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Detection of Tumor Cell Surface-reactive Antibodies
肿瘤细胞膜结合性抗体的检测   

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Abstract

Vaccine-based immunotherapy is being used to treat dogs with primary brain tumors. The vaccines are composed of a lysate of autologous tumor cells, which stimulate an immune response producing tumor specific antibodies that are capable of inducing antibody-dependent cell-mediated cytotoxicity to allogeneic, as well as autologous, tumor cells. This protocol will describe the tumor cell serum antibody-binding assay to measure the tumor-reactive IgG antibody response. Key features of this assay are that it is performed with sera collected from the canine patient prior to and following vaccination as the source of antibodies and canine brain tumor cells used as the target cells.

Materials and Reagents

  1. Tumor cells
    Note: Tumor sample is harvested from pet dogs during brain surgery.
  2. BD MatrigelTM (BD Bioscience, catalog number: 354230 )
  3. DMEM/F12 Medium with L-glutamine (Life Technologies, Gibco®, catalog number: 11320-033 )
  4. 50x B-27 supplement without vitamin A (Life Technologies, Gibco®, catalog number: 12587-010 )
  5. 100x N2 supplement (Life Technologies, Gibco®, catalog number: 17502-048 )
  6. Recombinant human Epidermal Growth Factor (Pepro Tech, catalog number: AF-100-15 )
  7. Recombinant human fibroblast growth factor-basic (Pepro Tech, catalog number: 100-18B )
  8. 50x Penicillin-streptomycin solution (Mediatech, Cellgro®, catalog number: 30-001-CI )
  9. Dispase (BD Bioscience, catalog number: 354235 )
  10. 1x Phosphate-Buffered Saline (Mediatech, Cellgro®, catalog number: 21-040-CV )
  11. MACS BSA stock solution (10% BSA) (Miltenyi Biotec, catalog number: 130-091-376 )
  12. 1x TrypLETM Express (Life Technologies, Gibco®, catalog number: 12605-010 )
  13. 0.4% Trypan Blue Solution (Life Technologies, Gibco®, catalog number: 15250-061 )
  14. Goat anti-canine IgG (H&L) f(ab’)2-fluorescein isothiocyanate (American Qualex, catalog number: F145FN )
  15. Neural Stem Cell (NSC) medium (see Recipes)
  16. Matrigel Coating solution (see Recipes)
  17. rh-EGF stock solution (see Recipes)
  18. rh-FGF basic stock solution (see Recipes)

Equipment

  1. 10 mm culture plates (Sigma-Aldrich, Corning® Costar®, catalog number: CLS430165 )
  2. Scalpel blade
  3. 100 μm filter (Corning Incorporated, catalog number: 352360 )
  4. BD FACS tubes (12 x75 mm) (BD Biosciences, Falcon®, catalog number: 352235 )
  5. 15 ml tubes (BD Biosciences, Falcon®, catalog number: 352096 )
  6. Culture dish (100 x 20 mm) (Sarstedt AG, catalog number: 83.1802 )
  7. 1.5 ml Eppendorf microcentrifuge tubes (Sigma-Aldrich, catalog number: T9661-500EA )
  8. Neubauer hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
  9. Incubator (5% CO2, 5% O2, 37 °C) (BioSpherix)
  10. Shaker table (Eppendorf, New Brunswick Excella® E25 incubator shaker)
  11. Centrifuge (Thermo Fisher Scientific, model: ST40R )
  12. Microcentrifuge (Thermo Fisher Scientific, model: MicroCL 17 )
  13. BD FACSCanto three-laser flow cytometer (BD)

Procedure

  1. Culture of tumor cells
    1. Coat the 10 mm culture plates with 3 ml of matrigel solution, gently rock to completely cover the plate and set aside for 30 min for gel polymerization.
    2. Mince canine tumor samples (2-5 cc depending on the size of the tumor) with a scalpel blade into 1 mm pieces, digest with 10 ml of TrypLE Express for 15 min at 37 °C on a shaker table, pass the suspension through a 100 μm filter, wash with 20 ml NSC medium centrifuge at 500 x g for 5 min, and resuspend with 1 ml of NSC medium.
    3. Add 1 ml of canine tumor cell suspension and 6 ml of NSC medium to the plate and place in a 37 °C humidified incubator with 5% O2 and 5% CO2.
    4. Supplement the NSC media plates with 10 μl each of rh-EGF stock solution and rh-FGF basic stock solution every Monday, Wednesday and Friday, and the media should be changed at least once a week or if the medium indicator changes to a orange-yellow color due to rapid cell proliferation.

