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Soft Agar Colony Formation Assay as a Hallmark of Carcinogenesis
软琼脂集落形成实验测定作为致癌性标志的集落形成   

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Abstract

Soft agar colony formation assay is established to estimate the anchorage-independent growth ability of cells. In this assay, a bottom layer of agar with complete media is poured and solidified first, followed by an upper layer containing a specified number of cells suspended in medium-agar mixture. After two weeks of incubation, the number of colonies will be counted, serving as an indicator of malignancy of tumor cells.

Keywords: Anchorage-independent growth(非贴壁依赖性生长), Colony formation(集落形成), Carcinogenesis(致癌性), Malignant phenotype(恶性表型), Agar(琼脂)

Background

Anchorage-independent growth is an ability of cells to grow independently on a solid surface, and is considered as a hallmark of carcinogenesis (de Larco and Todaro, 1978). Soft agar colony formation assay is a well-established method to evaluate cellular anchorage-independent growth for the detection of the tumorigenic potential of malignant cells (Roberts et al., 1985), which is developed from plate colony formation assay described by Puck et al. in 1956 where cells were seeded on to a culture plate to assay the ability of cells to form colonies (Puck et al., 1956). The limitation of plate colony formation assay is that it only displays cellular abilities for anchorage-dependent growth, by which normal cells can escape from anoikis (a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix) and survive (Taddei et al., 2012). In contrast, malignant cells are capable of proliferating and growing without attachment to a substrate. Therefore, soft agar colony formation assay is developed to characterize this ability in vitro (Hamburger and Salmon, 1977; Yuan et al., 2017). The soft agar colony formation assay has been widely adapted for researches on cell differentiation, transformation and tumorigenesis as well as the efficacy evaluation of anti-tumor treatment.

Materials and Reagents

  1. Cell culture disc (75-cm2) (Corning, catalog number: 430641 )
  2. Cell culture plate (12-well) (Corning, Costar®, catalog number: 3513 )
  3. Falcon 15 ml conical centrifuge tubes (Corning, catalog number: 430791 )
  4. Counting slides (Bio-Rad Laboratories, catalog number: 1450011 )
  5. 0.1-20 ml volume pipette tips (Eppendorf, catalog number: 22492012 )
  6. 5-200 ml volume pipette tips (Eppendorf, catalog number: 22492039 )
  7. 50-1,000 ml volume pipette tips (Eppendorf, catalog number: 22492055 )
  8. Human SGC7901 cell line (Cell Resource Center of the Chinese Academy of Sciences, catalog number: CC-Y1456 )
  9. Phosphate buffered saline (PBS) pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: C10010500BT )
  10. Trypsin-EDTA (0.25%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
  11. RPMI 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: C11875500BT )
  12. Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10099141 )
  13. Penicillin-streptomycin (5,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
  14. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  15. Agar (Biowest, catalog number: 111860 )
  16. Complete 1640 medium (see Recipes)
  17. 5% agar solution (see Recipes)

Equipment

  1. 2-20 μl pipettes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641060N )
  2. 20-200 μl pipettes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641080N )
  3. 100-1,000 μl pipettes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4641100N )
  4. Clean bench (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraguardTM ECO )
  5. Autoclave (TOMY DIGITAL BIOLOGY, model: SX-500 )
  6. Water-Jacketed CO2 incubators (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 , catalog number: 3131)
  7. Thermostat water bath (Prima Technology, model: YB12 )
  8. Centrifuge (Eppendorf, model: 5424 R )
  9. Automated cell counter (Bio-Rad Laboratories, model: TC20TM )
  10. Advanced microscopy group microscope (Thermo Fisher Scientific, model: EVOS )
  11. Gel count colony counter (Oxford optronix, model: GelCountTM )

