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3D Stroma Invasion Assay
三维基质侵袭实验   

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参见作者原研究论文

本实验方案简略版
Cell and Tissue Research
Nov 2011

Abstract

We have developed a 3D co-culture system composed of fibroblasts and colorectal cancer cells that enables us to study the desmoplastic reaction. This method also enables us to study the influence of the desmoplastic reaction on the migration of colorectal cancer cells through the surrounding stroma. This protocol has been previously published (Coulson-Thomas et al., 2011) and is described here in more detail.

Keywords: 3D culture (三维培养), Fibroblasts (成纤维细胞), Cancer cells (癌细胞), Desmoplastic reaction (促结缔组织增生性反应), Cancer cell invasion (癌细胞侵袭), Stromagenic system (基质生成系统)

Background

The progression of cancer relies on intricate cross-talk between the cancer cells and surrounding cells, such as fibroblasts, inflammatory cells and endothelial cells, which form the cancer microenvironment. Fibroblasts are the major extracellular matrix producing cells and are responsible for the structural formation of tissues. Fibroblasts surrounding tumors are ‘activated’ by cancer cells into tumor-associated fibroblasts (TAFs) and play key roles in tumorigenesis and metastasis. In some cancers, TAFs up-regulate extracellular matrix expression producing an unorganized matrix, consisting mainly of collagen fibers and proteoglycans, which affects cancer cell proliferation, migration and spread. This is called the desmoplastic reaction, and during cancer cell growth different tumors may exhibit various grades of desmoplasia.

Materials and Reagents

  1. NuncTM Lab-TekTM II Chamber SlideTM System with 2 wells ( Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 154461 )
  2. NuncTM cell culture dishes ( Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 172931 )
  3. Cotton swabs
  4. Human colorectal fibroblasts CCD-112CoN (ATCC, catalog number: CRL-1541 )
  5. Caco-2 and HCT 166 cancer cell lines isolated from primary colorectal tumors (ATCC, catalog numbers: HTB-37 and CCL-247 )
  6. pEGFP-N1 (TaKaRa Bio, Clontech)
  7. DMEM culture medium (Thermo Fisher Scientific, GibcoTM)
  8. RPMI culture medium (Thermo Fisher Scientific, GibcoTM)
  9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM)
  10. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25-030-081 )
  11. Penicillin/streptomycin (Thermo Fisher Scientific, InvitrogenTM)
  12. L-ascorbic acid (Sigma-Aldrich, catalog number: A4403 )
  13. Collagen I
  14. Anti-fibronectin (BD transduction laboratories)
  15. FuGENE® HD transfection reagent (Promega, catalog number: E2311 )
  16. Trypsin/EDTA (Thermo Fisher Scientific, GibcoTM)
  17. Paraformaldehyde, aqueous solution - 16% (Electron Microscopy Sciences, catalog number: 15700 )
  18. Complete media for Caco-2 and HCT 166 cells (see Recipes)
  19. Complete media for fibroblasts (see Recipes)
  20. Media for maintaining 3D cultures (see Recipes)

Equipment

  1. Ultra-fine forceps with a straight tip (Fine Science tools, catalog number: 11399-80 )
  2. CO2 cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i , catalog number: 51026280)
  3. Table top centrifuge (Eppendorf, model: 5702 RH )
  4. Vi-CELL XR cell counter (Beckman Coulter)
  5. Biological safety cabinets (Thermo Fisher Scientific, Thermo ScientificTM, model: Safe 2020 Class II , catalog number: 51026639)
  6. Scanning confocal inverted microscope (Zeiss, model: LSM 510 )
  7. Time-lapse confocal microscope (Zeiss, model: LSM 710 )

Software

  1. Java ImageJ and the Zen Imaging software from Zeiss
  2. Excel (Microsoft)
  3. GraphPad Prism (GraphPad Software)

Procedure

  1. Preparation of a 3D fibroblast-produced matrix
    1. Seed fibroblasts at a density of 3 x 106 cells per well of a 2 well-chamber slide to form the control 3D matrix and a mixture of fibroblasts and tumor cells at a density of 3 x 106 fibroblasts and 0.5 x 106 tumor cells per well to form the desmoplastic 3D matrix in DMEM and RPMI (1:1) containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.
    2. Incubate the cells at 37 °C in a 5% CO2 humidified environment until confluent.
    3. Once a confluent monolayer is formed, treat the cultures with 3 ml of 25 µg/ml ascorbic acid every other day for an additional 10 days to induce the production of a 3D matrix, maintaining the cells at 37 °C in a 5% CO2 humidified environment. The medium should be changed every other day with fresh medium containing 25 µg/ml ascorbic acid. The cells may be treated for longer than 10 days if a thicker and denser matrix is desired. Extracellular matrix components such as collagen I and fibronectin can be used to visualize the 3D matrix (Figure 1). All fibroblasts would respond to ascorbic acid treatment; however, the thickness, density and organization may vary among different types of fibroblasts.
      Note: Fresh ascorbic acid solutions should be prepared every other day since ascorbic acid in solution decays over time.


