Spin the cells at 300 x g for 5 min, count the cells and resuspend in RPMI or DMEM media containing 10% FCS and 1% Pen-Strep. If red blood cells are present, lyse with ACK buffer. Briefly, add 3-5 ml of ACK lysis buffer and incubate for 5 min. Quench the reaction by adding two times the volume of PBS. Spin for 5 min, 300 x g and repeat wash one more time. 
    4. Plate cells at 7.5 x 105 cells/well/500 μl of RPMI or DMEM/10% FCS and 1% Pen-Strep in 24-well plates, and incubate at 37 °C in 5% CO2 for 3 h.
    5. Remove non-adherent cells, and wash the attached cells once with RPMI or DMEM media. 
    6. Save an aliquot (to detach the cells add 2 mM EDTA in PBS for approximately 10-15 min, collect the cells and spin down for 5 min at 300 x g), resuspend in PBS and evaluate the purity of your macrophage population by staining with CD11b and F4/80 antibodies (1 μg of antibody per million of cells) by flow cytometry, as illustrated in Figure 1.


      Figure 1. Analysis of peritoneal macrophages

  2. Tumor derived blood MDSC isolation
    1. Inject 1 x 104-1 x 105 4T1 cells subcutaneously into the mammary fat pad of female Balb/c mice (Reuter, 2011). On day 21-28 almost 90% of white blood cells are MDSCs (doubled positive CD11b+Gr1+), as seen in Figure 2. Determine the percentage of Gr1 positive MDSCs by staining with anti Gr-1- APC and CD11b- PE antibodies and analyze by flow cytometry (1 μg of antibody per million of cells).


      Figure 2. Analysis of MDSCs in the blood of day 28 of 4T1-tumor bearing mice

    2. On day 21-28 after tumor implantation, collect 100-500 μl blood by cardiac puncture from tumor bearing animals and lyse red blood cells with ACK buffer for 3-5 min at RT (5 ml of ACK for 500 μl of blood). Wash cells twice with PBS by spinning two times at 300 x g for 5 min at RT, count cells and resuspend in complete media.
      Optional: If MDSCs represent a minor percentage of total white blood cells, purify MDSCs with myeloid-derived suppressor cell isolation kit.
    3. Co-culture MDSCs (1.5 x 106 MDSCs/well/500 μl of RPMI or DMEM/10% FCS and 1% Pen-Strep media) with 7.5 x 105 macrophages by mixing them together in the same well (or at with different ratios MDSCs:Macrophages, 2:1, 1:1, etc.) in triplicates, add 100 ng/ml of LPS and incubate for 16-18 h at 37 °C, 5% CO2. Include wells that contain only macrophages and only MDSCs as controls (as represented in Figure 3) as well as in the presence and absence of LPS, as illustrated in Figure 4.
    4. After 16-18 h, collect culture supernatants and measure IL-12 and IL-10 secretion by ELISA (Remove cell debris by spinning 3 min 800 x g). Optional is detection of IFN-γ secretion according to the manufacturer’s protocol.


      Figure 3. Representative scheme of the co-culture of macrophages with MDSC’s


      Figure 4. Representative analysis of a macrophage polarization by MDSCs assay

Acknowledgments

This work was supported by the Translational Cancer Award from Stanford Cancer Institute and the Breast Cancer Research program from the Department of Defense grant W81XWH-14-1-0397. This protocol was adapted from a previous study (Sinha et al., 2007).

References

  1. Gabrilovich, D. I., Ostrand, R. S. and Bronte, V. (2012). Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol 12(4): 253-268. 
  2. Lu, M. and Varley, A. W. (2013). Harvest and culture of mouse peritoneal macrophages. Bio-protocol 3(22): e976. 
  3. Reuter, J. (2011). Subcutaneous injection of tumor cells. Bio-protocol Bio101: e166.
  4. Sinha, P., Clements, V. K., Bunt, S. K., Albelda, S. M. and Ostrand-Rosenberg, S. (2007). Cross-talk between myeloid-derived suppressor cells and macrophages subverts tumor immunity toward a type 2 response. Journal of Immunology 179(2): 977-983. 
  5. Vences-Catalan, F., Rajapaksa, R., Srivastava, M. K., Marabelle, A., Kuo, C. C., Levy, R. and Levy, S. (2015). Tetraspanin CD81 promotes tumor growth and metastasis by modulating the functions of T regulatory and myeloid-derived suppressor cells. Cancer Res 75(21): 4517-4526.

