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Colon Cancer-associated Fibroblast Establishment and Culture Growth
结肠癌相关成纤维细胞的建立和培养生长    

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Abstract

Cancer-associated fibroblasts (CAFs) are one of the major players in tumor-stroma crosstalk. Findings in experimental studies suggest important roles for CAFs in regulation of tumor growth, metastasis and drug response (Hanahan and Coussens, 2012). Furthermore, their clinical relevance is supported by new findings from tumor analyses, demonstrating the prognostic and response-predictive significance of CAF-derived markers or gene signatures (Berdiel-hacer et al., 2014; Finak et al., 2008; Navab et al., 2011; Paulsson and Micke, 2014). CAFs are a heterogeneous pool of cell subsets with distinct functions which needs to be better defined by their marker expressions. The development of a methodology for the establishment of fibroblast primary cultures derived from human colon tumors allowed us to characterize their functional and molecular properties (Herrera et al., 2013). In addition, the different molecular mechanisms through which CAFs affect tumor growth and metastasis are still to be clarified. Therefore, functional and molecular characterization of the cancer-associated fibroblasts is essential to fully understand their role in tumor progression.

Materials and Reagents

  1. 50 ml Falcon tubes
  2. Sterile disposable plastic Pasteur pipettes or sterile forceps
  3. 10 cm tissue culture dishes
  4. T-25 tissue culture flasks
  5. MW6 tissue culture plates (Jet Biofil, catalog number: TCP-011-006 )
  6. Human tissue samples (colon normal and tumor tissue)
  7. Dulbecco’s Modified Eagle Medium (DMEM) high glucose without L-glutamine (Lonza, catalog number: BE12-614F )
  8. L-glutamine (Lonza, catalog number: BE17-605E )
  9. NormocinTM (InvivoGen, catalog number: ANT-NR-2 )
  10. Penicillin/Streptomycin (Lonza, catalog number: BE17-602E )
  11. Amphotericin B (Carl Roth GmbH + Co., catalog number: 0246.1 )
  12. Gentamicin (Carl Roth GmbH + Co., catalog number: 0233.2 )
  13. Phosphate-buffered saline (PBS) (Lonza, catalog number: BE17-516F )
  14. Fetal Bovine Serum medium (FBS) (Biowest, catalog number: S181B-500 )
  15. Trypsin/EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056 )
  16. FBM & FGM-2 Bullet kit [Lonza, catalog number:( CC-3131 ) and ( CC-4126 )]
    Note: FBM & FGM-2 Bullet kit is on the list of Fibroblast Growth Media (FGM) Kits] (Lonza, catalog number: CC-3132 ).
  17. DMEM (see Recipes)
  18. FBS with high concentration of antibiotics (see Recipes)
  19. FBS with normal concentration of antibiotics (see Recipes)

Equipment

  1. Laminar flow tissue culture hood
  2. Scalpels
  3. 37 °C shaker
  4. 37 °C and 5% CO2 cell culture incubator
  5. Centrifuge

Procedure

  1. Before starting
    1. Immediately after surgery, colon normal and tumor tissues are preserved in Falcon tubes with 10 ml of DMEM with a high concentration of antibiotics to avoid bacterial or fungal contamination. To this end, DMEM is supplemented with 5% FBS, L-glutamine (1%), 300 U/ml Penicillin, 300 µg/ml Streptomycin and 5 µg/ml Amphotericin B.
    2. Tissue samples are kept at 4 °C until use. It is recommended to minimize the time between the colon surgery and the processing of the sample.
    3. Make sure that you have all the necessary materials, reagents and equipment.

  2. Washing samples: Normal mucosa and tumor material
    1. Falcon tubes with human tissue samples from colon tumor or normal mucosa are placed into a laminar flow tissue culture hood under sterile conditions. The average size of tissues required is 1 cm2.


      Figure 1. Surgical colon human samples. Representative images of tumor tissue sample to show the used average size.

    2. Perform two quick washes of the tissue samples with 10 ml PBS each time using Falcon tubes and Pasteur pipettes to handle the samples.
    3. Then, incubate the samples for 30 min in 10 ml of PBS with 0.5 mg/ml Normocin, 0.5 µg/ml Amphotericin B and 0.1 mg/ml Gentamicin in the Falcon tubes. Shake gently in a 37 °C shaker.

