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

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Experimental Cell Research
Nov 2010



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), Stromal reaction (基质反应), Fibroblasts (成纤维细胞), Extracellular matrix and proteoglycans (细胞外基质和蛋白多糖)


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)


  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 )


  1. Prism-GraphPad software


  1. Preparing the O-ring co-culture system
    1. The fibroblasts and prostate cancer cells should be maintained as separate cell lines, according to the supplier’s guidelines.
      Note: We maintained PC3 cells and fibroblasts in DMEM complete medium. The cells were passaged at 80-90% confluence and centrifuged at 300 x g.
    2. For the heterotypic co-culture system the fibroblasts and prostate cancer cells are removed from the culture dishes with trypsin/EDTA and centrifuged.
    3. The cell pellets are suspended in complete culture medium.
    4. A sterile O-ring is placed on top of glass coverslips in 24-well polystyrene culture dishes.
      Note: No silicone should be used to secure the O-ring to the coverslip since it would after affect the migration of the cells over the cell-free area. The O-ring simply sits on the cover slip; therefore care should be taken while transporting the microplate into the incubator.
    5. Fibroblasts (1.5 x 104 in 350 μl) are placed around the O-ring and prostate cancer cells (0.5 x 104 in 100 μl) are placed inside the O-ring, as represented in Figure 1.
      Note: Steps A1 to A7 must be carried out in a biosafety cabinet to ensure culture sterility and operator’s safety.

      Figure 1. O-ring co-culture technique. Fibroblasts are seeded around the O-ring and cancer cells are seeded inside the O-ring.

    6. The cells are maintained in culture with the O-rings for 24 h at 37 °C and 5% CO2.
    7. The O-rings are then removed with a forceps and the cells are washed twice with complete medium to remove any cells that have not adhered, and then left in culture for a further 48 h at 37 °C and 5% CO2.
      Note: Upon the removal of the O-ring the investigator can ascertain the O-ring area is cell-free, which is an indication that there was no cross-contamination between cell lines. Over the next 24 h the two cell lines readily migrate into the cell-free area forming a zone containing both cell lines in direct contact.
    8. For immunocytochemistry the cells are fixed in 4% paraformaldehyde prepared in PBS after 24 or 48 h.
      Note: The investigators should monitor their cells under a microscope in order to determine the optimal time for fixing their specific cell types. We determined 24 h was optimal for most cells types.

  2. Immunofluorescence staining of the ECM molecules
    1. The cells are washed with PBS and incubated in blocking solution (5% FBS) for 1 h at room temperature.
    2. The cells are then incubated with primary antibodies overnight at 4 °C. The primary antibodies used in our published work were: anti-collagen I (Calbiochem I-8H5 and Sigma COL1), anti-collagen IV (against α1 type IV) and two distinct anti-collagen V (Chemicon AB763P and Abcam Clone V-3 C9), rabbit anti-fibromodulin (Santa Cruz H-50), rabbit anti-biglycan (Santa Cruz H-150), mouse anti-fibronectin, goat anti-perlecan (Santa Cruz L-20), anti-versican (Santa Cruz H-56) and finally, mouse anti-smooth muscle α actin conjugated with Cy3 (clone 1A4).
    3. The coverslips are then washed three times for 15 min and incubated for 1 h at room temperature with appropriate fluorescent secondary antibodies conjugated to Alexa Fluor® 488 or Alexa Fluor® 594.
    4. The coverslips are then washed with PBS three times, 15 min for each wash and mounted on glass slides in Fluoromount G (2:1 in PBS).
    5. Negative controls must be done for immunostaining experiments. These were done in our set of experiments by omitting the primary antibody. Coverslips are examined using scanning confocal inverted microscope (Zeiss LSM510) and fluorescence quantified using a LSM image browser.

Data analysis

This method has previously been used to analyse the prostate and colorectal stromal reaction (Coulson-Thomas et al., 2010 and 2011). In these studies the extracellular matrix (ECM) production was analysed in the fibroblast only region, cancer cell only region and interface which contained both the fibroblasts and cancer cells. The interface containing both fibroblasts and cancer cells can be viewed in Figure 2. The ECM was analysed by immunocytochemistry using a range of antibodies against different ECM components. We repeated the experiments at least three times in triplicates which yielded statistically significant results, with the Student’s t-test using the Prism-GraphPad software.