简介

骨髓衍生的抑制细胞(MDSC)是粒细胞(未成熟​​骨髓细胞)的子集,其利用多种机制调节先天和适应性免疫系统。 MDSC通常存在于体内,但它们的数量在炎症和癌症期间增加,从而促进免疫抑制微环境。除了MDSC,巨噬细胞也在癌症发展中发挥重要作用。肿瘤相关巨噬细胞(TAM)有两个亚类:M1和M2。 M1是由干扰素γ(IFN-γ)和/或脂多糖(LPS)活化并分泌大量白细胞介素12(IL-12)从而诱导Th1抗肿瘤免疫应答的"抗肿瘤"巨噬细胞。 M2或"致肿瘤发生"巨噬细胞被白介素4(IL-4)和白细胞介素10(IL-10)激活并分泌大量的IL-10,这促进肿瘤进展(Gabrilovich等人, 。,2012)。在肿瘤微环境中MDSC和巨噬细胞之间的相互作用显示增强这些亚群介导的免疫抑制。 MDSC通过产生IL-10而影响TAM,其继而诱导IL-12的下调并将M1极化为M2巨噬细胞。在我们的研究中,我们使用以下方案来评价肿瘤诱导的MDSC将LPS活化的M1极化为M2巨噬细胞的能力(Vences-Catalan等人,2015)。该方案改编自先前的研究(Sinha等人,2007)。

关键字:肌源性干细胞, 巨噬细胞, 肿瘤, 巨噬细胞极化, 细胞因子

材料和试剂

  1. 70μM细胞过滤器(Corning,Falcon ,目录号:352350)
  2. 1和10ml注射器(BD,目录号:309659和309604)
  3. 15和50ml聚丙烯锥形管(Corning,Falcon ,目录号:352096和352070)
  4. 18纱布针(BD,目录号:305196)
  5. 24孔板(Corning,Falcon ,目录号:351147)
  6. 小鼠
    注意:我们使用6-8周龄的雌性Balb/c小鼠,但是可以使用任何小鼠品系,只要巨噬细胞和MDSC来自相同的遗传背景即可。
  7. 单元格
    注意:我们使用4T1乳腺癌细胞系与Balb/c同源; 已知这种肿瘤模型在血液,脾和肿瘤中诱导MDSC的强积聚
  8. 硫代乙醇酸酯(BD,目录号:211716)
    注意:使用3%的水溶液并在121℃的高压灭菌器中灭菌15分钟。
  9. PBS(Corning,Cellgro,目录号:21-031-CV)
  10. RPMI 1640(Corning,Cellgro,目录号:10-040-CV)
  11. DMEM培养基(Corning,Cellgro,目录号:10-017-CV)
  12. 青霉素 - 链霉素(Pen-Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140-22)
    注意:将5ml该溶液用于500ml RPMI 1640培养基中。
  13. 胎牛血清(FCS)(GE Life Sciences,HyClone ,FetalClone III,目录号:SH30109.03)
  14. ACK缓冲液(Quality Biological,目录号:118-156-101)
  15. IL-12p70和IL-10ELISA试剂盒(Biolegend,Legend Max TM,目录号:431417和433607)
  16. CD11b PE(克隆:M1/70)(BD,目录号:553311)
  17. F4/80 APC(克隆:BM8)(eBioscience,目录号:17-4801-82)
  18. 抗Ly6G和Ly6c(Gr1)APC(克隆:RB6-8C5)(BD,目录号:553129)
  19. LPS 1mg/ml(Sigma-Aldrich,目录号:L3012-5MG) 注意:如果使用不同的肿瘤模型,其中血液或脾脏中的MDSC分别代表总外周血单核细胞或脾细胞的小百分比,用骨髓衍生的抑制细胞分离试剂盒纯化MDSC,并根据制造商方案 Miltenyi biotec,目录号:130-094-538)。

设备

  1. 离心机(Eppendorf,型号:5810R)