  3. Dissecting and seeding samples: Normal mucosa and tumor material
    1. Tissue samples are transferred to a 10 cm tissue culture dish. Do not let the samples dry out at this point.
    2. The samples are carefully analyzed: normal tissue is fatty and soft, while tumor tissue is stiff, multimodular to variable degrees, and sometimes displays more irrigated areas.
    3. Cut the tissue samples into pieces of approximately 2-3 mm3 by scalpels.
    4. Use Pasteur pipettes or sterile forceps to seed tissue pieces in T-25 tissue culture flasks with 2 ml of FBS supplemented with 0.2 mg/ml Normocin, 200 U/ml Penicillin, 200 µg/ml Streptomycin and 0.1 mg/ml Gentamicin (Hanahan and Coussens, 2012). We call this medium "FBS with high concentration of antibiotics". Incubate the T-25 flasks at 37 °C in a humid atmosphere containing 5% CO2.
    5. After 24 h, it is quite useful to remove culture medium from the T-25 flasks, wash twice with PBS, and add fresh “FBS with high concentration of antibiotics” medium. When changing the medium, be careful and try to keep the pieces stuck on the plate. If after two days bacterial or fungal contamination does not appear, it is optional to change the medium to "FBS with normal concentration of antibiotics" (Berdiel-hacer et al., 2014): FBS with 0.1 mg/ml Normocin, 100 U/ml Penicillin and 100 µg/ml Streptomycin. However, if contamination persists, continue using "FBS with high concentration of antibiotics". If after five days contamination has not disappeared, discard the samples.
    6. Normally, outgrowths of fibroblasts appear after 10 days and the culture medium is replaced by FBM supplemented with FGM-2 Bulletkit to facilitate fibroblast growth (Finak et al., 2008). The fibroblastic cells are routinely maintained in FBM medium at 37 °C in a humid atmosphere containing 5% CO2. Change the medium every two days.

  4. Expansion of fibroblast primary cell cultures
    1. After 30-40 days, when the cell culture is usually quite large (see Figure 1), the remnants of the tissue are removed giving a light tap to the flask.
    2. Fibroblasts are washed twice with PBS and trypsinized by Trypsin/EDTA for 5 min at 37 °C. Use serum medium (FBS) to block the trypsin activity and centrifuge at 1,200 rpm during 5 min. After that, seed the fibroblasts cells in MW6 tissue plates using FBM medium.
    3. Then, fibroblast primary cultures are subcultured at a 1 to 2 ratio (Navab et al., 2011) in FBM medium and maintained at 37 °C in a humid atmosphere containing 5% CO2. Change the medium every two days. Primary CAFs initially show bi- and/or multi-polar morphology and then acquired a uniform spindle-shaped morphotype and formed parallel arrays and whorls at confluence. Expression of α-sma and Vimentin and the absence of Pan Cytokeratin (PanCK) could be analyzed by immunofluorescence to verify the CAF enrichment in the cell culture as was done in Herrera et al. (2013).


      Figure 2. Establishment and characterization of primary CAFs from colon cancer patients. Representative images of CAF cultures along the establishment time. Scale bar=50 μm

Notes

  1. We recommend not putting more than 5-6 pieces in each T25 flask, which helps control contamination. Use several flasks per sample if you need to do so.
  2. We prefer to use the medium "FBS with high concentration of antibiotics" for 7-10 days (replace with fresh medium every two days).
  3. We recommend changing the medium to FBM after 12-14 days even if fibroblasts are not seen.
  4. Cell numbers can be expanded minimally, though bear in mind that primary cells enter senescence after prolonged in vitro culture. Immortalization can be performed using adequate procedures (Navab et al., 2011).

Recipes

  1. DMEM
    5% FBS
    L-glutamine (1%)
    300 U/ml Penicillin
    300 µg/ml Streptomycin
  2. 5 µg/ml Amphotericin BFBS with high concentration of antibiotics
    FBS supplemented with 0.2 mg/ml Normocin, 200 U/ml Penicillin, 200 µg/ml Streptomycin and 0.1 mg/ml Gentamicin (Hanahan and Coussens, 2012)
  3. FBS with normal concentration of antibiotics
    FBS with 0.1 mg/ml Normocin, 100 U/ml Penicillin and 100 µg/ml Streptomycin

Acknowledgments

This protocol is an extended version of the one described in Herrera et al. (2013) and Stanisavljevic et al. (2015).
M. Eaude helped with the English text. This research is supported by PI12/02037, RD12/0036/0041 and RD12/0036/0021 from the Instituto de Salud Carlos III-FEDER; by the Fundación Científica AECC; by SAF2010-20750 from the Ministerio de Economía y Competitividad of Spain-FEDER; by S2010/BMD-2344 from the Comunidad de Madrid; and by the Fundación Banco Santander. Antonio García de Herreros’ laboratory is supported by RD12/0036/0005 (from the Instituto de Salud Carlos III-FEDER) and SAF2013-48849-C2-1-R (from the Ministerio de Economía y Competitividad of Spain-FEDER). Cristina Peña is a recipient of a Miguel Servet Contract from the Instituto de Salud Carlos III. All authors state no conflicts of interest. We thank lab members for help and advice throughout this research.