Software

  1. Statistical Program for Social Sciences 17.0 software (SPSS)

Procedure

  1. Preparing 5% agar solution (see Recipes).
  2. Production of the bottom layer of agar.
    1. Add 9 ml complete medium (37 °C) to 1 ml 5% agar solution (50 °C) and mix thoroughly.
    2. Pipette 0.8 ml mixture to each well of 12-well plate and allow it to solidify for 30 min at room temperature.
  3. Preparation of cell suspension.
    1. Remove the complete medium from culture dish and wash cells with 1x PBS.
    2. Add 0.5 ml 0.25% trypsin (37 °C) for 3-5 min and collect detacted cells by adding complete medium.
    3. Spin cells at 60 x g for 5 min and resuspend cells in complete medium, then count cells and adjust the concentration of cells to 1 x 103 cells/ml.
  4. Production of the upper layer of agar.
    1. Add 9.4 ml re-suspended cells (37 °C) to 0.6 ml 5% agar solution (50 °C) and mix homogeneously.
    2. Pipette 0.8 ml the cell-agar mixture onto the solidified bottom layer of agar in 12-well plate and allow it to solidify for 30 min at room temperature.
    3. Add 800 μl complete medium on top to prevent drying of agar and then cells were maintained in a 37 °C humidified incubator with a mixture of 95% air and 5% CO2.
  5. Clone counting
    1. Monitor colony formation for 2-3 weeks before counting.
    2. All colonies per well were counted with a gel count colony counter, and then determine the average number of colonies of the three replicates for each group.
    3. Capture images of colonies at room temperature using an advanced microscopy group microscope (Figure 1).


      Figure 1. Photographs of representative colonies from SGC7901 cell lines. SGC7901 cells were cultured in the upper layer of agar and the formation of colonies was captured at 2-3 weeks after culture. Scale bars: A, 400 μm; B, 100 μm. 

Data analysis

All statistical data were analyzed with the Statistical Program for Social Sciences 17.0 software (SPSS). The experiments were conducted in triplicates. The results are presented as the mean ± SD. Differences between means were assessed using Student’s t-test or one-way analysis of variance. P < 0.05 was considered to be statistically significant.

Notes

  1. This assay was conducted using human SGC7901 cell line and is applicable to other cancer cell lines.
  2. Incubate cells typically 2-3 weeks and adjust the incubation period according to the tumorgenecity of the cell line.
  3. After autoclaving, agar solution should be kept sterile during the following operation.
  4. Pay attention to the temperature of agar solution and complete medium. It is recommended to keep agar solution and complete medium at 50 °C and 37 °C, respectively, and mix them as soon as possible to avoid in-homogenous agglomeration (Puck et al., 1956).
  5. Do not pour the upper layer of agar until the bottom layer of agar is coagulated completely.
  6. Details of the instruction of gel count colony counter can be obtained from the website: http://www.oxfordoptronix.com/product17/page501/menu2/Colony_Counting/GelCount_/GelCount_.html

Recipes

  1. Complete 1640 medium
    10% FBS
    1% penicillin-streptomycin
    2 mM glutamine
  2. 5% agar solution
    1. Dissolve 5 g agar powder in 100 ml saline and autoclave at 121 °C for 15 min
    2. Place the sterile 5% agar solution in 50 °C water bath to keep it in liquid phase

Acknowledgments

This work was funded by NSFC grants 81602641 to Dr. Xiaodi Zhao.

References

  1. de Larco, J. E. and Todaro, G. J. (1978). Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci U S A 75(8): 4001-4005.
  2. Hamburger, A. W. and Salmon, S. E. (1977). Primary bioassay of human tumor stem cells. Science 197(4302): 461-463.
  3. Puck, T. T., Marcus, P. I. and Cieciura, S. J. (1956). Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer. J Exp Med 103(2): 273-283.
  4. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D. F. and Sporn, M. B. (1985). Type β transforming growth factor: a bifunctional regulator of cellular growth. Proc Natl Acad Sci U S A 82(1): 119-123.
  5. Taddei, M. L., Giannoni, E., Fiaschi, T. and Chiarugi, P. (2012). Anoikis: an emerging hallmark in health and diseases. J Pathol 226(2): 380-393.
  6. Yuan, P., He, X. H., Rong, Y. F., Cao, J., Li, Y., Hu, Y. P., Liu, Y., Li, D., Lou, W. and Liu, M. F. (2017). KRAS/NF-κB/YY1/miR-489 signaling axis controls pancreatic cancer metastasis. Cancer Res 77(1): 100-111.