      Figure 1. Fibronectin immunostaining with mouse anti-fibronectin evidencing 3D matrix formation in a 3D stromagenic model. Scale bar = 20 μm.

  2. Cell invasion assay
    1. Transfect colorectal tumor cells with the GFP plasmid using FuGENE® HD transfection reagent (500 µg of plasmid in a total volume of 2 ml) and incubate the cells at 37 °C in a 5% CO2 humidified environment for 48 h, according to the manufacturer’s instructions.
      Note: It is highly recommended for the first experiment to test the best confluence at which the cells should be transfected. This would entail seeding cells at increasing densities, for example at 30%, 50%, 75% and 90% confluence, in order to test which confluence provides the highest transfection rate. We established that for us 75% confluence provided the highest transfection rate.
    2. Remove the GFP transfected cells from the culture dish using trypsin/EDTA and seed 1 x 106 tumor cells onto the 3D stromagenic systems prepared in item A (both experimental and control systems – see Notes below).
    3. Allow the GFP positive cells to migrate for 1 h, maintaining the cells at 37 °C in a 5% CO2 humidified environment after which the invading cells can be imaged using time-lapse confocal microscopy.
    4. Carry out the time-lapse analysis using a scanning confocal inverted microscope. Time-lapse images can be captured every minute for a total of 15 to 30 min to enable the investigator to see the movement of the tumor cells invading the 3D matrix.
    5. Z-stacks should be obtained throughout the thickness of the 3D matrix at the end of the 2 h period to verify the depth to which the cancer cells invaded, as previously described (Coulson-Thomas et al., 2011; de Paula et al., 2012). Z-stack projections using the z-axis as reference can be made using any imaging software, such as Image Processing and Analysis with Java ImageJ and the Zen Imaging software from Zeiss.

Data analysis

The time-lapse images can be assembled into a video using the LSM or ZEN software. A z-stack of the images may also be projected using the LSM or ZEN software in order to estimate the depth of the invading cell. We carried out our experiments three times in triplicate, and our statistical analyses were calculated using the t-test in Excel (Microsoft) and GraphPad Prism (GraphPad Software).

Notes

  1. Experimental groups consist of seeding GFP positive colorectal tumor cells onto the desmoplastic 3D stromagenic system composed of fibroblasts and colorectal cancer cells, while control groups consist of seeding GFP positive colorectal tumor cells onto the 3D stromagenic system composed of solely fibroblasts.
  2. This protocol was developed to study the invasion of colorectal tumor cells, but any other cancer cell type could be tested.
  3. The two-hour migration period should be tested and adapted if necessary when using different cancer cell lines.
  4. The same procedure can be used to induce the production of a 3D fibroblast-produced matrix in culture plate inserts with a pore size of 8 µm (30 mm, Millicell®-PCF, Millipore Corp). In this case, the GFP positive colorectal tumor cells are seeded on the 3D stromagenic matrix in the culture plate insert in serum free medium and allowed to migrate for 8 h to the lower chamber containing serum supplemented with 10% FBS. Once fixed using 10% paraformaldehyde, cells are removed from the upper compartment using a cotton swab and the GFP positive cells that migrated are analyzed.

Recipes

  1. Complete media for Caco-2 and HCT 166 cells
    RPMI medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin
  2. Complete media for fibroblasts
    DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin
  3. Media for maintaining 3D cultures
    A mixture of RPMI and DMEM media (1:1) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 25 μg/ml ascorbic acid

Acknowledgments

This protocol is from Coulson-Thomas et al., 2011. This work was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo).

References

  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. de Paula, C. A., Coulson-Thomas, V. J., Ferreira, J. G., Maza, P. K., Suzuki, E., Nakahata, A. M., Nader, H. B., Sampaio, M. U. and Oliva, M. L. (2012). Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways. J Biol Chem 287(1): 170-182.