  2. Harvest of tumor cells
    1. When the tumor cells have grown to 100% confluency, the cells should be harvested.
    2. Aspirate all of the remaining NSC medium, then add 3 ml of medium back to the plate.
    3. Add 500 μl of Dispase to the plate and gently rock to spread over the cells to solubilize the matrigel coating and detach the cells. Prepare 1 ml aliquots of the Dispase to prevent excessive freeze-thaw cycles.
    4. Incubate the plate at 37 °C for 20-30 min or until all cells are detached.
    5. Decant the tumor cell suspension into a 15-ml conical tube and add 1x PBS to a final volume of 10 ml.
    6. Centrifuge the cells at 500 x g for 5 min, decant supernatant, resuspend in 1x PBS (1 ml).
    7. The cells are counted by placing 10 μl of cell suspension into an Eppendorf microcentrifuge tube and adding 10 μl of Trypan Blue.
    8. Count cells using a Neubauer hemocytometer.

  3. Serum antibody: tumor cell binding experiment
    1. Place 105 tumor cells into a FACS tube and add PBS to a total volume of 200 μl (produce triplicate samples).
    2. Add 2 μl of serum (pre or post-treatment sera from the canine patient) into the cell suspension (1:100 dilution of serum to cells).
    3. Incubate at 4 °C for 30 min.
    4. After incubation, dilute cell suspension with 2 ml of FACS buffer, vortex, and centrifuge at 1,000 rpm for 5 min.
    5. Decant the supernatant, repeat the wash with FACS buffer and decant.
    6. Add 1 μl of the conjugated 2° antibody [goat anti-canine IgG (H&L) f(ab’)2-fluorescein isothiocyanate] and incubate at 4 °C for 30 min.
    7. After incubation, add 2 ml of FACS buffer, vortex, and centrifuge at 1,000 rpm for 5 min, decant, repeat the wash and decant.
    8. Resuspend the labeled cells in 200 μl FACS buffer and analyze using flow cytometer.

  4. Observations
    Use as controls tubes:
    1. 2° Ab only - tumor cells + conjugated 2° antibody (no serum)
    2. Unstained - tumor cells + serum (no 2° conjugated Ab)
    3. Normal dog serum - tumor cells + serum from normal dog + 2° conjugated Ab

Recipes

  1. Neural Stem Cell medium
    500 ml DMEM/F12
    100x 5 ml N2
    50x 10 ml B27
    5 ml Penicillin-streptomycin solution
  2. Matrigel Coating solution
    2.7 ml Neural Stem Cell medium
    0.3 ml BD MatrigelTM (0.3 ml aliquots in Eppendorf tubes stored at -20 °C)
  3. rh-EGF stock solution (20 μg/ml)
    20 μg recombinant human EGF
    1 ml 0.1% BSA in PBS stock solution
    Stored at -20 °C
  4. rh-FGF basic stock solution (20 μg/ml)
    20 μg recombinant human FGF basic
    1 ml 0.1% BSA stock solution
    Stored at -20 °C

Acknowledgments

This protocol was adapted from procedures in the following manuscript: Andersen et al. (2013). This work was supported by funding to B.M. Andersen from Torske Klubben Fellowship for Minnesota Residents, Medical Scientist Training Program Grant T32 GM008244, the Cancer Biology Training Grant T32 CA009138—36, and F30 CA167912; to M.A. Hunt from the American Brain Tumor Association Discovery Grant supported by the Anonymous Family Foundation; to J.R. Ohlfest from 1R21NS070955-01, R01 CA154345, R01 CA160782, the American Cancer Society grant RSG-09-189-01-LIB, Minnesota Medical Foundation, the Hedberg Family Foundation, and the Children's Cancer Research Fund.

References

  1. Andersen, B. M., Pluhar, G. E., Seiler, C. E., Goulart, M. R., SantaCruz, K. S., Schutten, M. M., Meints, J. P., O'Sullivan, M. G., Bentley, R. T., Packer, R. A., Thomovsky, S. A., Chen, A. V., Faissler, D., Chen, W., Hunt, M. A., Olin, M. R. and Ohlfest, J. R. (2013). Vaccination for invasive canine meningioma induces in situ production of antibodies capable of antibody-dependent cell-mediated cytotoxicity. Cancer Res 73(10): 2987-2997.