简介

建立软琼脂集落形成测定法来估计细胞的锚定依赖性生长能力。 在该测定中,首先将具有完全培养基的琼脂的底层倾倒并固化,然后将含有特定数量的悬浮于中等琼脂混合物中的细胞的上层。 孵化两周后,将计数菌落数,作为肿瘤细胞恶性肿瘤的指标。
【背景】无固定生长是细胞在固体表面独立生长的能力,被认为是致癌作用的标志(de Larco和Todaro,1978)。软琼脂集落形成测定是评估细胞粘附非依赖性生长以检测恶性细胞的致瘤性潜力的良好方法(Roberts等人,1985),其由Puck等人描述的板集落形成测定。在1956年,细胞接种到培养板上以测定细胞形成菌落的能力(Puck等,1956)。板集落形成测定的局限性在于它仅显示针对锚定依赖性生长的细胞能力,正常细胞可以从其脱离(一种在锚定依赖性细胞脱离周围的细胞外基质时发生的程序性细胞死亡形式)并生存(Taddei等,2012)。相比之下,恶性细胞能够不附着于底物而增殖和生长。因此,开发了软琼脂集落形成测定法来表征体外这种能力(Hamburger and Salmon,1977; Yuan et al。,2017)。软琼脂集落形成实验广泛适用于细胞分化,转化和肿瘤发生的研究以及抗肿瘤治疗的疗效评价。

关键字:非贴壁依赖性生长, 集落形成, 致癌性, 恶性表型, 琼脂

材料和试剂

  1. 细胞培养盘(75厘米 2 )(康宁,目录号:430641)
  2. 细胞培养板(12孔)(Corning,Costar ®,目录号:3513)
  3. Falcon 15 ml锥形离心管(Corning,目录号:430791)
  4. 计数片(Bio-Rad Laboratories,目录号:1450011)
  5. 0.1-20 ml体积移液器吸头(Eppendorf,目录号:22492012)
  6. 5-200 ml体积移液器吸头(Eppendorf,目录号:22492039)
  7. 50-1,000毫升体积移液器吸头(Eppendorf,目录号:22492055)
  8. 人SGC7901细胞系(中国科学院细胞资源中心,目录号:CC-Y1456)
  9. 磷酸盐缓冲盐水(PBS)pH 7.4(Thermo Fisher Scientific,Gibco TM,目录号:C10010500BT)
  10. 胰蛋白酶-EDTA(0.25%)(Thermo Fisher Scientific,Gibco TM,目录号:25200072)
  11. RPMI 1640培养基(Thermo Fisher Scientific,Gibco TM,目录号:C11875500BT)
  12. 胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:10099141)
  13. 青霉素 - 链霉素(5,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
  14. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  15. 琼脂(Biowest,目录号:111860)
  16. 完成1640培养基(见食谱)
  17. 5%琼脂溶液(参见食谱)

设备

  1. 2-20μl移液管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:4641060N)
  2. 20-200μl移液器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4641080N)
  3. 100-1,000μl移液器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4641100N)
  4. 清洁台(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Heraguard TM ECO)
  5. 高压釜(TOMY DIGITAL BIOLOGY,型号:SX-500)
  6. 将水包覆的CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific,购自,型号:Forma TM系列II 3110,目录号:3131) br />
  7. 恒温水浴(Prima Technology,型号:YB12)
  8. 离心机(Eppendorf,型号:5424 R)
  9. 自动细胞计数器(Bio-Rad Laboratories,型号:TC20 TM
  10. 高级显微镜组显微镜(Thermo Fisher Scientific,型号:EVOS)
  11. 凝胶计数菌落计数器(Oxford optronix,型号:GelCount TM

软件

  1. 社会科学统计计划17.0软件(SPSS)

程序

  1. 准备5%琼脂溶液(参见食谱)。
  2. 生产底层琼脂。
    1. 将9ml完全培养基(37℃)加入到1ml 5%琼脂溶液(50℃)中并充分混合。
    2. 将0.8ml混合物移至12孔板的每个孔中,并使其在室温下固化30分钟。
  3. 细胞悬浮液的制备
    1. 从培养皿中取出完整培养基,用1x PBS洗涤细胞
    2. 加入0.5ml 0.25%胰蛋白酶(37℃)3-5分钟,并通过加入完全培养基收集去细胞
    3. 60℃的旋转细胞5分钟,并将细胞重新悬浮于完全培养基中,然后计数细胞并将细胞浓度调节至1×10 3个细胞/ml。 >
  4. 生产上层琼脂。
    1. 将9.4ml重悬浮细胞(37℃)加入0.6ml 5%琼脂溶液(50℃)中,并均匀混合。
    2. 将细胞琼脂混合物移至凝胶底层的琼脂12孔板中,使其在室温下固化30分钟。
    3. 在顶部加入800μl完全培养基以防止琼脂干燥,然后将细胞保持在37℃加湿培养箱中,混合物为95%空气和5%CO 2。
  5. 克隆计数
    1. 计数前2-3周监测菌落形成。
    2. 用凝胶计数菌落计数器对每孔的每个菌落进行计数,然后确定每组的三次重复的平均菌落数。
    3. 使用高级显微镜组显微镜在室温下拍摄菌落图像(图1)