References

  1. Berdiel-Acer, M., Berenguer, A., Sanz-Pamplona, R., Cuadras, D., Sanjuan, X., Paules, M. J., Santos, C., Salazar, R., Moreno, V., Capella, G., Villanueva, A. and Mollevi, D. G. (2014). A 5-gene classifier from the carcinoma-associated fibroblast transcriptomic profile and clinical outcome in colorectal cancer. Oncotarget 5(15): 6437-6452.
  2. Finak, G., Bertos, N., Pepin, F., Sadekova, S., Souleimanova, M., Zhao, H., Chen, H., Omeroglu, G., Meterissian, S., Omeroglu, A., Hallett, M. and Park, M. (2008). Stromal gene expression predicts clinical outcome in breast cancer. Nat Med 14(5): 518-527.
  3. Hanahan, D. and Coussens, L. M. (2012). Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21(3): 309-322.
  4. Herrera, M., Islam, A. B., Herrera, A., Martin, P., Garcia, V., Silva, J., Garcia, J. M., Salas, C., Casal, I., de Herreros, A. G., Bonilla, F. and Pena, C. (2013). Functional heterogeneity of cancer-associated fibroblasts from human colon tumors shows specific prognostic gene expression signature. Clin Cancer Res 19(21): 5914-5926.
  5. Navab, R., Strumpf, D., Bandarchi, B., Zhu, C. Q., Pintilie, M., Ramnarine, V. R., Ibrahimov, E., Radulovich, N., Leung, L., Barczyk, M., Panchal, D., To, C., Yun, J. J., Der, S., Shepherd, F. A., Jurisica, I. and Tsao, M. S. (2011). Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer. Proc Natl Acad Sci U S A 108(17): 7160-7165.
  6. Paulsson, J. and Micke, P. (2014). Prognostic relevance of cancer-associated fibroblasts in human cancer. Semin Cancer Biol 25: 61-68.

简介

癌症相关成纤维细胞(CAF)是肿瘤 -​​ 基质串扰的主要参与者之一。实验研究的结果表明CAFs在肿瘤生长,转移和药物反应的调节中的重要作用(Hanahan和Coussens,2012)。此外,它们的临床相关性得到来自肿瘤分析的新发现的支持,表明CAF衍生的标记或基因特征的预后和应答预测显着性(Berdiel-hacer等人,2014; Finak 等人,2008; Navab等人,2011; Paulsson和Micke,2014)。 CAF是具有不同功能的细胞亚群的异质池,其需要由它们的标记表达更好地定义。开发用于建立源自人结肠肿瘤的成纤维细胞原代培养物的方法允许我们表征其功能和分子性质(Herrera等人,2013)。此外,CAF影响肿瘤生长和转移的不同分子机制仍有待澄清。因此,癌症相关成纤维细胞的功能和分子表征是完全了解他们在肿瘤进展中的作用的必要条件。

材料和试剂

  1. 50ml Falcon管
  2. 无菌一次性塑料巴斯德移液器或无菌镊子
  3. 10厘米组织培养皿
  4. T-25组织培养瓶
  5. MW6组织培养板(Jet Biofil,目录号:TCP-011-006)
  6. 人体组织样本(结肠正常和肿瘤组织)
  7. 不含L-谷氨酰胺的Dulbecco's Modified Eagle Medium(DMEM)高葡萄糖(Lonza,目录号:BE12-614F)
  8. L-谷氨酰胺(Lonza,目录号:BE17-605E)
  9. Normocin TM (InvivoGen,目录号:ANT-NR-2)
  10. 青霉素/链霉素(Lonza,目录号:BE17-602E)
  11. 两性霉素B(Carl Roth GmbH + Co.,目录号:0246.1)
  12. 庆大霉素(Carl Roth GmbH + Co.,目录号:0233.2)
  13. 磷酸盐缓冲盐水(PBS)(Lonza,目录号:BE17-516F)
  14. 胎牛血清培养基(FBS)(Biowest,目录号:S181B-500)
  15. 胰蛋白酶/EDTA(Thermo Fisher Scientific,Gibco TM ,目录号:25200-056)
  16. FBM& FGM-2子弹套件[Lonza,目录号:(CC-3131)和(CC-4126)]
    注意:FBM& FGM-2 Bullet试剂盒在成纤维细胞生长培养基(FGM)试剂盒列表中(Lonza,目录号:CC-3132)。
  17. DMEM(参见配方)
  18. FBS与高浓度的抗生素(见配方)
  19. FBS与正常浓度的抗生素(见配方)

