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In vitro Flow Adhesion Assay for Analyzing Shear-resistant Adhesion of Metastatic Cancer Cells to Endothelial Cells
体外流式粘附实验法分析转移癌细胞到内皮细胞的抗剪粘附性   

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Abstract

Hematogenous metastasis is a primary cause of mortality from metastatic cancer. The shear-resistant adhesion of circulating tumor cells to the vascular endothelial cell surface under blood flow is an essential step in cell extravasation and further tissue invasion. This is similar to a process exploited by leukocytes for adhesion to inflamed blood vessels (leukocyte mimicry). The shear resistant adhesion is mediated by high affinity interactions between endothelial adhesion molecules and their counter receptor ligand expressed on circulating cells. Thus, weak interaction results in a rapid detachment of circulating cells from endothelium. Despite the critical role of vascular adhesion of cancer cells in hematogenous metastasis, our knowledge regarding this process has been limited due to the difficulty of mimicking dynamic flow conditions in vitro. In order to gain better insight into the shear-resistant adhesion of cancer cells to the endothelium, we developed a protocol for measuring the shear resistant adhesion of circulating tumor cells to endothelial cells under physiologic flow conditions by adapting a well established flow adhesion assay for inflammatory cells. This technique is useful to evaluate 1) the shear resistant adhesion competency of cancer cells and 2) the endothelial adhesion molecules necessary to support cancer cell adhesion (Kang et al., 2015).

Materials and Reagents

  1. Flow chamber: µ-Slide I 0.4 Luer (ibidi, catalog number: 80176 )


    Figure 1. Flow chamber: µ-Slide I 0.4 Luer (modified the picture from www.ibidi.com)

  2. Serial Connector for µ-Slides (ibidi, catalog number: 10830 )
  3. Syringe (10 ml, 30 ml)
  4. Silicone Tubing (0.8 mm ID) (ibidi, catalog number: 10841 )
  5. 50 ml tube with line connection (SARSTEDT AG & Co, catalog number: 60.596 )
  6. Y tube fitting (ibidi, catalog number: 10828 )
  7. Weight (SP Scienceware, Bel Art, catalog number: F183240000 )
  8. Hose clips (ibidi, catalog number: 10821 )
  9. Human microvascular endothelial cells (HMVEC) (Lonza, catalog number: CC2543 )
  10. Cancer cells
    Cell line or primary cancer cells [e.g., human breast cancer cell line, MDA-MB-231 (ATCC, catalog number: CRM-HTB-26 )]
  11. Endothelial basal medium-2 (Lonza, catalog number: CC-3156 )
  12. EGM SingleQuot Kit (Lonza, catalog number: CC-4133 )
  13. Dulbecco's Modification of Eagle's Medium (DMEM) (Thermo Fisher Scientific, Mediatech, Cellgro®, catalog number: 13-013 ) supplemented with 1% fetal bovine serum (FBS)
  14. Heat Inactivated Fetal Bovine Serum (FBS) (Life Technologies, catalog number: 10082-147 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 10082-147”.
  15. Glutamax (Life Technologies, catalog number: 35050 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 35050 ”.
  16. Antibiotic-antimycotic (Life Technologies, catalog number: 15240 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15240 ”.
  17. Collagen I, Rat Tail (Life Technologies, catalog number: A1048301 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: A1048301”.
  18. Fibronectin from human plasma (Sigma-Aldrich, catalog number: F0895 )
  19. Cell culture medium for HMVEC (see Recipes)
  20. Cell culture medium for Cancer cells (see Recipes)

Equipment

  1. Glass beaker (1,000 ml)
  2. Stir bar
  3. 37 °C, 5% CO2 cell culture incubator (NuAire, model: In-VitroCell ES NU-5800 )
  4. Cell culture centrifuge (Eppendorf, model: 5702 )
  5. Inverted Microscope with camera (Nikon, model: Eclipse TS100 )
  6. Camera (McCrone, model: MicroPublisher 3.3 RTV )
  7. KDS LegatoTM 200 syringe pump (KD Scientific)


    Figure 2. KDS LegatoTM 200 syringe pump (modified picture from http://www.kdscientific.com/products/pumps/Legato200.asp)

  8. Ibidi Heating System [Ibidi Temperature Controller ©ibidimodel (ibidi, model: Version 3.3)]


    Figure 3. Ibidi Heating System (modified picture from http://ibidi.com/xtproducts/en/Instruments-Accessories/Heating-Incubation-Systems/ibidi-Heating-System-Universal-Fit)