简介

我们开发了一种由成纤维细胞和结肠直肠癌细胞组成的3D共培养系统,使我们能够研究脱发反应。这种方法还使我们能够研究脱发反应对结肠直肠癌细胞通过周围基质的迁移的影响。以前已经公布了该协议(Coulson-Thomas等人,2011),并且在此更详细地描述。

背景 癌症的进展依赖于癌细胞和周围细胞如成纤维细胞,炎性细胞和内皮细胞之间的复杂的串扰,其形成癌症微环境。成纤维细胞是主要的细胞外基质产生细胞并且负责组织的结构形成。肿瘤周围的成纤维细胞被癌细胞“激活”成肿瘤相关成纤维细胞(TAFs),并在肿瘤发生和转移中发挥关键作用。在一些癌症中,TAF上调细胞外基质表达,产生一种主要由胶原纤维和蛋白多糖组成的无组织基质,其影响癌细胞增殖,迁移和扩散。这被称为脱发反应,并且在癌细胞生长期间,不同的肿瘤可能表现出各种等级的发育不良。

关键字:三维培养, 成纤维细胞, 癌细胞, 促结缔组织增生性反应, 癌细胞侵袭, 基质生成系统

材料和试剂

  1. 使用2孔(Thermo Fisher Scientific,Thermo Scientific TM 的Nunc TM II-Room TM ,目录号:154461)
  2. Nunc TM 细胞培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:172931)
  3. 棉签
  4. 人结肠直肠成纤维细胞CCD-112CoN(ATCC,目录号:CRL-1541)
  5. 从原发性结肠直肠肿瘤(ATCC,目录号:HTB-37和CCL-247)分离的Caco-2和HCT166癌细胞系
  6. pEGFP-N1(TaKaRa Bio,Clontech)
  7. DMEM培养基(Thermo Fisher Scientific,Gibco TM
  8. RPMI培养基(Thermo Fisher Scientific,Gibco TM
  9. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM
  10. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25-030-081)
  11. 青霉素/链霉素(Thermo Fisher Scientific,Invitrogen TM
  12. L-抗坏血酸(Sigma-Aldrich,目录号:A4403)
  13. 胶原蛋白I
  14. 抗纤连蛋白(BD转导实验室)
  15. FuGENE ® HD转染试剂(Promega,目录号:E2311)
  16. 胰蛋白酶/EDTA(Thermo Fisher Scientific,Gibco TM
  17. 多聚甲醛,水溶液-16%(电子显微镜科学,目录号:15700)
  18. Caco-2和HCT 166细胞的完整培养基(参见食谱)
  19. 成纤维细胞的完整培养基(见食谱)
  20. 用于维护3D文化的媒体(请参阅食谱)

设备

  1. 超细镊子用直的尖端(精细科学工具,目录号:11399-80)
  2. CO 2细胞培养箱(Thermo Fisher Scientific,Thermo Scientific,Superson TM,型号:Heracell TM 150,目录号:51026280)
  3. 台式离心机(Eppendorf,型号:5702 RH)
  4. Vi-CELL XR细胞计数器(Beckman Coulter)
  5. 生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:Safe 2020 Class II,目录号:51026639)
  6. 扫描共焦倒置显微镜(Zeiss,型号:LSM 510)
  7. 延时共焦显微镜(Zeiss,型号:LSM 710)

软件

  1. 来自Zeiss的Java ImageJ和Zen Imaging软件
  2. Excel(Microsoft)
  3. GraphPad Prism(GraphPad软件)

程序

  1. 制备3D成纤维细胞产生的基质
    1. 以每孔3×10 6个细胞/孔的2个孔室载玻片的种子成纤维细胞形成对照3D基质,并以3×10 6个成纤维细胞和0.5×10 6个肿瘤细胞,以在含有10%FBS,2mM L-谷氨酰胺,100μM的DMEM和RPMI(1:1)中形成去塑性3D基质U/ml青霉素和100μg/ml链霉素。
    2. 将细胞在37℃下在5%CO 2加湿环境中孵育直至汇合。
    3. 一旦形成汇合单层,每隔一天用3ml25μg/ml抗坏血酸处理培养物10天以诱导3D基质的产生,将细胞保持在37℃,5%CO < sub> 2 加湿环境。培养基应每隔一天更换一次含有25μg/ml抗坏血酸的新鲜培养基。如果需要更厚和更致密的基质,细胞可以处理超过10天。可以使用细胞外基质成分如胶原蛋白I和纤连蛋白来显现3D基质(图1)。所有成纤维细胞都会对抗坏血酸的治疗作出反应;然而,不同类型的成纤维细胞的厚度,密度和组织可能不同。
      注意:抗坏血酸在溶液中随着时间的推移衰变,每隔一天应制备新鲜的抗坏血酸溶液。