设备

  1. 层流组织培养罩
  2. 解释器
  3. 37℃摇床
  4. 37℃和5%CO 2细胞培养箱中培养
  5. 离心机

程序

  1. 开始之前
    1. 手术后立即保留结肠正常和肿瘤组织 在Falcon管中用10ml高浓度的DMEM 抗生素以避免细菌或真菌污染。为此, DMEM补充有5%FBS,L-谷氨酰胺(1%),300U/ml青霉素, ?300μg/ml链霉素和5μg/ml两性霉素B
    2. 组织 将样品保持在4℃直至使用。建议最小化 结肠手术和样品处理之间的时间
    3. 确保您拥有所有必要的材料,试剂和设备。

  2. 洗涤样品:正常粘膜和肿瘤材料
    1. Falcon管与来自结肠肿瘤或正常粘膜的人组织样品 ?在无菌下置于层流组织培养罩中 条件。所需的组织的平均尺寸为1cm 。


      图1.手术结肠人类样品。肿瘤组织样品的代表性图像显示使用的平均尺寸。

    2. 执行两次快速洗涤的组织样本用10毫升PBS每 时间使用Falcon管和巴斯德移液管处理样品
    3. 然后,将样品在10ml含有0.5mg/ml Normocin,0.5μg/ml两性霉素B和0.1mg/ml庆大霉素的PBS中孵育30分钟 猎鹰管。在37℃振荡器中轻轻摇动。

  3. 解剖和种子样品:正常粘膜和肿瘤材料
    1. 将组织样品转移至10cm组织培养皿。此时不要让样品变干。
    2. 仔细分析样品:正常组织是脂肪和软的, ?而肿瘤组织是僵硬的,多模态到可变程度,和 有时显示更多灌溉区域。
    3. 用手术刀将组织样本切成约2-3毫米的小片。
    4. 使用巴斯德移液管或无菌镊子种子组织片 T-25组织培养瓶与2ml补充有0.2的FBS mg/ml诺霉素,200U/ml青霉素,200μg/ml链霉素和0.1mg/ml 庆大霉素(Hanahan和Coussens,2012)。我们将这种媒介称为"FBS" 高浓度的抗生素"。在37℃下孵育T-25烧瓶 ?含有5%CO 2的潮湿气氛
    5. 24小时后,它是相当 用于从T-25烧瓶中除去培养基,用洗涤两次 PBS,并加入新鲜的"具有高浓度抗生素的FBS"培养基。 当更换介质时,小心,并尽量保持片断 ?碟子。如果两天后细菌或真菌污染没有 ?出现,可选择将介质更换为"FBS与正常 浓度的抗生素"(Berdiel-hacer等人,2014):用0.1mg/ml诺霉素,100U/ml青霉素和100μg/ml链霉素的FBS, ?如果污染持续,继续使用"高浓度的FBS 的抗生素",如果五天后污染没有消失, 丢弃样品。
    6. 通常,出现成纤维细胞的生长 10天后,用补充的FBM替换培养基 使用FGM-2 Bulletkit以促进成纤维细胞生长(Finak等人 2008)。成纤维细胞常规地保持在FBM培养基中 37℃,在含有5%CO 2的潮湿气氛中。更换介质 两天。

  4. 成纤维细胞原代细胞培养物的扩增
    1. 30-40天后,细胞培养通常相当大 图1),除去组织的残余物,得到轻拍 烧瓶
    2. 成纤维细胞用PBS洗涤两次并胰蛋白酶化 通过胰蛋白酶/EDTA在37℃下5分钟。使用血清培养基(FBS)阻断 胰蛋白酶活性并在1,200rpm离心5分钟。之后, 使用FBM培养基将成纤维细胞在MW6组织平板中接种
    3. 然后,将成纤维细胞原代培养物以1比2传代培养 (Navab等人,2011)在FBM培养基中并在37℃下在潮湿环境中维持 含有5%CO 2的气氛。每两天更换一次培养基。主 CAF最初显示双极和/或多极形态,然后获得a ?均匀纺锤形形态并形成平行阵列和螺旋 ?在汇合。 α-sma和波形蛋白的表达和不存在 可以通过免疫荧光分析细胞角蛋白(PanCK)以验证 如在Herrera等人(2013)中所做的细胞培养物中的CAF富集。


