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Three Dimensional Spheroid Co-culture Invasion Assay
三维球状体共培养侵袭研究   

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Abstract

The assay was developed to investigate the impact of stromal cells of different types (in our case breast cancer associated fibroblasts stably manipulated to modify expression of genes of interest) on the invasive capacity of epithelial cancer cells (in our case breast cancer cell lines) (Verghese et al., 2013). Typical two dimensional invasion assays do necessarily account for the presence of extracellular matrix that is present around the stromal and tumour cells in vivo and therefore cellular behaviour within these cultures may be non-physiological. This spheroid assay was developed to attempt to replicate more closely the environment that is present around breast cancer stromal and tumour cells in actual tumours (Verghese et al., 2013). Extra cellular matrix composed of both collagen IV and collagen I is included and fibroblasts and epithelial cells are given the opportunity to develop “physiological” interactions (Verghese et al., 2013; Hooper et al., 2006). The method was developed from Nowicki et al. (2008), and we have published data using it in Verghese et al. (2013).

Materials and Reagents

  1. Epithelial cells and fibroblasts [see Verghese et al. (2013) for the type we have used, although many others may be applicable]
  2. Growth factor reduced matrigel (BD, catalogue number: 356231 )
  3. Collagen-1 (BD, catalog number: 354236 )
  4. Fetal calf serum (FCS) (Sigma-Aldrich, catalog number: F7524 )
  5. DMEM (Life Technologies, InvitrogenTM, catalog number: 31966 )
  6. MEM powder (Sigma-Aldrich, catalog number: M0268 )
  7. 1 M N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) (pH 7.5)
  8. NaHCO3
  9. Rabbit anti-cytokeratin (Abcam, catalog number: Ab9377 )
  10. Zymed antibody diluent (Life Technologies, catalog number: 003218 )
  11. DPX (Sigma-Aldrich, catalog number: 317616 ) (a mounting agent)
  12. Mayer’s haematoxylin (any supplier)
  13. Scott’s tap water substitute (any supplier)
  14. Eosin (any supplier)
  15. Formalin (any supplier)
  16. Ethanol (any supplier)
  17. Xylene (any supplier)
  18. Collagen-1/Matrigel mix (see Recipes)
  19. Collagen medium (see Recipes)

Equipment

  1. Tissue culture incubator (37 °C, humidified air/5% CO2) (Panasonic Corporation, Sanyo)
  2. 35 mm petri dish (ibidi GmbH, catalog number: 81158 )
  3. E1000 Eclipse microscope (Nikon Corporation)
  4. Superfrost Plus slides and cover slips (Menzel Glaser)
  5. Access retrieval unit (The Menarini Group) (a machine performing automated antigen retrieval giving enhanced reproducibility)
  6. Automated stainer (IntelliPATH) (a machine performing automated immunohistochemistry protocols giving enhanced reproducibility)

Software

  1. Photoshop (ADOBE)

Procedure

  1. Spheroid formation
    1. Equal numbers of epithelial cells (MCF-7 cells) and fibroblasts that are stably manipulated were suspended in DMEM medium/10% FCS at a final concentration of 1 x 106 /ml. 25 µl of this was placed in the centre of a 35 mm petri dish and inverted.
    2. 5 ml of DMEM with 10% fetal calf serum was added to the lid of the petri dish to prevent drying.
    3. This was incubated at 37 °C (humidified air/5% CO2) for 24 h to allow formation of spheroids.
    4. The next day 300 µl of a collagen-1/Matrigel mix (maintained on ice) was added to each well of a 24 well plate and incubated for 1 h for the gel to solidify.
    5. Cells that had formed tight spheroids (defined as those that could easily be ”picked” in their entirety using a pipette) were harvested using a 200 µl pipette and placed in the centre of the well containing the gel.
      Note: Modifications to cells that reduce the strength of their cell-cell interactions may impact negatively on this process.
    6. An additional 300 µl of collagen-1/Matrigel mix was added on top of the spheroid and incubated for 1 h at 37 °C.
    7. Finally 300 µl of DMEM was added to the well. This was then incubated for 48 to 96 h (37 °C; humidified air/5% CO2).