  9. Magnetic hot stir (Thermo Fisher Scientific, catalog number: 200-48SH)
  10. Thermometer (Thermo Fisher Scientific, catalog number: 13-201-695 )
  11. Level (Stanley Black & Decker, catalog number: 43-511)

Software

  1. Q-capture pro 7.0 (Qimaging)

Procedure

  1. Grow HMVEC monolayer in flow chamber (µ-Slide I 0.4 Luer)
    1. Dilute collagen (250 µg/ml) and fibronectin (75 µg/ml) with sterilized PBS. Fill the flow chamber with the collagen and fibronectin mixture solution and incubate for 1-3 h in a culture hood at RT (e.g., 100 µl of collagen and fibronectin mixture solution for µ-Slide I 0.4 Luer).
    2. To wash the flow chamber, add sterilized water into one channel end of flow chamber and take out the same volume from the other side channel end. Use 5 times the volume of flow chamber (e.g.,500 µl for µ-Slide I 0.4 Luer).
    3. Fill the chamber with 100 µl of fresh culture medium.
    4. Infuse HMVEC cells (1 x 105 of HMVEC cells in 100 μl HMVEC cells culture medium) into the collagen/fibronectin-coated flow chamber and remove pre-filled culture medium from the opposite side channel end. Do not remove culture medium from step 3 before infusing HMVEC cells to avoid the formation of air bubbles. Fill both Luer reservoirs of flow chamber (Figure 1). Incubate culture until HMVEC cells have grown to 100% confluence, approximately 2-3 days. It is critical to use 100% confluent endothelial monolayer to avoid cancer cells to be stuck in between the endothelial cells. To prevent drying of medium and keep sterile conditions, put caps (provided with flow chamber from manufacturer) on both Luer reservoirs. To change the culture medium inside the flow chamber, first, remove the old medium from both Luer reservoirs not inside the channel. Then, add fresh culture medium into one channel end of the flow chamber and remove the same volume of old medium from the opposite side channel end. Refill both Luer reservoirs of flow chamber (Figure 2).


      Figure 4. Cell seeding in flow chamber-Modified from Ibid Application Note 3 (http://ibidi.com/fileadmin/support/application_notes/AN03_Growing_cells.pdf)


      Figure 5. Change medium in flow chamber. (http://ibidi.com/fileadmin/support/application_notes/AN03_Growing_cells.pdf)

  2. Assemble the flow chamber with pump and perform the flow adhesion assay


    Figure 6. The diagram of overall setting for flow adhesion assay

    1. Assemble reservoirs and turn on the hot plate to warm the water to 37 °C.


      Figure 7. Assemble reservoirs (step B1)

    2. Set the syringe pump next to the inverted microscope and turn ibidi heating system on at 37 °C.
    3. Fill reservoirs A and B with tissue culture medium and wash the line by plunging out with DMEM containing 1% FBS, ensuring there are no air bubbles. Close both tubings with hose clips.
    4. Prepare single cell suspension of cancer cells (MDA-MB-231 cells: 1 x 105 cell/ml in 10 ml of DMEM containing 1% FBS) and add to reservoir A with magnetic stir bar. (Concentration of cells may vary).


      Figure 8. Addition of cells into Reservoir A, corresponding with step B4

    5. Place the flow chamber with HMVEC monolayer onto the ibidi heating system.


      Figure 9. The process of line washing with culture medium via line connected to Reservoir B

    6. Release the hose clip for reservoir B and drip culture media from this reservoir. Attach the serial connector end of tubing to Luer connector (IN). Avoid letting any air bubbles enter the channel.


      Figure 10. Connecting the line to the syringe pump (step B6)

    7. Once the channel is filled with culture media, attach the serial connector from the syringe pump to Luer connector (OUT).
    8. Set the pump for syringe withdrawal at a constant flow rate. The flow rate will vary depending on shear stress calculations for each type of flow chamber. (e.g., for shear stress of 1 dyn/cm2 using µ-Slide I 0.4 Luer, the flow rate setting is 0.76 ml/min. For more information about flow rate and shear stress in different types of flow chambers, check Application Note 11 in http://ibidi.com/fileadmin/support/application_notes/AN11_Shear_stress.pdf). Different shear stresses should be tested. (*See Notes following procedure)