Figure 2. Image of the O-ring area containing both the prostate cancer cells and fibroblasts. Fibroblasts are evidenced by vimentin staining in green and the prostate cancer cells are evidenced by tubulin staining in red. Scale bar = 20 μm.


  1. This protocol was developed to study the inter-play between stromal cells and tumour cells; however, it could be used with any two cell types.
  2. This co-culture technique is an interesting platform for testing the effect of anti-tumour drugs on tumour cells, activated fibroblasts and tumour cells within the activated fibroblast microenvironment, which is closer to the physiological environment.
  3. The cell numbers seeded within the O-ring and around the O-ring should be adapted when using different cell lines according to their proliferation.
  4. The O-rings and coverslips were autoclaved prior to use.


  1. Complete culture medium
    Fetal bovine serum (FBS) (final concentration: 10%)
    L-glutamine-penicillin-streptomycin (1x)
  2. 4% paraformaldehyde
    Paraformaldehyde (final concentration: 4%)


This protocol was from Coulson-Thomas et al. (2010). This work was funded by FAPESP, CAPES and CNPq.


  1. Coulson-Thomas, V. J., Coulson-Thomas, Y. M., Gesteira, T. F., de Paula, C. A., Mader, A. M., Waisberg, J., Pinhal, M. A., Friedl, A., Toma, L. and Nader, H. B. (2011). Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion. Cell Tissue Res 346(2): 223-236.
  2. Coulson-Thomas, V. J., Coulson-Thomas, Y. M., Gesteira, T. F., Andrade de Paula, C. A., Carneiro, C. R., Ortiz, V., Toma, L., Kao, W. W. and Nader, H. B. (2013). Lumican expression, localization and antitumor activity in prostate cancer. Exp Cell Res 319(7): 967-981.
  3. Coulson-Thomas, V. J., Gesteira, T. F., Coulson-Thomas, Y. M., Vicente, C. M., Tersariol, I. L., Nader, H. B. and Toma, L. (2010). Fibroblast and prostate tumor cell cross-talk: fibroblast differentiation, TGF-β, and extracellular matrix down-regulation. Exp Cell Res 316(19): 3207-3226.
  4. Coulson-Thomas, Y. M., Gesteira, T. F., Norton, A. L., Kao, W. W., Nader, H. B. and Coulson-Thomas, V. J. (2015). The role of proteoglycans in the reactive stroma on tumor growth and progression. Histol Histopathol 30(1): 33-41.


我们在成纤维细胞和癌细胞之间开发了二维异型共培养技术,可以研究基质反应。为此,将基质细胞接种并立即在肿瘤细胞系周围培养,并且细胞建立细胞 - 细胞接触以及遍及基质细胞的可溶性因子的梯度,类似于在组织中发现的。因此,该系统还使得研究者能够区分由直接的细胞 - 细胞接触和分泌因子引起的事件。

背景 组织内肿瘤的生长和存活取决于与周围基质细胞(如成纤维细胞,炎性细胞,内皮细胞和淋巴细胞)的相互作用。研究表明,随着肿瘤生长,癌细胞与周围成纤维细胞之间存在广泛的串扰。此外,肿瘤细胞可以将这些成纤维细胞激活成肿瘤相关成纤维细胞(TAF)。在某些情况下,这些成纤维细胞可能会限制肿瘤生长(Coulson-Thomas等人,2011和2013);然而,在许多情况下,这些TAF帮助肿瘤细胞生长和存活(Coulson-Thomas等,2010和2015)。因此,体外癌症研究也应考虑到TAF对癌细胞的保护作用。考虑到这一点,我们在成纤维细胞和癌细胞之间开发了2D异型共培养技术,可以在同一系统中研究TAF和癌细胞。