      图1.来自SGC7901细胞系的代表性菌落的照片。 SGC7901细胞在琼脂上层培养,培养2-3周后捕获菌落形成。刻度棒:A,400μm; B,100μm。 

数据分析

所有统计数据均用社会科学统计学17.0软件(SPSS)进行分析。实验一式三份进行。结果表示为平均值±SD。手段之间的差异使用Student's 测试或单向方差分析进行评估。 0.05被认为具有统计学意义。

笔记

  1. 该测定使用人SGC7901细胞系进行,适用于其他癌细胞系
  2. 孵育细胞通常2-3周,并根据细胞系的肿瘤发生率调整潜伏期
  3. 高压灭菌后,琼脂溶液在下列操作中应保持灭菌
  4. 注意琼脂溶液和完全培养基的温度。建议分别在50°C和37°C保存琼脂溶液和完全培养基,并尽快混合,以避免均匀聚集(Puck等人,1956)。
  5. 不要倒入琼脂的上层,直到琼脂的底层完全凝结。
  6. 凝胶计数菌落计数器说明的细节可从网站获得: http://www.oxfordoptronix.com/product17/page501/menu2/Colony_Counting/GelCount_/GelCount_.html

食谱

  1. 完成1640媒体
    10%FBS
    1%青霉素 - 链霉素
    2 mM谷氨酰胺
  2. 5%琼脂溶液
    1. 将5克琼脂粉末溶于100ml盐水中,121℃高压灭菌15分钟
    2. 将无菌的5%琼脂溶液置于50°C水浴中,使其保持液相

致谢

这项工作由国家自然科学基金委员会拨款81602641资助给赵小迪博士。

参考

  1. de Larco,JE和Todaro,GJ(1978)。  来自鼠肉瘤病毒转化细胞的生长因子。
    Proc Natl Acad Sci USA 75(8):4001-4005。
  2. Hamburger,AW和Salmon,SE(1977)。主要人类肿瘤干细胞的生物测定。 科学 197(4302):461-463。
  3. Puck,TT,Marcus,PI和Cieciura,SJ(1956)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/13286432"target ="哺乳动物细胞在体外的克隆生长;来自具有和不具有饲养层的单个HeLa细胞的菌落的生长特性。实验医学103(2):273-283。
  4. Roberts,AB,Anzano,MA,Wakefield,LM,Roche,NS,Stern,DF and Sporn,MB(1985)。  类型β转化生长因子:细胞生长的双功能调节因子。美国Proc Natl Acad Sci USA 82(1):119 -123。
  5. Taddei,ML,Giannoni,E.,Fiaschi,T.和Chiarugi,P。(2012)。 Anoikis:健康和疾病的新兴标志。 226(2):380-393。
  6. Yuan,P.,He,XH,Rong,YF,Cao,J.,Li,Y.,Hu,YP,Liu,Y.,Li,D.,Lou,W.and Liu,MF(2017) ; KRAS/NF-κB/YY1/miR-489信号轴控制胰腺癌转移。癌症研究 77(1):100-111。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Du, F., Zhao, X. and Fan, D. (2017). Soft Agar Colony Formation Assay as a Hallmark of Carcinogenesis. Bio-protocol 7(12): e2351. DOI: 10.21769/BioProtoc.2351.
  2. Zhao, X. D., Lu, Y. Y., Guo, H., Xie, H. H., He, L. J., Shen, G. F., Zhou, J. F., Li, T., Hu, S. J., Zhou, L., Han, Y. N., Liang, S. L., Wang, X., Wu, K. C., Shi, Y. Q., Nie, Y. Z. and Fan, D. M. (2015). MicroRNA-7/NF-kappaB signaling regulatory feedback circuit regulates gastric carcinogenesis. J Cell Biol 210(4): 613-627.
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