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Jul 2018

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Analysis of Random Migration of Cancer Cells in 3D
癌细胞随机迁移的3D分析   

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Abstract

The ability of cancer cells to migrate through a complex three-dimensional (3D) environment is a hallmark event of cancer metastasis. Therefore, an in vitro migration assay to evaluate cancer cell migration in a 3D setting is valuable to examine cancer progression. Here, we describe such a simple migration assay in a 3D collagen-fibronectin gel for observing cell morphology and comparing the migration abilities of cancer cells. We describe below how to prepare the collagen-fibronectin gel castings, how to set up time-lapse recording, how to draw single-cell trajectories from movies and extract key parameters that characterize cell motility, such as cell speed, directionality, mean square displacement, and directional persistence. In our set-up, cells are sandwiched in a single plane between two collagen-fibronectin gels. This trick facilitates the analysis of cell tracks, which are for the most part 2D, at least in the beginning, but in a 3D environment. This protocol has been previously published in Visweshwaran et al. (2018) and is described here in more detail.

Keywords: Collagen gel (胶原凝胶), 3D migration (三维迁移), Cell motility (细胞运动), MDA-MB-231 (MDA-MB-231), Cell tracking (细胞示踪), MSD (MSD), Directionality (方向性), Cell speed (细胞速度)

Background

The migration of cells within our body is an essential process and driven by complex underlying cellular functions. It plays an important role in many physiological processes, including development, immune responses, and tissue regeneration (Kunwar et al., 2006; Friedl and Weigelin, 2008; Reig et al., 2014). In addition, certain pathological situations such as tumor invasion and metastasis rely on cell motility (Thiery, 2002). Consequently, cell migration has become a major field of study in the context of both fundamental and translational research. Although in the last decade we have witnessed enormous advances in the understanding of the mechanisms underlying the highly plastic process of cell migration, many questions remain open. Especially, the complex regulation of migration is still unclear within differently composed microenvironments that are crowded by cells and extracellular matrix (Petrie et al., 2009; Friedl and Wolf, 2010).

To analyze cell migration, several in vitro and in vivo cell migration assays have been developed over the years. Thein vivo cell migration assays most closely mimic the real physiological situation and observe cells within their natural environment with its complexities of variable extracellular matrix (ECM) composition, geometry, topography and pore size. However, performing such experiments is labor- and cost-intensive, time-consuming, tough to control and requires advanced imaging techniques and animal experiments. Due to such practical challenges, cell migration has traditionally been studied on two-dimensional (2D) surfaces (Dang and Gautreau, 2018), e.g., in the context of wound-healing assays (Molinie and Gautreau, 2018). While this works to some extent for adherent cells such as breast epithelial carcinoma cells, 2D migration assays have little physiological relevance and thus little predictive value. Moreover, the striking difference between the 2D and the 3D settings becomes understandable in the light of recent studies of cell motility (Lämmermann et al., 2009; Friedl et al., 2012; Petrie and Yamada, 2016). These recent studies demonstrate that cell migration is a very plastic process and the cells embedded in 3D matrices composed of collagens or matrigel employ a very different locomotory machinery than cells on 2D surfaces. Therefore, studying the migration of cells embedded within a physiological-like 3D environment could lead to the results that have more significance and better predictive value.

Apart from being easier to perform than true in vivo migration experiments, 3D migration assays with their simpler matrix composition offer the advantage of a controlled, easily manipulable environment. Thus, it facilitates easy dissection of molecular mechanisms and the interpretation of experimental results. The 3D migration studies are usually done with the help of Boyden chambers (e.g., transwell assays [Visweshwaran et al., 2018]). However, these assays typically provide only an endpoint readout of cell migration efficiency, thereby not much information derived for an in-depth analysis. In contrast, real-time microscopy-based 3D assays allow one to observe and track individual cells, and thus the analysis of various parameters such as cell morphology during migration, cell speed, and directionality.

To our knowledge, there are not many optimal methods available for 3D cell migration analysis. All existing methods have their limitations. They either allow the experimenter to use a set-up where the embedded cells in the collagen gel are prone to migrate randomly in all directions including Z-axis, which render them hard to track and quantify migration parameters, or compel the experimenter to use complex, hard-to-handle and often costly setups (Rommerswinkel et al., 2014; Biswenger et al., 2018) to perform 3D migration assays. To overcome these limitations, we have developed a set-up where cells are sandwiched in a single plane between two collagen-fibronectin gels. This procedure facilitates the analysis of cell tracks, which are for the most part 2D, at least in the beginning, but in a 3D environment. In essence, here we describe an easy method for performing and analyzing 3D migration assays based on a home-made set-up and an easy analysis pipeline.