程序

注意:腹膜巨噬细胞以及MDSC的分离应在无菌条件下进行。

  1. 巨噬细胞分离
    1. 在巨噬细胞分离前4-5天腹膜内(ip)腹腔注射3-4天的Balb/c小鼠与1ml无菌的3%巯基乙酸盐。
    2. 4-5天后,通过颈椎脱臼使安乐死小鼠(AVMA Guide for the Euthanasia of Animals:2013 Edition)。小心地在皮肤上做一个小切口以暴露腹膜壁,避免切开腹膜壁,以防止PBS的渗漏。通过注射5-10毫升无菌冷PBS腹膜腔收获腹膜细胞;给小的按摩以脱落腹膜细胞(使用18纱布针提取,用PBS洗涤几次)。每次洗腹膜时收集PBS(Lu,2013)。
    3. 在300×g下旋转细胞5分钟,计数细胞并重悬于含有10%FCS和1%Pen-Strep的RPMI或DMEM培养基中。如果存在红细胞,用ACK缓冲液裂解。简而言之, 加入3-5ml ACK裂解缓冲液并孵育5分钟。通过加入两倍体积的PBS淬灭反应。旋转5分钟,300 x g ,然后重复洗一次。
    4. 以7.5×10 5个细胞/孔/500μlRPMI或DMEM/10%FCS和1%Pen-Strep在24孔板中的平板细胞,并在37℃在5%CO 2 3小时。
    5. 去除非粘附细胞,并用RPMI或DMEM培养基洗涤附着的细胞一次。
    6. 保存等分试样(分离细胞加入PBS中的2mM EDTA约10-15分钟,收集细胞并以300×g离心5分钟)重悬于PBS中,并评估您的纯度通过流式细胞术用CD11b和F4/80抗体(每百万个细胞1μg抗体)染色的巨噬细胞群,如图1所示。


      图1.腹膜巨噬细胞分析

  2. 肿瘤来源的血液MDSC分离
    1. 将1×10 4个-1×10 5个的4T1细胞皮下注射到雌性Balb/c小鼠的乳腺脂肪垫中(Reuter,2011)。在第21-28天,几乎90%的白细胞是MDSC(双重阳性CD11b + Gr1 + ),如图2所示。确定Gr1阳性MDSC的百分比通过用抗Gr-1-APC和CD11b-PE抗体染色并通过流式细胞术分析(每百万个细胞1μg抗体)。

      图2.在4T1-肿瘤小鼠的第28天的血液中分析MDSC

    2. 在肿瘤植入后第21-28天,通过心脏穿刺从携带肿瘤的动物收集100-500μl血液,并用ACK缓冲液在RT下裂解红细胞3-5分钟(对于500μl血液,5ml的ACK)。用PBS洗涤细胞两次,在室温下以300×g旋转两次,每次5分钟,计数细胞,并重悬于完全培养基中。
      可选:如果MDSC占总白细胞的较小百分比,则用骨髓衍生抑制细胞分离试剂盒纯化MDSC。
    3. 将具有7.5×10 5个细胞培养物的共培养MDSC(1.5×10 6个MDSC /孔/500μlRPMI或DMEM/10%FCS和1%Pen-Strep培养基)巨噬细胞,通过将它们在相同的孔中混合在一起(或以不同比例MDSC:巨噬细胞,2:1,1:1等)一式三份,加入100ng/ml LPS并孵育在37℃,5%CO 2下16-18小时。包括只含有巨噬细胞和仅MDSC作为对照的孔(如 如图3所示)以及在存在和不存在LPS的情况下,如图4所示。
    4. 16-18小时后收集培养物上清液,并通过ELISA(通过旋转3分钟800×g除去细胞碎片)测量IL-12和IL-10分泌。 任选地是根据制造商的方案检测IFN-γ分泌

      图3.巨噬细胞与MDSC的共培养的代表性方案


      图4.通过MDSCs测定法代表的巨噬细胞极化分析

致谢

这项工作得到斯坦福癌症研究所的翻译癌症奖和国防部授予的乳腺癌研究计划W81XWH-14-1-0397的支持。该方案改编自先前的研究(Sinha等人,2007)。

参考文献

  1. Gabrilovich,D.I.,Ostrand,R.S.and Bronte,V。(2012)。 肿瘤对骨髓细胞的协调调节。 Nat Rev Immunol 12(4):253-268。
  2. Lu,M。和Varley,A.W。(2013)。 小鼠腹膜巨噬细胞的收获和培养。生物协议 3(22):e976。
  3. Reuter,J。(2011)。 皮下注射肿瘤细胞 生物方案 Bio101:e166。
  4. Sinha,P.,Clements,V.K.,Bunt,S.K.,Albelda,S.M.and Ostrand-Rosenberg,S。(2007)。 骨髓衍生的抑制细胞和巨噬细胞之间的串扰破坏肿瘤免疫 朝向2型反应。 免疫学杂志 179(2):977-983。
  5. Vences-Catalan,F.,Rajapaksa,R.,Srivastava,M.K.,Marabelle,A.,Kuo,C.C.,Levy,R.and Levy,S。 Tetraspanin CD81通过调节T调节功能来促进肿瘤生长和转移 和髓样衍生的抑制细胞。 Cancer Res 75(21):4517-4526。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vences-Catalán, F., Srivastava, M. K. and Levy, S. (2016). Macrophage Polarization by Tumor-induced MDSCs Assay. Bio-protocol 6(16): e1900. DOI: 10.21769/BioProtoc.1900.
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