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Three-dimensional Invasion Assay
三维侵袭实验   

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Abstract

The invasive ability of cancer cells is a crucial function for cancer metastasis and the surrounding microenvironment of cancer cells in living tissues is three-dimension (3D). Therefore, to establish an in vitro invasion assay in a 3D system to predict cancer invasive ability is valuable in the research for cancer metastasis. Here, we describe a 3D invasion assay for observing the morphology and comparing the invasive ability of cancer cells in artificial 3D environments (Yang et al., 2012). Collagen I gels are used to cover on the top of cancer cells attached on coverslip glass dish and medium containing FBS is added as a chemoattractant. After incubation for a suitable time, the cells are fixed and stained. The invasion index can be calculated and the morphology can be imaged with a laser confocal microscope.

Keywords: 3D invasion(3d入侵), Collagen gel(胶原凝胶), Invasion index(侵袭指数)

Materials and Reagents

  1. Cell lines: OECM-1(Huang et al., 2004) and FaDu (ATCC® HTB-43 TM)
  2. 0.1% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 15400 )
  3. 0.1 mg/ml poly-L lysine (Sigma-Aldrich, catalog number: P9404-25MG )
  4. PBS
  5. FBS (Thermo Fisher Scientific, catalog number: SH30071.03 )
  6. PureCor® bovine collagen solution (Advance Biomatrix Inc., catalog number: 5005-B )
  7. 1 M NaOH solution
  8. 5x RPMI medium
  9. 3% Paraformaldehyde (Sigma-Aldrich, catalog number: P6148-500G )
  10. 0.5% Triton X100 (Bionovas, catalog number: 56-81-5 )
  11. Alexa Fluor® 488 Phalloidin (Life Technologies, catalog number: A12379 )
  12. DAPI (Sigma-Aldrich, catalog number: D8417 )
  13. 1% BSA in PBS
  14. 1.8 mg/ml collagen I mix solution (see Recipes)

Equipment

  1. Lab-Tek® chambered #1.0 coverglass system (NUNC, catalog number: 155383 )
  2. Laser confocal microscope with 60x oil lens (Olympus, model: FV1000 )
  3. CO2 incubator

Software

  1. Olympus FV10-ASW 1.7 software

Procedure

     Day 1

  1. Treat Lab-Tek® chambered #1.0 coverglass system with 300 μl of 0.1 mg/ml poly-L lysine solution for one hour at 37 °C.
  2. Aspirate the poly-L lysine solution and wash one time with PBS.
  3. Trypsinize cells and 2 x 105 cells in 500 μl medium were plated on coverglass system for attachment.
  4. After attachment time for 3 to 6 h, prepare the appropriate volume of collagen I mix solution (final concentration 1.8 mg/ml) on ice then carefully remove the medium from coverglass system (avoid to wash cells again) and add 500 μl of collagen I mix solution to coverglass system.
  5. Cells were incubated at 37 °C, 5% CO2 for 2 h.
  6. Overlay with 400 μl of medium containing with 15% FBS on collagen gels.
  7. Incubate at 37 °C, 5% CO2 for 24 to 48 h.


     Day 2 or 3

  1. Carefully aspirate medium from wells and rinse wells including collagen gel invaded by cells with PBS once.
  2. Carefully pour 400 μl of 3% paraformaldehyde in PBS for 40 min at RT to fix cells.
  3. Carefully rinse two times with 400 μl PBS.
  4. Permeabilization in 400 μl of 0.5% Triton X-100 in PBS for 40 min at RT.
  5. Carefully rinse two times with 400 μl PBS.
  6. Incubate cells with 400 μl of 1% BSA in PBS for 40 min at RT.
  7. Stain cells with 500 μl of Alexa Fluor® 488 Phalloidin diluted to 1 units/ml in PBS for 90 min at RT.
  8. Carefully rinse two times with PBS.
  9. Stain cells with 500 μl of 2 μg/ml DAPI in PBS for 30 min.
  10. Wash three times with 400 μl PBS and aspirate all PBS.
  11. Samples can be stored at 4 °C for 2 weeks or ready to be imaged by a laser confocal microscope. Imaging and quantification.
  12. Use an Olympus FV1000 laser confocal microscope with 60x oil lens to capture images. The volume of observation is xyz = 210 x 210 x 50 μm3.
  13. Confocal Z slices are collected each well at 50 μm from the bottom of the well and z interval is set to 1 μm.
  14. Images of sequential Z sections were obtained and reconstructed by Olympus FV10-ASW 1.7 software.