      Figure 11. Leveling the heights of chamber and reservoir

    9. To avoid gravity-based infusion, use a level to ensure the bottom of reservoir A and the flow are at the same height.


      Figure 12. Start of flow adhesion assay

    10. Run the syringe pump and check fluid flow inside the channel under microscope. Ensure there are no bubbles in the route or channel.
    11. Open the hose clip for reservoir A and close the hose clip for reservoir B. Infuse cancer cells (MDA-MB-231 cells: 1 x 105 cell/ml in 10 ml of DMEM containing 1% FBS) for 5 min.
    12. Close reservoir A and open B to wash the line with culture medium.
    13. Count the cancer cells adherent to HUMVEC under the inverted microscope or take photos (at least 5 fields) using Q-capture pro 7.0 and count the adherent cancer cells. Red arrow in Figure 13 shows MDA-MB-231 cells adhering to human endothelial cells at 1 dyn/cm2 for 5 min at 37 °C.


      Figure 13. Image of cancer cell adhesion to monolayer of endothelial cells. Red arrow indicates cancer cells adhering to endothelial cells.

Notes

  1. The adhesion of each cell line is different. Experimental conditions may need to be optimized based on the cells of interest.
  2. It is worthwhile to test different flow rates. We usually use 1-5 dyn/cm2.
  3. It is important that endothelial cells are fully confluent with no empty space in between cells.

Recipes

  1. Cell culture medium for HMVEC
    Endothelial basal medium-2 supplemented with 2% FBS
    EGM SingleQuot Kit containing:
    Epidermal growth factor (EGF) 10 ng/ml
    Hydrocortisone 1 μg/ml
    Gentamicin 30 μg/ml
    Amphotericin B (GA-1000) 15 ng/ml
  2. Cell culture medium for cancer cells (e.g., MDA-MB-231)
    Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
    1% glutamax
    1% antibiotic-antimycotic

Acknowledgments

This work was supported by the Department of Defense (W81XWH-11-1-0238 to T. T), the National Institutes of Health (1R01CA160271-01A1 to T. T), the American Cancer Society (IRG-08-060-04), and the Pennsylvania Breast Cancer Coalition (T. T). Images were shared by ibidi USA, Inc., Madison WI (ibidi.com).

References

  1. Kang, S. A., Hasan, N., Mann, A. P., Zheng, W., Zhao, L., Morris, L., Zhu, W., Zhao, Y. D., Suh, K. S., Dooley, W. C., Volk, D., Gorenstein, D. G., Cristofanilli, M., Rui, H. and Tanaka, T. (2015). Blocking the adhesion cascade at the premetastatic niche for prevention of breast cancer metastasis. Mol Ther 23(6): 1044-1054.

简介

血液转移是转移性癌症死亡的主要原因。循环肿瘤细胞对血流内的血管内皮细胞表面的剪切粘附是细胞外渗和进一步组织侵入的必要步骤。这类似于白细胞用于粘附到发炎血管的方法(白细胞模拟)。剪切抗性粘附由内皮粘附分子和其在循环细胞上表达的其受体配体之间的高亲和力相互作用介导。因此,弱相互作用导致循环细胞从内皮快速分离。尽管癌细胞在血源性转移中的血管粘附的关键作用,我们关于该过程的知识由于难以在体外模拟动态流动条件而受到限制。为了更好地了解癌细胞对内皮的剪切抗性粘附,我们开发了一种用于测量在生理流动条件下循环肿瘤细胞对内皮细胞的剪切抗性粘附的方案,通过适应良好确立的流动粘附测定炎症细胞。该技术可用于评价1)癌细胞的抗剪切粘附能力和2)支持癌细胞粘附所必需的内皮粘附分子(Kang等人,2015)。

材料和试剂

  1. 流动室:μ-Slide I 0.4 Luer(ibidi,目录号:80176)