简介

基于疫苗的免疫疗法被用于治疗具有原发性脑肿瘤的狗。 疫苗由自体肿瘤细胞的裂解物组成,其刺激产生免疫应答的肿瘤特异性抗体,其能够诱导对同种异体以及自体的肿瘤细胞的抗体依赖性细胞介导的细胞毒性。 该方案将描述肿瘤细胞血清抗体结合测定以测量肿瘤反应性IgG抗体应答。 该测定的主要特征是,在接种疫苗之前和之后从犬患者收集的血清作为抗体的来源进行,并且使用犬脑肿瘤细胞作为靶细胞。

材料和试剂

  1. 肿瘤细胞
    注意:在大脑手术期间从宠物狗收获肿瘤样品。
  2. BD Matrigel (BD Bioscience,目录号:354230)
  3. 具有L-谷氨酰胺的DMEM/F12培养基(Life Technologies,Gibco ,目录号:11320-033)
  4. 50×不含维生素A的B-27补充物(Life Technologies,Gibco ,目录号:12587-010)
  5. 100x N2补充剂(Life Technologies,Gibco ,目录号:17502-048)
  6. 重组人表皮生长因子(Pepro Tech,目录号:AF-100-15)
  7. 重组人成纤维细胞生长因子碱性(Pepro Tech,目录号:100-18B)
  8. 50x青霉素 - 链霉素溶液(Mediatech,Cellgro ,目录号:30-001-CI)
  9. Dispase(BD Bioscience,目录号:354235)
  10. 1x磷酸盐缓冲盐水(Mediatech,Cellgro ,目录号:21-040-CV)
  11. MACS BSA储备溶液(10%BSA)(Miltenyi Biotec,目录号:130-091-376)
  12. 1x TrypLE TM Express(Life Technologies,Gibco ®,目录号:12605-010)
  13. 0.4%台盼蓝溶液(Life Technologies,Gibco ,目录号:15250-061)
  14. 山羊抗犬IgG(H& L)f(ab')2-异硫氰酸荧光素(American Qualex,目录号:F145FN)
  15. 神经干细胞(NSC)培养基(见配方)
  16. Matrigel涂层溶液(参见配方)
  17. rh-EGF储备溶液(见配方)
  18. rh-FGF碱性储备液(参见配方)

设备

  1. 10mm的培养板(Sigma-Aldrich,Corning公司,目录号:CLS430165)。
  2. 刮刀刀片
  3. 100μm滤器(Corning Incorporated,目录号:352360)
  4. BD FACS管(12×75mm)(BD Biosciences,Falcon ,目录号:352235)
  5. 15ml管(BD Biosciences,Falcon ,目录号:352096)
  6. 培养皿(100×20mm)(Sarstedt AG,目录号:83.1802)
  7. 1.5ml Eppendorf微量离心管(Sigma-Aldrich,目录号:T9661-500EA)
  8. Neubauer血细胞计数器(Sigma-Aldrich,目录号:Z359629)
  9. 培养箱(5%CO 2,5%O 2,37℃)(BioSpherix)
  10. 振动台(Eppendorf,New Brunswick Excella E25培养摇床)
  11. 离心机(Thermo Fisher Scientific,型号:ST40R)
  12. 微量离心机(Thermo Fisher Scientific,型号:MicroCL 17)
  13. BD FACSCanto三激光流式细胞仪(BD)

程序

  1. 肿瘤细胞的培养
    1. 轻轻地用3ml Matrigel溶液涂覆10mm培养板 岩石完全覆盖板,并放置30分钟的凝胶 聚合
    2. 剁碎犬肿瘤样品(2-5cc,取决于   肿瘤的大小)用手术刀切成1mm片,消化 用10ml TrypLE Express在37℃在摇床上15分钟,通过   悬浮液通过100μm过滤器,用20ml NSC培养基洗涤 在500×g离心5分钟,并用1ml NSC培养基重悬。
    3. 加入1毫升犬肿瘤细胞悬浮液和6毫升NSC培养基   并置于具有5%O 2和5%CO 2的37℃湿润培养箱中。
    4. 补充NSC培养基板每个rh-EGF股票10微升 溶液和rh-FGF碱性储液每周一,周三和 星期五,媒体应至少每周更换一次或如果 中等指示剂由于快速细胞而变为橙黄色 增殖。