关键字:癌症, 基质反应, 成纤维细胞, 细胞外基质和蛋白多糖


  1. 24孔透明平底TC处理的多孔细胞培养板(Corning,Falcon ®,目录号:353047)
  2. 圆形玻璃盖玻片,Fisherbrand TM 覆盖玻璃,直径(公制)12mm(Thermo Fisher Scientific,Fisher Scientific,目录号:12-545-80)
  3. O型圈,PYREX ®克隆圆筒(10×10mm)(Corning,目录号:3166-10)
  4. 基质细胞,前列腺成纤维细胞(WPMY-1)(ATCC,目录号:CRL-2854 TM)
  5. 前列腺癌PC-3细胞(ATCC,目录号:CRL-1435 TM
  6. 胰蛋白酶/EDTA 0.25%(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  7. 磷酸盐缓冲盐水(PBS)pH 7.4,不含钙,镁和酚红
  8. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM
  9. 一抗
    1. 抗胶原I(Calbiochem I-8H5,San Diego,CA和Sigma COL1,Sigma-Aldrich,目录号:C2456)
    2. 抗胶原IV,抗α1型IV(Santa Cruz Biotechnology,目录号:sc-29010)
    3. 抗胶原V(EMD Millipore,目录号:AB763P和Abcam,目录号:ab134800)
    4. 兔抗纤维蛋白调节蛋白H-50(Santa Cruz Biotechnology,目录号:sc-33772)
    5. 兔抗双低聚糖H-150(Santa Cruz Biotechnology,目录号:sc-33788)
    6. 小鼠抗纤连蛋白(BD,BD Transduction Laboratories TM,目录号:610077)
    7. 山羊抗perlecan L-20(Santa Cruz Biotechnology,目录号:sc-27449)
    8. Anti-versican H-56(Santa Cruz Biotechnology,目录号:sc-25831)
    9. 与Cy3缀合的小鼠抗平滑肌α肌动蛋白(Sigma-Aldrich,目录号:克隆1A4)
  10. (Thermo Fisher Scientific,Molecular Probes/Invitrogen,Eugene,OR)的

  11. Dulbecco改良的Eagle培养基(DMEM)培养基(Thermo Fisher Scientific,Gibco TM,目录号:11965092)
  12. L-谷氨酰胺 - 青霉素 - 链霉素(100x)(Thermo Fisher Scientific,Gibco TM,目录号:10378016)
  13. 多聚甲醛(水溶液:16%)(电子显微镜科学,目录号:15700)
  14. 完成培养基(见食谱)
  15. 4%多聚甲醛(见食谱)


  1. 超细镊子用直的尖端(灭菌)(精细科学工具,目录号:11399-80)
  2. CO 2细胞培养培养箱(Thermo Fisher Scientific,Thermo Scientific TM,目录号:51026280)
  3. 台式离心机(Eppendorf,型号:5702RH)
  4. 自动细胞计数器或血细胞计数器
  5. 生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:51026639)
  6. 倒置荧光显微镜(Zeiss,型号:Observer.Z1 with Apotome)


  1. Prism-GraphPad软件


  1. 准备O型圈共培养系统
    1. 根据供应商的指导原则,成纤维细胞和前列腺癌细胞应保持分开的细胞系。
    2. 对于异型共培养系统,用胰蛋白酶/EDTA从培养皿中除去成纤维细胞和前列腺癌细胞,并离心。
    3. 将细胞沉淀悬浮于完全培养基中
    4. 将无菌O型圈放置在24孔聚苯乙烯培养皿中的玻璃盖玻片的顶部。
      注意:不要使用硅胶将O形圈固定到盖玻片上,因为它会影响细胞在无细胞区域的迁移。 O型圈只是坐在盖子上;因此,在将微孔板运送到培养箱中时应小心。
    5. 将成纤维细胞(350μl中的1.5×10 4个)放置在O形环周围,并将前列腺癌细胞(0.5×10 4)在100μl中放置)环,如图1所示。

      图1. O型圈共培养技术将成纤维细胞接种在O型圈周围,将癌细胞接种在O型圈内。

    6. 将细胞在37℃和5%CO 2维持与O形环培养24小时。
    7. 然后用钳子去除O形环,并用完全培养基洗涤细胞两次以除去任何未附着的细胞,然后在37℃和5%CO 2下再培养48小时 注意:在移除O型圈后,研究人员可以确定O型圈面积是无细胞的,这表明细胞系之间没有交叉污染。在接下来的24小时内,两条细胞系容易迁移到无细胞区域,形成一个包含两个直接接触的细胞系的区域。
    8. 对于免疫细胞化学,在24或48小时后,将细胞固定在PBS中制备的4%多聚甲醛中 注意:研究人员应在显微镜下监测其细胞,以确定固定其特定细胞类型的最佳时间。我们确定24小时是大多数细胞类型的最佳选择。