    图1.流动室:μ-Slide I 0.4 Luer(修改了来自 www.ibidi.com

  2. μ-Slides串行连接器(ibidi,目录号:10830)
  3. 注射器(10ml,30ml)
  4. 硅胶管(0.8mm ID)(ibidi,目录号:10841)
  5. 50ml具有管线连接的管(SARSTEDT AG& Co,目录号:60.596)
  6. Y管配件(ibidi,目录号:10828)
  7. 重量(SP Scienceware,Bel Art,目录号:F183240000)
  8. 软管夹(ibidi,目录号:10821)
  9. 人微血管内皮细胞(HMVEC)(Lonza,目录号:CC2543)
  10. 癌细胞
    细胞系或原发性癌细胞[例如人乳腺癌细胞系MDA-MB-231(ATCC,目录号:CRM-HTB-26)]
  11. 内皮基础培养基-2(Lonza,目录号:CC-3156)
  12. EGM SingleQuot Kit(Lonza,目录号:CC-4133)
  13. 补充有1%胎牛血清(FBS)的Dulbecco's Modification of Eagle's Medium(DMEM)(Thermo Fisher Scientific,Mediatech,Cellgro ,目录号:13-013)
  14. 热灭活胎牛血清(FBS)(Life Technologies,目录号:10082-1??47)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:10082-1??47。
  15. Glutamax(Life Technologies,目录号:35050)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:35050" />
  16. 抗生素 - 抗真菌剂(Life Technologies,目录号:15240)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:15240" />
  17. 胶原I,鼠尾(Life technologies,目录号:A1048301)
    注意:目前,"赛默飞世尔科技,Gibco TM ,目录号:A1048301" />
  18. 来自人血浆的纤连蛋白(Sigma-Aldrich,目录号:F0895)
  19. 用于HMVEC的细胞培养基(参见配方)
  20. 癌细胞的细胞培养基(参见配方)

设备

  1. 玻璃烧杯(1000ml)
  2. 搅拌棒
  3. 37℃,5%CO 2细胞培养箱(NuAire,型号:In-VitroCell ES NU-5800)中培养。
  4. 细胞培养离心机(Eppendorf,型号:5702)
  5. 带相机的倒置显微镜(尼康,型号:Eclipse TS100)
  6. 相机(McCrone,型号:MicroPublisher 3.3 RTV)
  7. KDS Legato TM 200注射泵(KD Scientific)


    图2. KDS Legato TM 200注射泵(http://www.kdscientific.com/products/pumps/Legato200.asp

  8. Ibidi加热系统[Ibidi Temperature Controller© ibidimodel(ibidi,model:Version 3.3)]


    图3. Ibidi供热系统( http://ibidi.com/xtproducts/en/Instruments-Accessories/Heating-Incubation-Systems/ibidi-Heating-System-Universal-Fit

  9. 磁力搅拌(Thermo Fisher Scientific,目录号:200-48SH)
  10. 温度计(Thermo Fisher Scientific,目录号:13-201-695)
  11. Level(Stanley Black& Decker,目录号:43-511)

软件

  1. Q捕获pro 7.0(Qimaging)

程序

  1. 在流动室中生长HMVEC单层(μ-Slide I 0.4 Luer)
    1. 稀释胶原(250μg/ml)和纤维连接蛋白(75μg/ml),灭菌 PBS。用胶原和纤连蛋白混合物填充流动室 溶液中,并在RT下在培养罩中孵育1-3小时(例如100μl 胶原和纤连蛋白混合物溶液用于μ-Slide I 0.4 Luer)。
    2. 要清洗流动室,请将无菌水加入一个通道末端 并从另一侧通道取出相同的体积 ?结束。对于μ-Slide I,使用5倍体积的流动室(例如500μl) 0.4 Luer)。
    3. 填充室100μl的新鲜培养基。
    4. 在100μlHMVEC细胞中浸泡HMVEC细胞(1×10 5个HMVEC细胞) 培养基)注射到涂布有胶原/纤连蛋白的流动室中 从相对侧通道末端除去预填充的培养基。做 在输注HMVEC细胞之前不从步骤3除去培养基 避免气泡的形成。填充两个鲁尔油藏 (图1)。孵育培养物直至HMVEC细胞生长至 100%汇合,约2-3天。关键是使用100% 汇合的内皮单层以避免癌细胞被卡住 在内皮细胞之间。防止介质干燥和保持 无菌条件下,放上盖子(提供流动室 制造商)在两个鲁尔水库。更换培养基 在流动室内,首先,从两个鲁尔去除旧介质 水库不在通道内。然后,加入新鲜培养基 一个通道端的流动室和去除相同体积的旧 介质从相对侧通道端。重新填充两个鲁尔水库 的流动室(图2)。


      图4.流动中的细胞接种 chamber - 修改自Ibid Application Note 3 ( http://ibidi.com/) fileadmin/support/application_notes/AN03_Growing_cells.pdf


      图5.更改流动室中的介质。 ( http://ibidi.com/fileadmin/support/application_notes/AN03_Growing_cells。 pdf

  2. 用泵装配流动室,进行流动粘附试验


    图6.流动粘附测定的整体设置图

    1. 装配水库,打开热板,将水加热至37°C

      图7.装配油藏(步骤B1)