    Figure 1. Representative image of FaDu overexpressing Twist1 cells that invaded into collagen after 24 h (please refer to Reference 1 for the detail)

  15. The invasion index is quantified as the number of cells existing at the distance from the bottom of slide between 30 to 50 μm divided by the total number of cells.
    Note: The cells that are partially fallen into the range of 30-50 μm can also be counted.
  16. The data are presented as the percentage of the invasion index of the control sample and representative vertical sections.

Recipes

  1. 1.8 mg/ml Collagen I mix solution
    1.7 ml PureCor® bovine collagen solution (3 mg/ml)
    0.6 ml 5x RPMI
    18 μl 1 M NaOH
    Add dH2O to 3 ml
    All buffers must be on ice before polymerization in the tissue culture incubator. This mix solution must be prepared freshly to use.

Acknowledgments

This protocol was carried out as described in Sanz-Moreno et al. (2008) with minor modifications. The establishment of this protocol was funded by the National Health Research Institutes (NHRI-EX100-10037BI to M-H.Y.; NHRI-EX100-9931BI to K-J.W.); the National Science Council (NSC 99-2314-B-010-007-MY3 and 100-2321-B-010-015 to M-H.Y.; NSC 100-2321-B-039-002 to M-C.H.); Taipei Veterans General Hospital (VGH 100-C-088, 101-C-005 to M-H.Y.); Veterans General Hospitals University System of Taiwan Joint Research Program (VGHUST101-G7-4-1 to M-H.Y.); a grant from the Ministry of Education, Aim for the Top University Plan (to M-H.Y.); a grant from the Department of Health, Center of Excellence for Cancer Research (DOH100-TD-C-111-007 to M-H.Y; DOH101-TD-C-111-005 to M-C.H.); and the Sister Institution Fund of China Medical University and Hospital and MD Anderson Cancer Center.

References

  1. Huang, G. C., Liu, S. Y., Lin, M. H., Kuo, Y. Y. and Liu, Y. C. (2004). The synergistic cytotoxicity of cisplatin and taxol in killing oral squamous cell carcinoma. Jpn J Clin Oncol 34(9): 499-504.
  2. Yang, W. H., Lan, H. Y., Huang, C. H., Tai, S. K., Tzeng, C. H., Kao, S. Y., Wu, K. J., Hung, M. C. and Yang, M. H. (2012). RAC1 activation mediates Twist1-induced cancer cell migration. Nat Cell Biol 14(4): 366-374.

简介

癌细胞的侵袭能力是癌症转移的关键功能,活组织中癌细胞的周围微环境是三维的(3D)。 因此,在3D系统中建立体外侵袭测定以预测癌症侵袭能力在癌症转移的研究中是有价值的。 在这里,我们描述了用于观察形态学并比较人造3D环境中癌细胞的侵袭能力的3D侵袭测定(Yang等人,2012)。 胶原I凝胶用于覆盖附着在盖玻片玻璃皿上的癌细胞的顶部,并且添加含有FBS的培养基作为化学引诱物。 在孵育合适的时间后,将细胞固定并染色。 可以计算入侵指数,并且可以用激光共聚焦显微镜成像形态

关键字:3d入侵, 胶原凝胶, 侵袭指数

材料和试剂

  1. 细胞系:OECM-1(Huang等人,2004)和FaDu(ATCC HTB-43 TM
  2. 0.1%胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:15400)
  3. 0.1mg/ml聚-L赖氨酸(Sigma-Aldrich,目录号:P9404-25MG)
  4. PBS
  5. FBS(Thermo Fisher Scientific,目录号:SH30071.03)
  6. 牛胶原溶液(Advance Biomatrix Inc.,目录号:5005-B)
  7. 1M NaOH溶液
  8. 5x RPMI培养基
  9. 3%多聚甲醛(Sigma-Aldrich,目录号:P6148-500G)
  10. 0.5%Triton X100(Bionovas,目录号:56-81-5)
  11. Alexa Fluor 488鬼笔环肽(Life Technologies,目录号:A12379)
  12. DAPI(Sigma-Aldrich,目录号:D8417)
  13. 1%BSA的PBS溶液中
  14. 1.8 mg/ml胶原I混合溶液(见配方)