  2. 收获肿瘤细胞
    1. 当肿瘤细胞生长至100%汇合时,应收获细胞
    2. 吸出所有剩余的NSC培养基,然后加3毫升培养基回到板
    3. 加入500微升Dispase的板,轻轻摇滚,以扩散 细胞以溶解基质胶涂层并分离细胞。 准备1   ml等分试样,以防止过度的冻融循环
    4. 孵育板在37℃下20-30分钟或直到所有细胞分离
    5. 将肿瘤细胞悬浮液倒入15-ml锥形管中,加入1×PBS至终体积为10ml
    6. 将细胞以500×g离心5分钟,倾析上清液,重悬于1×PBS(1ml)中。
    7. 通过将10μl细胞悬浮液置于中来计数细胞 Eppendorf微量离心管并加入10μl台盼蓝
    8. 使用Neubauer血细胞计数器计数细胞
  3. 血清抗体:肿瘤细胞结合实验
    1. 将10 5肿瘤细胞放入FACS管中,加入PBS至总体积为200μl(产生一式三份样品)。
    2. 加入2μl血清(来自犬的前或后处理血清 患者)进入细胞悬浮液(血清对细胞稀释1:100)。
    3. 在4℃孵育30分钟。
    4. 孵育后,用2ml FACS缓冲液稀释细胞悬浮液,涡旋,并以1,000rpm离心5分钟。
    5. 倾析上清液,用FACS缓冲液重复洗涤并倾析
    6. 加入1μl的缀合的2°抗体[山羊抗犬IgG(H& L)   f(ab')2-异硫氰酸荧光素]并在4℃下孵育30分钟。
    7. 孵育后,加入2ml FACS缓冲液,涡旋,并在37℃离心 1,000 rpm,5分钟,倾析,重复洗涤和倾析
    8. 将标记的细胞在200μlFACS缓冲液中重悬,并使用流式细胞仪进行分析

  4. 观察值
    用作对照管:
    1. 2°Ab - 肿瘤细胞+共轭2°抗体(无血清)
    2. 未染色 - 肿瘤细胞+血清(无2°缀合的Ab)
    3. 正常狗血清 - 肿瘤细胞+正常狗的血清+ 2°结合的抗体

食谱

  1. 神经干细胞培养基
    500 ml DMEM/F12
    100x 5 ml N2
    50x 10ml B27
    5ml青霉素 - 链霉素溶液
  2. Matrigel涂料溶液
    2.7 ml神经干细胞培养基
    将0.3ml BD Matrigel TM(在Eppendorf管中保存于-20℃下的0.3ml等分试样)
  3. rh-EGF储备溶液(20μg/ml) 20μg重组人EGF 1ml 0.1%BSA的PBS储液
    储存于-20°C
  4. rh-FGF碱性储备溶液(20μg/ml) 20μg重组人类FGF碱性 1ml 0.1%BSA储备液
    储存于-20°C

致谢

该协议改编自以下手稿中的程序:Andersen等人(2013)。这项工作得到了B.M.的资助。 Andersen来自Torske Klubben明尼苏达居民奖学金,医学科学家培训计划G32 T32 GM008244,癌症生物学培训授权T32 CA009138-36和F30 CA167912;来自美国脑肿瘤协会发现资助的匿名家庭基金会支持的M.A.Hunt;来自1R21NS070955-01,R01CA154345,R01CA160782,美国癌症协会授予RSG-09-189-01-LIB,明尼苏达医学基金会,Hedberg家族基金会和儿童癌症研究基金的J.R.Ohlfest。

参考文献

  1. Andersen,BM,Pluhar,GE,Seiler,CE,Goulart,MR,SantaCruz,KS,Schutten,MM,Meints,JP,O'Sullivan,MG,Bentley,RT,Packer,RA,Thomovsky,SA, Faissler,D.,Chen,W.,Hunt,MA,Olin,MR和Ohlfest,JR(2013)。 针对入侵性犬脑膜炎的疫苗接种可以原位产生能够抗体的抗体 - 依赖性细胞介导的细胞毒性。癌症研究73(10):2987-2997。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Andersen, B. M., Goulart, M. R., Olin, M. R. and Pluhar, G. E. (2014). Detection of Tumor Cell Surface-reactive Antibodies. Bio-protocol 4(9): e1114. DOI: 10.21769/BioProtoc.1114.
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