    2. 将注射泵旁边的倒置显微镜,并在37℃开启ibidi加热系统
    3. 填充容器A和B与组织培养基和洗线 ?通过用含有1%FBS的DMEM刺入,确保没有空气 气泡。用软管夹关闭两个管。
    4. 准备单细胞 悬浮的癌细胞(MDA-MB-231细胞:1×10 5个细胞/ml,在10ml 的含有1%FBS的DMEM),并加入到磁力搅拌的储存器A中 酒吧。 (细胞浓度可能不同)。


      图8.将细胞添加到水库A中,对应于步骤B4

    5. 将具有HMVEC单层的流动室置于ibidi加热系统上。


      图9.使用培养基通过连接到水库B的线进行线洗的过程

    6. 释放储存器B的软管夹,并从中取出培养基 这个水库。将管道的串行连接器端连接到鲁尔 连接器(IN)。避免让任何气泡进入通道。


      图10.将管线连接到注射泵(步骤B6)

    7. 一旦通道充满培养基,将注射器泵的串行连接器连接到Luer连接器(OUT)
    8. 以恒定流速设置注射器取出泵。的 流速将根据每种类型的剪切应力计算而变化 ?的流动室。 (例如,对于使用μ-滑动I的剪切应力为1达因/厘米<2> 0.4 Luer,流速设定为0.76ml/min。了解更多信息 关于不同类型流动室中的流速和剪切应力, 请检查 http://ibidi.com/fileadmin/support/application_notes/AN11_Shear_stress中的应用笔记11。 pdf )。 ?应测试不同的剪切应力。 (*见下面的注释 程序)


      图11.平衡腔室和水库的高度

    9. 为了避免基于重力的输注,使用水平仪来确保储存器A的底部和流动处于相同的高度。


      图12.开始流动粘附测定

    10. 运行注射器泵并检查下面通道内的流体流量 显微镜。确保路径或通道中没有气泡。
    11. 打开储液罐A的软管夹,并关闭软管夹 输注癌细胞(MDA-MB-231细胞:1×10 5个细胞/ml 10ml含1%FBS的DMEM)中5分钟
    12. 关闭水库A,打开B,用培养基冲洗管道。
    13. 计数粘附在HUMVEC下的癌细胞 显微镜或拍照(至少5个字段)使用Q捕获pro 7.0 并计数粘附的癌细胞。图13中的红色箭头显示 MDA-MB-231细胞在1dyn/cm 2下粘附于人内皮细胞5天 min在37℃

      图13.癌细胞粘附至单层的图像 的内皮细胞。 红色箭头表示癌细胞粘附 内皮细胞。

笔记

  1. 每个细胞系的粘附是不同的。实验条件可能需要基于感兴趣的细胞进行优化。
  2. 测试不同流速是值得的。我们通常使用1-5 dyn/cm 2
  3. 重要的是内皮细胞完全融合,在细胞之间没有空间。

食谱

  1. HMVEC的细胞培养基
    补充有2%FBS的内皮基础培养基-2 EGM SingleQuot套件包含:
    表皮生长因子(EGF)10ng/ml
    氢化可的松1μg/ml
    庆大霉素30μg/ml
    两性霉素B(GA-1000)15ng/ml
  2. 用于癌细胞的细胞培养基(例如MDA-MB-231)
    补充有10%胎牛血清(FBS)的Eagle培养基(DMEM)的Dulbecco's修饰
    1%glutamax
    1%抗生素 - 抗真菌剂

致谢

这项工作由国防部(W81XWH-11-1-0238到T.T),国家卫生研究院(1R01CA160271-01A1到T.T),美国癌症协会(IRG-08-060-04 )和宾夕法尼亚州乳腺癌联盟(T.T)。图像由ibidi USA,Inc.,Madison WI(ibidi.com)共享。

参考文献

  1. Kang,SA,Hasan,N.,Mann,AP,Zheng,W.,Zhao,L.,Morris,L.,Zhu,W.,Zhao,YD,Suh,KS,Dooley,WC,Volk, Gorenstein,DG,Cristofanilli,M.,Rui,H。和Tanaka,T。(2015)。 阻断前转移间隙处的粘连级联,用于预防乳腺癌转移。 Mol Ther 23(6):1044-1054。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kang, S., Bajana, S. and Tanaka, T. (2016). In vitro Flow Adhesion Assay for Analyzing Shear-resistant Adhesion of Metastatic Cancer Cells to Endothelial Cells. Bio-protocol 6(4): e1731. DOI: 10.21769/BioProtoc.1731.
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