设备

  1. Lab-Tek 腔室#1.0盖玻片系统(NUNC,目录号:155383)
  2. 具有60x油透镜(Olympus,型号:FV1000)的激光共聚焦显微镜
  3. CO <2>孵化器

软件

  1. Olympus FV10-ASW 1.7软件

程序

     第1天

  1. 在37℃下用300μl的0.1mg/ml聚-L赖氨酸溶液处理Lab-Tek 腔室#1.0盖玻片系统一小时。
  2. 吸出聚-L赖氨酸溶液,用PBS洗一次。
  3. 将在500μl培养基中的胰蛋白酶化细胞和2×10 5个细胞接种在盖玻片系统上用于附着。
  4. 在附着时间3至6小时后,在冰上准备适当体积的胶原I混合溶液(终浓度1.8毫克/毫升),然后小心地从盖玻片系统中除去培养基(避免再次洗涤细胞),并加入500微升胶原蛋白I 混合溶液到盖玻片系统
  5. 将细胞在37℃,5%CO 2下孵育2小时
  6. 用400μl含有15%FBS的培养基在胶原凝胶上覆盖
  7. 在37℃,5%CO 2下孵育24至48小时


     第2或3天

  1. 小心地从孔中吸出培养基并冲洗孔,包括用PBS浸润的胶原蛋白凝胶一次
  2. 小心地倒入400微升3%多聚甲醛的PBS溶液中40分钟,以固定细胞
  3. 用400μlPBS小心冲洗两次。
  4. 在400μl0.5%Triton X-100的PBS中在室温下透化40分钟
  5. 用400μlPBS小心冲洗两次。
  6. 用400μl1%BSA的PBS溶液在室温下孵育细胞40分钟
  7. 用500μl在PBS中稀释至1单位/ml的Alexa Fluor?488鬼笔环素在室温下染色细胞90分钟。
  8. 用PBS小心冲洗两次。
  9. 用500μl2μg/ml DAPI在PBS中染色细胞30分钟
  10. 用400μlPBS洗涤三次,并吸出所有PBS
  11. 样品可以在4℃下储存2周或者准备通过激光共聚焦显微镜成像。 成像和定量。
  12. 使用Olympus FV1000激光共聚焦显微镜与60x油镜头捕获图像。 观察的体积为xyz = 210×210×50μm 3
  13. 共聚焦Z切片被收集每个井从井底部50μm,z间隔设置为1微米
  14. 获得顺序Z切片的图像,并通过Olympus FV10-ASW 1.7软件重建。


    图1.24小时后侵入胶原蛋白的FaDu过表达Twist1细胞的代表性图像(详情请参阅参考文献1)

  15. 入侵指数被定量为在30至50μm之间的从载玻片的底部的距离处除以总细胞数所存在的细胞数。
    注意:部分落入30-50微米范围内的细胞也可以计数。
  16. 数据表示为对照样品和代表性垂直切片的侵入指数的百分比。

食谱

  1. 1.8mg/ml胶原I混合溶液
    1.7ml PureCor 牛胶原溶液(3mg/ml) 0.6ml 5x RPMI
    18μl1M NaOH
    将dH sub 2 O加到3ml
    中 所有缓冲液必须在冰上,然后在组织培养箱中聚合。 此混合溶液必须新鲜配制使用。

致谢

该协议如Sanz-Moreno等人(2008)中所述进行,进行了微小的修改。该方案的建立由国家健康研究所(NHRI-EX100-10037BI至M-H.Y .; NHRI-EX100-9931BI至K-J.W。)资助;国家科学委员会(NSC 99-2314-B-010-007-MY3和100-2321-B-010-015至M-H.Y .; NSC 100-2321-B-039-002至M-C.H。台北退伍军人综合医院(VGH 100-C-088,101-C-005至M-H.Y。);退伍军人综合医院大学台湾系统联合研究计划(VGHUST101-G7-4-1至M-H.Y。);教育部赠款,顶尖大学计划(至M-H.Y。);卫生部,癌症研究卓越中心(DOH100-TD-C-111-007至M-H.Y; DOH101-TD-C-111-005至M-C.H。)的赠款;中国医科大学和医院的姐妹机构基金和MD安德森癌症中心。

参考文献

  1. Huang,G.C.,Liu,S.Y.,Lin,M.H.,Kuo,Y.Y.and Liu,Y.C。(2004)。 顺铂和紫杉醇在杀死口腔鳞状细胞癌中的协同细胞毒性 Jpn J Clin Oncol 34(9):499-504。
  2. Yang,W. H.,Lan,H. Y.,Huang,C. H.,Tai,S. K.,Tzeng,C. H.,Kao,S. Y.,Wu,K. J.,Hung,M. C. and Yang,M. H.(2012)。 RAC1激活介导Twist1诱导的癌细胞迁移 Nat Cell Biol 14(4):366-374。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yang, W. and Yang, M. (2013). Three-dimensional Invasion Assay. Bio-protocol 3(17): e885. DOI: 10.21769/BioProtoc.885.
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