      图2.建立和表征 来自结肠癌患者的原发性CAF。 CAF的代表性图像 文化沿着建立时间。比例尺=50μm

笔记

  1. 我们建议不要在每个T25烧瓶中放置超过5-6个,这有助于控制污染。如果需要,每个样品使用几个烧瓶。
  2. 我们优选使用具有高浓度抗生素的培养基"FBS"7-10天(每两天更换新鲜培养基)。
  3. 我们建议在12-14天后更换培养基到FBM,即使没有看到成纤维细胞
  4. 细胞数目可以最小化地扩展,尽管记住原代细胞在延长的体外培养后进入衰老。永生化可以使用适当的程序进行(Navab等人,2011)。

食谱

  1. DMEM
    5%FBS
    L-谷氨酰胺(1%) 300 U/ml青霉素
    300μg/ml链霉素
  2. 5μg/ml两性霉素BFBS,含高浓度抗生素
    补充有0.2mg/ml诺霉素,200U/ml青霉素,200μg/ml链霉素和0.1mg/ml庆大霉素的培养基(Hanahan和Coussens,2012)。
  3. FBS与正常浓度的抗生素
    FBS与0.1mg/ml Normocin,100U/ml青霉素和100μg/ml链霉素的缓冲液

致谢

此协议是Herrera等人(2013年)和Stanisavljevic等人(2015年)所描述的协议的扩展版本。
M. Eaude帮助了英文文本。该研究由来自Salud Carlos III-FEDER研究所的PI12/02037,RD12/0036/0041和RD12/0036/0021支持;由基金会西班牙 - FEDER竞争力部长SAF2010-20750;来自Comunidad de Madrid的S2010/BMD-2344;和桑坦德银行。 AntonioGarcíade Herreros的实验室得到RD12/0036/0005(来自Salud Carlos III-FEDER研究所)和SAF2013-48849-C2-1-R(西班牙经济部长FEDER的资助)的支持。 CristinaPe?a是来自Salud Carlos III学院的Miguel Servet合同的收件人。所有作者不存在利益冲突。我们感谢实验室成员在整个研究中的帮助和建议。

参考文献

  1. Berdiel-Acer,M.,Berenguer,A.,Sanz-Pamplona,R.,Cuadras,D.,Sanjuan,X.,Paules,MJ,Santos,C.,Salazar,R.,Moreno,V.,Capella, G.,Villanueva,A。和Mollevi,DG(2014)。 来自癌相关成纤维细胞转录组概况和结肠直肠癌临床结果的5基因分类。/a> 5(15):6437-6452。
  2. Finak,G.,Bertos,N.,Pepin,F.,Sadekova,S.,Souleimanova,M.,Zhao,H.,Chen,H.,Omeroglu,G.,Meterissian,S.,Omeroglu, Hallett,M。和Park,M。(2008)。 基质基因表达预测乳腺癌的临床结果 em> 14(5):518-527
  3. Hanahan,D。和Coussens,L.M。(2012)。 犯罪附件:招募到肿瘤微环境的细胞的功能。 Cancer Cell 21(3):309-322。
  4. Herrera,M.,Islam,AB,Herrera,A.,Martin,P.,Garcia,V.,Silva,J.,Garcia,JM,Salas,C.,Casal,I.,de Herreros,AG,Bonilla, F.和Pena,C。(2013)。 来自人类结肠肿瘤的癌症相关成纤维细胞的功能异质性显示特异性预后基因表达特征。 Clin Cancer Res 19(21):5914-5926
  5. Navab,R.,Strumpf,D.,Bandarchi,B.,Zhu,CQ,Pintilie,M.,Ramnarine,VR,Ibrahimov,E.,Radulovich,N.,Leung,L.,Barczyk,M.,Panchal, D.,To,C.,Yun,JJ,Der,S.,Shepherd,FA,Jurisica,I.and Tsao,MS(2011)。 非小细胞肺癌中癌相关成纤维细胞的预后基因表达特征。 Proc Natl Acad Sci USA 108(17):7160-7165。
  6. Paulsson,J.和Micke,P。(2014)。 癌症相关成纤维细胞在人类癌症中的预后相关性 癌症生物 25:61-68。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Herrera, M., Herrera, A., Larriba, M. J., Ferrer-Mayorga, G., Herreros, A. G., Bonilla, F., Baulida, J. and Peña, C. (2016). Colon Cancer-associated Fibroblast Establishment and Culture Growth. Bio-protocol 6(7): e1773. DOI: 10.21769/BioProtoc.1773.
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