  2. ECM分子的免疫荧光染色
    1. 细胞用PBS洗涤,并在封闭溶液(5%FBS)中在室温下孵育1小时
    2. 然后将细胞与一抗在4℃孵育过夜。我们发表的工作中使用的主要抗体是:抗胶原I(Calbiochem I-8H5和Sigma COL1),抗胶原IV(抗α1型IV)和两种不同的抗胶原V(Chemicon AB763P和Abcam克隆V-3 C9),兔抗纤维蛋白调节蛋白(Santa Cruz H-50),兔抗三聚糖(Santa Cruz H-150),小鼠抗纤维连接蛋白,山羊抗perlecan(Santa Cruz L-20),anti-versican(Santa Cruz H-56),最后,与Cy3缀合的小鼠抗平滑肌α肌动蛋白(克隆1A4)
    3. 然后将盖玻片洗涤三次15分钟,并在室温下用与Alexa Fluor 488或Alexa Fluor 594缀合的合适的荧光二抗孵育1小时。 br />
    4. 然后将盖玻片用PBS洗涤三次,每次洗涤15分钟,并在Fluoromount G(PBS中的2:1)中载玻片上。
    5. 必须对免疫染色实验进行阴性对照。这些在我们的一组实验中完成,省略了一级抗体。使用扫描共焦倒置显微镜(Zeiss LSM510)检查盖片,并使用LSM图像浏览器进行荧光定量。


此方法以前用于分析前列腺和结肠直肠基质反应(Coulson-Thomas等,2010和2011)。在这些研究中,仅在成纤维细胞区域,仅含有癌细胞的区域和界面中分析细胞外基质(ECM)产生,其中包含成纤维细胞和癌细胞。包含成纤维细胞和癌细胞的界面可以在图2中看到。通过免疫细胞化学分析ECM,使用针对不同ECM组分的一系列抗体。我们重复实验至少三次,一式三份,产生统计学显着的结果,使用Prism-GraphPad软件的Student's 测试。



  1. 该方案被开发用于研究基质细胞和肿瘤细胞之间的间作;然而,它可以与任何两种单元格类型一起使用。
  2. 这种共培养技术是一种有趣的平台,用于测试抗肿瘤药物对激活的成纤维细胞微环境内的肿瘤细胞,活化的成纤维细胞和肿瘤细胞的影响,其更接近生理环境。
  3. 根据其增殖情况,使用不同的细胞系时,应适应在O型圈内和O型圈周围种植的细胞数。
  4. O型圈和盖玻片在使用前进行高压灭菌。


  1. 完成培养基
    L-谷氨酰胺 - 青霉素 - 链霉素(1x)
  2. 4%多聚甲醛


该协议来自Coulson-Thomas等人。 (2010)。这项工作由FAPESP,CAPES和CNPq资助。


  1. Coulson-Thomas,VJ,Coulson-Thomas,YM,Gesteira,TF,de Paula,CA,Mader,AM,Waisberg,J.,Pinhal,MA,Friedl,A.,Toma,L.and Nader,HB(2011) 。结肠直肠癌发育不良反应上调胶原合成和限制癌细胞侵袭。细胞组织研究 346(2):223-236。
  2. Coulson-Thomas,VJ,Coulson-Thomas,YM,Gesteira,TF,Andrade de Paula,CA,Carneiro,CR,Ortiz,V.,Toma,L.,Kao,WW和Nader,HB(2013)一个class ="ke-insertfile"href =""target ="_ blank">前列腺癌的Lumican表达,定位和抗肿瘤活性。 Exp Cell Res 319(7):967-981。
  3. Coulson-Thomas,VJ,Gesteira,TF,Coulson-Thomas,YM,Vicente,CM,Tersariol,IL,Nader,HB and Toma,L。(2010)。< a class ="ke-insertfile"href ="target ="_ blank">成纤维细胞和前列腺肿瘤细胞的串扰:成纤维细胞分化,TGF-β和细胞外基质下调。 > Exp Cell Res 316(19):3207-3226。
  4. Coulson-Thomas,YM,Gesteira,TF,Norton,AL,Kao,WW,Nader,HB and Coulson-Thomas,VJ(2015)。 
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引用:Coulson-Thomas, V. . (2017). Analysis of Cancer Stromal Reaction Using an O-ring Co-culture Assay. Bio-protocol 7(4): e2131. DOI: 10.21769/BioProtoc.2131.