  2. Quantifying invasion
    1. Phase-contrast images were obtained at 24, 48 and 96 h using an E1000 Eclipse microscope at a predefined magnification.
    2. The invasive area of cells outgrowing from the spheroids was quantified relative to the area of the central spheroidal itself. Quantification of the area was performed using Photoshop.
    3. The identity of invasive cells (epithelial or fibroblast) was assessed using immunohistochemistry.
    4. For representative images, illustrative examples of quantification, and assessment of variation between individual assays, please see Verghese et al. (2013).

  3. Assessing expression/cell identity within spheroids
    1. The collagen gels containing the spheroids were removed from the wells and fixed in 4% formalin overnight.
    2. They were transferred to 70% ethanol and subsequently processed for paraffin embedding.
    3. Multiple 5 µm sections of the collagen gel spheroids were prepared on to Superfrost Plus slides.
    4. Every 5th section from each spheroid was dewaxed in xylene and rehydrated with graded ethanol.
    5. They were then stained in Mayer’s haematoxylin, Scott’s tap water substitute, and eosin.
    6. Following this, sections were dehydrated in ethanol and xylene, and mounted in DPX. They were examined to identify the level at which the spheroids has the greatest diameter.
    7. The sections before and after this level were used to perform immunohistochemistry to examine spheroid histology and expression within cell types of interest.
      1. We used rabbit anti-cytokeratin, a broad-spectrum anti-cytokeratin antibody, to label specifically the epithelial cells.
      2. The antibody was diluted at 1:100 in Zymed antibody diluent.
      3. Antigen retrieval was performed using Access Revelation Solution in the Access retrieval unit and staining using an automated stainer (IntelliPATH running a standard X-Cell plus protocol). (Other antigen retrieval protocols are also suitable.)
      4. Slides were counterstained in haematoxylin, mounted in DPX and images taken using the E1000 Eclipse microscope.
      5. See Verghese et al. (2013) for illustrative examples.

Recipes

  1. Collagen-1/Matrigel mix
    The following components were maintained on ice and mixed for a final Collagen-1 concentration of approximately 4.6 mg/ml and a final Matrigel concentration of approximately 2.2 mg. The final concentrations of collagen and matrigel were based on previous work (Gaggioli et al., 2007). During the preparation of the mixture one should ensure all of the material (particularly collagen and matrigel) is pipetted successfully, rather than retained within the pipette tip. The final mix should be maintained on ice. We prepared this mixture freshly for every experiment and attempt to use only single batches of reagents since batch-to-batch variation can be an issue.
    200 µl Matrigel
    400 µl Collagen-1
    80 µl collagen medium
    100 µl fetal calf serum (FCS)
    220 µl DMEM
    Up to 1,000 µl with water
  2. Collagen medium
    2.5 g MEM powder (5x)
    5 ml 1 M HEPES (pH 7.5)
    1 g NaHCO3
    Up to 50 ml with water
    Mixed and then sterilised by passing it through a 0.2 µm filter

Acknowledgments

We thank Steve Hooper and Eric Sahai (CRUK London Research Institute) for help in the development of this method. The work was supported by the MRC.

References

  1. Gaggioli, C., Hooper, S., Hidalgo-Carcedo, C., Grosse, R., Marshall, J. F., Harrington, K. and Sahai, E. (2007). Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol 9(12): 1392-1400.
  2. Hooper, S., Marshall, J. F. and Sahai, E. (2006). Tumor cell migration in three dimensions. Method Enzymol 406: 625-643.
  3. Nowicki, M. O., Dmitrieva, N., Stein, A. M., Cutter, J. L., Godlewski, J., Saeki, Y., Nita, M., Berens, M. E., Sander, L. M., Newton, H. B., Chiocca, E. A. and Lawler, S. (2008). Lithium inhibits invasion of glioma cells; possible involvement of glycogen synthase kinase-3. Neuro Oncol 10(5): 690-699.
  4. Verghese, E. T., Drury, R., Green, C. A., Holliday, D. L., Lu, X., Nash, C., Speirs, V., Thorne, J. L., Thygesen, H. H., Zougman, A., Hull, M. A., Hanby, A. M. and Hughes, T. A. (2013). MiR-26b is down-regulated in carcinoma-associated fibroblasts from ER-positive breast cancers leading to enhanced cell migration and invasion. J Pathol 231(3): 388-399.

