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Analysis of Cancer Stromal Reaction Using an O-ring Co-culture Assay

使用O形环共培养分析法分析癌症基质反应

Vivien Jane Coulson-Thomas Vivien Jane Coulson-Thomas

发布: 2017年02月20日第7卷第4期 DOI: 10.21769/BioProtoc.2131 浏览次数: 9546

评审: Guillermo GomezAndrea IntroiniJingli Cao

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Abstract

We have developed a 2D heterotypic co-culture technique between fibroblasts and cancer cells that enables the study of the stromal reaction. For such, stromal cells are seeded and cultured immediately around a tumour cell line, and the cells establish cell-cell contacts, as well as a gradient of soluble factors throughout the stromal cells, similar to that found in tissues. Thus, this system also enables the researcher to distinguish between events that are caused by direct cell-cell contact and secreted factors.

Keywords: Cancer (癌症) search Stromal reaction (基质反应) search Fibroblasts (成纤维细胞) search Extracellular matrix and proteoglycans (细胞外基质和蛋白多糖) search

Background

The growth and survival of a tumour within a tissue depends upon interactions with surrounding stromal cells, such as fibroblasts, inflammatory cells, endothelial cells and lymphatic cells. Research has shown that as tumours grow there is extensive cross-talk between the cancer cells and the surrounding fibroblasts. Moreover, the tumour cells may activate these fibroblasts into tumour-associated fibroblasts (TAFs). In some instances, these fibroblasts may restrict tumour growth (Coulson-Thomas et al., 2011 and 2013); however, in many cases these TAFs aid tumour cell growth and survival (Coulson-Thomas et al., 2010 and 2015). Therefore, in vitro cancer studies should also take into account the protective effects TAFs can have on cancer cells. Taking this into account, we developed a 2D heterotypic co-culture technique between fibroblasts and cancer cells that enables the study of TAFs and cancer cells in the same system.

Materials and Reagents

  1. 24-well clear flat bottom TC-treated Multiwell cell culture plate (Corning, Falcon®, catalog number: 353047 )
  2. Circular glass coverslips, FisherbrandTM cover glasses, diameter (metric) 12 mm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-545-80 )
  3. O-Rings, PYREX® cloning cylinder (10 x 10 mm) (Corning, catalog number: 3166-10 )
  4. Stromal cells, prostate fibroblasts (WPMY-1) (ATCC, catalog number: CRL-2854TM )
  5. Prostate cancer PC-3 cells (ATCC, catalog number: CRL-1435TM )
  6. Trypsin/EDTA 0.25% (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  7. Phosphate buffer saline (PBS) pH 7.4, without calcium, magnesium and phenol red
  8. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM)
  9. Primary antibodies
    1. Anti-collagen I (Calbiochem I-8H5, San Diego, CA and Sigma COL1, Sigma-Aldrich, catalog number: C2456 )
    2. Anti-collagen IV, against α1 type IV (Santa Cruz Biotechnology, catalog number: sc-29010 )
    3. Anti-collagen V (EMD Millipore, catalog number: AB763P and Abcam, catalog number: ab134800 )
    4. Rabbit anti-fibromodulin H-50 (Santa Cruz Biotechnology, catalog number: sc-33772 )
    5. Rabbit anti-biglycan H-150 (Santa Cruz Biotechnology, catalog number: sc-33788 )
    6. Mouse anti-fibronectin (BD, BD Transduction LaboratoriesTM, catalog number: 610077 )
    7. Goat anti-perlecan L-20 (Santa Cruz Biotechnology, catalog number: sc-27449 )
    8. Anti-versican H-56 (Santa Cruz Biotechnology, catalog number: sc-25831 )
    9. Mouse anti-smooth muscle α actin conjugated with Cy3 (Sigma-Aldrich, catalog number: clone 1A4 )
  10. Alexa Fluor® 488 or Alexa Fluor® 594 (Thermo Fisher Scientific, Molecular Probes/Invitrogen, Eugene, OR)
  11. Dulbecco’s modified Eagle medium (DMEM) culture medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  12. L-glutamine-penicillin-streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 10378016 )
  13. Paraformaldehyde (aqueous solution: 16%) (Electron Microscopy Sciences, catalog number: 15700 )
  14. Complete culture medium (see Recipes)
  15. 4% paraformaldehyde (see Recipes)

Equipment

  1. Ultra-Fine forceps with a straight tip (Sterilized) (Fine Science Tools, catalog number: 11399-80 )
  2. CO2 cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 51026280 )
  3. Table top centrifuge (Eppendorf, model: 5702RH )
  4. Automatic cell counter or hemocytometer
  5. Biological safety cabinets (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 51026639 )
    Note: The cell culture experiments must be carried out in sterile conditions.
  6. Inverted fluorescence microscope (Zeiss, model: Observer.Z1 with Apotome )

Software

  1. Prism-GraphPad software

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Coulson-Thomas, V. J. (2017). Analysis of Cancer Stromal Reaction Using an O-ring Co-culture Assay. Bio-protocol 7(4): e2131. DOI: 10.21769/BioProtoc.2131.

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分类

癌症生物学 > 侵袭和转移 > 肿瘤微环境

细胞生物学 > 细胞分离和培养 > 细胞分化

细胞生物学 > 细胞分离和培养 > 共培养

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