      图1.使用小鼠抗纤维连接蛋白进行纤维连接蛋白免疫染色,证明三维基质模型中的3D基质形成。比例尺= 20μm。

  2. 细胞侵袭分析
    1. 使用高效转染试剂(500μg总体积为2ml的质粒),用GFP质粒转染结肠直肠肿瘤细胞,并将细胞在37℃下于5%CO 2 2 加湿环境48小时,根据制造商的说明。
      注意:强烈建议第一个实验测试细胞应该转染的最佳汇合点。这将需要以增加的密度进行接种细胞,例如在30%,50%,75%和90%汇合处,以测试哪个汇合提供最高的转染率。我们确定,对于我们来说,75%的融合提供了最高的转染率。
    2. 从培养皿中使用胰蛋白酶/EDTA和种子1×10 6个肿瘤细胞将GFP转染的细胞从项目A(实验和对照系统 - 见下面的注释)中制备的3D间质系统上除去。 br />
    3. 允许GFP阳性细胞迁移1小时,将细胞保持在37℃的5%CO 2加湿环境中,之后可以使用延时共聚焦显微镜对入侵的细胞进行成像。 />
    4. 使用扫描共焦倒置显微镜进行延时分析。可以每分钟捕获延时图像,总共15到30分钟,使研究者能够看到入侵3D矩阵的肿瘤细胞的运动。
    5. 应在2 h期结束时,在整个3D矩阵的厚度上获得Z-叠层,以验证癌细胞入侵的深度,如前所述(Coulson-Thomas等人, 2011; de Paula等人,2012)。使用z轴作为参考的Z-stack投影可以使用任何成像软件进行,例如使用Java ImageJ的图像处理和分析以及来自Zeiss的Zen Imaging软件。

数据分析

延时图像可以使用LSM或ZEN软件组装成视频。也可以使用LSM或ZEN软件投影图像的z叠层,以便估计入侵单元的深度。我们三次进行了三次实验,我们的统计分析使用Excel(Microsoft)和GraphPad Prism(GraphPad Software)中的 t 测试进行计算。

笔记

  1. 实验组包括将GFP阳性结肠直肠肿瘤细胞接种在由成纤维细胞和结肠直肠癌细胞组成的脱颗粒3D造粒系统上,而对照组由将GFP阳性结肠直肠肿瘤细胞接种到仅由成纤维细胞组成的3D间质系统上。
  2. 该方案被开发用于研究结肠直肠肿瘤细胞的侵袭,但可以测试任何其他癌细胞类型。
  3. 如果需要,使用不同的癌细胞系时,应对两小时的迁移期进行测试和调整
  4. 可以使用相同的程序来诱导在孔径为8μm(30mm,Millicell-MCF,Millipore Corp)的培养板插入物中生产3D成纤维细胞产生的基质。在这种情况下,将GFP阳性结肠直肠肿瘤细胞接种在无血清培养基中的培养板插入物中的3D基质基质上,并允许迁移8小时至含有补充有10%FBS的血清的下室。一旦使用10%多聚甲醛固定,使用棉签将细胞从上隔室中取出,并分析迁移的GFP阳性细胞。

食谱

  1. Caco-2和HCT 166细胞的完整培养基
    补充有10%FBS,2mM L-谷氨酰胺,100U/ml青霉素和100μg/ml链霉素的RPMI培养基
  2. 完整的成纤维细胞培养基
    补充有10%FBS,2mM L-谷氨酰胺,100U/ml青霉素和100μg/ml链霉素的DMEM培养基
  3. 用于维护3D文化的媒体
    补充有10%FBS,2mM L-谷氨酰胺,100U/ml青霉素,100μg/ml链霉素和25μg/ml抗坏血酸的RPMI和DMEM培养基(1:1)的混合物

致谢

该协议来自于2011年的科尔逊·托马斯(Coulson-Thomas),这项工作得到了CAPES(协调中心),CNPq(Conselho Nacional de DesenvolvimentoCientíficoeTecnológico)和FAPESP(保加利亚圣保罗圣保罗圣保罗圣保罗教堂)。

参考文献

  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. (Pa,CA,Coulson-Thomas,VJ,Ferreira,JG,Maza,PK,Suzuki,E.,Nakahata,AM,Nader,HB,Sampaio,MU和Oliva,ML(2012)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22039045"target ="_ blank"> 胰蛋白酶抑制剂(EcTI),一种植物蛋白酶抑制剂,通过抑制Src蛋白 - 粘着激酶(FAK)信号通路,减少体外细胞粘附和侵袭。 J Biol Chem 287(1):170 -182。
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引用:Coulson-Thomas, Y. M. and Coulson-Thomas, V. . (2017). 3D Stroma Invasion Assay. Bio-protocol 7(6): e2195. DOI: 10.21769/BioProtoc.2195.
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