简介

开发该测定以研究不同类型(在我们的情况下乳腺癌相关的成纤维细胞稳定操纵以修饰目的基因的表达)的基质细胞对上皮癌细胞(在我们的乳腺癌细胞系)的侵袭能力的影响( Verghese ,,2013)。典型的二维侵袭测定确实需要考虑在体内基质和肿瘤细胞周围存在的细胞外基质的存在,因此这些培养物中的细胞行为可能是非生理的。开发这种球状体测定以尝试更密切地复制存在于实际肿瘤中的乳腺癌基质和肿瘤细胞周围的环境(Verghese等人,2013)。包括由胶原IV和胶原I组成的细胞外基质,并且成纤维细胞和上皮细胞被给予发展"生理"相互作用的机会(Verghese等人,2013; Hooper等人, ,2006)。该方法由Nowicki等人(2008)开发,并且在Verghese等人(2013)中使用它来公开数据。

材料和试剂

  1. 上皮细胞和成纤维细胞[参见Verghese et al。 (2013),我们使用的类型,但许多其他可能适用]
  2. 生长因子减少基质胶(BD,目录号:356231)
  3. 胶原蛋白-1(BD,目录号:354236)
  4. 胎牛血清(FCS)(Sigma-Aldrich,目录号:F7524)
  5. DMEM(Life Technologies,Invitrogen TM ,目录号:31966)
  6. MEM粉末(Sigma-Aldrich,目录号:M0268)
  7. 1 M N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)(HEPES)(pH7.5)
  8. NaHCO 3
  9. 兔抗细胞角蛋白(Abcam,目录号:Ab9377)
  10. Zymed抗体稀释液(Life Technologies,目录号:003218)
  11. DPX(Sigma-Aldrich,目录号:317616)(固定剂)
  12. Mayer的苏木精(任何供应商)
  13. 斯科特的自来水替代品(任何供应商)
  14. Eosin(任何供应商)
  15. 福尔马林(任何供应商)
  16. 乙醇(任何供应商)
  17. 二甲苯(任何供应商)
  18. 胶原-1/Matrigel混合(见配方)
  19. 胶原培养基(见配方)

设备

  1. 组织培养孵育器(37℃,潮湿空气/5%CO 2)(Panasonic Corporation,Sanyo)
  2. 35mm培养皿(ibidi GmbH,目录号:81158)
  3. E1000 Eclipse显微镜(尼康公司)
  4. Superfrost Plus幻灯片和盖玻片(Menzel Glaser)
  5. 访问检索单元(Menarini集团)(执行自动化抗原检索的机器,具有增强的再现性)
  6. 自动染色机(IntelliPATH)(执行自动化免疫组织化学方案的机器,提供更高的再现性)

软件

  1. Photoshop(ADOBE)

程序

  1. 球体形成
    1. 等量的上皮细胞(MCF-7细胞)和成纤维细胞   稳定操作的细胞悬浮在DMEM培养基/10%FCS中 浓度为1×10 6个/ml。 将25μl放置在a的中心   35毫米培养皿,倒置。
    2. 将5ml具有10%胎牛血清的DMEM加入培养皿的盖子中以防止干燥。
    3. 将其在37℃(潮湿空气/5%CO 2)下温育24小时以允许球体形成。
    4. 第二天将300μl胶原-1/Matrigel混合物(保持在冰上) 加入24孔板的每个孔中并孵育1小时   凝胶固化。
    5. 已形成紧密球体的细胞(定义   作为那些可以很容易地"挑选"整体使用 移液管),使用200μl移液管并置于中心 的含有凝胶的孔。
      注意:修改细胞会降低细胞与细胞间的相互作用,可能会对此过程产生负面影响。
    6. 在球体的顶部再加入300μl胶原-1/Matrigel混合物,并在37℃下孵育1小时。
    7. 最后,将300μlDMEM加入到孔中。 然后将其孵育48至96小时(37℃;加湿空气/5%CO 2)。

  2. 量化入侵
    1. 使用E1000 Eclipse显微镜以预定的放大倍数在24,48和96小时获得相差图像
    2. 细胞从球状体生长的侵入区域是 相对于中心球状体本身的面积定量。 使用Photoshop进行面积的定量。
    3. 使用免疫组织化学评估侵袭性细胞(上皮或成纤维细胞)的身份
    4. 对于代表性图像,量化的说明性示例, 和评估各个测定之间的差异,请参阅 Verghese (2013)。

  3. 评估球体内的表达/细胞身份
    1. 从孔中除去含有球状体的胶原凝胶,并在4%福尔马林中固定过夜。
    2. 将它们转移至70%乙醇中,随后加工用于石蜡包埋。
    3. 将多个5μm的胶原凝胶球体切片制备到Superfrost Plus载玻片上。
    4. 每个球体的每5个切片在二甲苯中脱蜡并用分级乙醇再水合。
    5. 然后他们在梅尔的苏木精,斯科特的自来水替代品和曙红中染色。
    6. 此后,将切片在乙醇和二甲苯中脱水, 安装在DPX。 他们被检查以确定的水平 球体具有最大的直径。
    7. 前面和后面的部分 之后该水平用于进行免疫组织化学检查 球状体组织学和感兴趣的细胞类型内的表达。
      1. 我们使用兔抗细胞角蛋白,广谱抗细胞角蛋白抗体,以特异性标记上皮细胞。
      2. 将抗体在Zymed抗体稀释液中以1:100稀释。
      3. 使用Access Revelation Solution in进行抗原检索 Access检索单元和染色使用自动染色机 (运行标准X-Cell plus方案的IntelliPATH)。 (其他抗原 检索协议也是适合的。)
      4. 将载玻片在苏木精中复染色,安装在DPX中,使用E1000 Eclipse显微镜拍摄图像。
      5. 有关说明性示例,请参见Verghese (2013)。

食谱

  1. 胶原-1/Matrigel混合物
    将以下组分保持在冰上并混合,最终的胶原-1浓度为约4.6mg/ml,最终的Matrigel浓度为约2.2mg。胶原和基质胶的最终浓度基于以前的工作(Gaggioli等人,2007)。在混合物的制备期间,应当确保所有材料(特别是胶原和基质胶)被成功地移液,而不是保留在移液管尖端内。最终混合物应保持在冰上。我们为每个实验新鲜准备了该混合物,并试图仅使用单批次的试剂,因为批次间的变化可能是一个问题。
    200μlMatrigel
    400μl胶原蛋白-1
    80μl胶原培养基
    100μl胎牛血清(FCS)
    220微升DMEM
    用水加至1000μl
  2. 胶原培养基
    2.5克MEM粉末(5x)
    5ml 1 M HEPES(pH 7.5)
    1g NaHCO 3 3/h 用水冲洗至50 ml
    混合,然后通过0.2μm过滤器
    灭菌

致谢

我们感谢Steve Hooper和Eric Sahai(CRUK伦敦研究所)帮助开发这种方法。 这项工作得到了MRC的支持。

参考文献

  1. Gaggioli,C.,Hooper,S.,Hidalgo-Carcedo,C.,Grosse,R.,Marshall,J.F.,Harrington,K.and Sahai,E。 成纤维细胞引起的癌细胞的集体侵袭,对于前导细胞和随后的细胞中的RhoGTP酶具有不同的作用。 a> Nat Cell Biol 9(12):1392-1400。
  2. Hooper,S.,Marshall,J.F。和Sahai,E。(2006)。 三维肿瘤细胞迁移 方法酶 406 : 625-643。
  3. 本文的研究结果表明,该方法可以有效地抑制细胞凋亡,增加细胞凋亡,增加细胞凋亡, S.(2008)。 锂抑制胶质瘤细胞的侵袭; 可能涉及糖原合酶激酶-3。 Neuro Oncol 10(5):690-699。
  4. Verghese,ET,Drury,R.,Green,CA,Holliday,DL,Lu,X.,Nash,C.,Speirs,V.,Thorne,JL,Thygesen,HH,Zougman,A.,Hull,MA,Hanby ,AM和Hughes,TA(2013)。 MiR-26b在来自ER阳性乳腺癌的癌相关成纤维细胞中下调,导致增强 细胞迁移和侵袭。 231(3):388-399。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Verghese, E. T. and Hughes, T. A. (2015). Three Dimensional Spheroid Co-culture Invasion Assay. Bio-protocol 5(1): e1373. DOI: 10.21769/BioProtoc.1373.
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