Materials and Reagents

  1. µ-slide 8 well with a glass bottom (Ibidi, catalog number: 80827)
  2. Cell line: MDA-MB-231 (ATCC, catalog number: HTB-26)
  3. L-15 medium (Thermo Fisher Scientific, catalog number: 11415049)
  4. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: A31605)
  5. Penicillin-Streptomycin (100x) (Thermo Fisher Scientific, catalog number: 15140122)
  6. Trypsin (0.25%) (Thermo Fisher Scientific, catalog number: 15050065)
  7. DPBS (Thermo Fisher Scientific, catalog number: 14190250)
  8. 10x MEM (Thermo Fisher Scientific, catalog number: 11430030)
  9. HEPES (Thermo Fisher Scientific, catalog number: 15630080)
  10. Collagen I (Rat-tail, 4.41 mg/ml) (BD Biosciences, catalog number: 354236)
  11. Fibronectin (5 mg) (FN) (Sigma, catalog number: F1141)
  12. MDA-MB-231 culture medium (see Recipes)
  13. Collagen-Fibronectin gel mix (see Recipes)

Equipment

  1. Phase contrast microscope with 20x air objective, temperature module, CO2 module and auto focus module (Zeiss Axio Observer microscope)
  2. 37 °C cell culture incubator
  3. Laminar airflow hood

Software

  1. Fiji: ImageJ with MTrackJ plugin
    Note: Fiji: ImageJ, software could be downloaded from https://imagej.net/Fiji/Downloads and for MTrackJ plugin information go to https://imagescience.org/meijering/software/mtrackj/.
  2. Microsoft (MS) Excel 2007 or later

Procedure

  1. Prepare the appropriate volume of collagen-fibronectin mix as described in Recipe 2.
  2. Add 100 µl of gel mix per well of µ-Slide 8 well and let it polymerize at 37 °C for 1 h (Figure 1A).
    Note: After gelification, collagen-fibronectin gel becomes slightly milky white and collagen fibers become uniformly visible.
  3. During this gelification period, trypsinize the cells and count them. Set the cell concentration to 104 cells per ml.
  4. After gel polymerizes, put 200 µl of medium containing cells (i.e., 2,000 cells) on top of the gel.
  5. Put µ-slide 8 well in the incubator at 37 °C and allow the cells to adhere on the gel surface for about 2 h (Figure 1A).
  6. Once cells have adhered, remove the medium gently. This can be done by slowly pipetting out the medium from the corners of the µ-slide well.
    Note: This step is delicate. While removing the medium, extra care must be taken not to disturb the gel and cells. If the gel gets disturbed/damaged, then they are not good for imaging.
  7. After removing the medium, keep the µ-slide aside in the laminar airflow hood and quickly, re-prepare the collagen-fibronectin mix.
  8. Put 100 µl of gel mix on the top of the cells adhering to the previously casted gel (Figure 1A).
  9. Incubate for 1 h at 37 °C in the incubator for the gel to polymerize.
    Note: After gelification of the second layer of gel, one can observe cells that are sandwiched between two gels in a single z plane.
  10. Meanwhile, switch on the microscope and the microscope stage heater. Adjust the temperature to 37 °C.
  11. After gelification of the second layer gel, add 150 µl of complete medium on top of the second gel to ensure the gels do not dry out (Figure 1A).
  12. Carefully place the µ-slide into a microscope insert designed to hold the slides and allowing for temperature, humidity, and CO2 control.
  13. Place the insert onto the microscope stage. Set the focus on the cells that are at the center of well. Use a 20x objective. Use an objective with phase contrast (Figure 1B).
    Note: The center of the µ-slide wells is best for imaging to avoid aberrations close to the sides of the wells.
  14. Acquire time-lapse images for 60 h with a time-lapse interval of 20 min.


    Figure 1. Steps in 3D migration setup. A. Schematic overview depicting the steps involved in the protocol. B. Exemplary time-lapse images of the MDA-MB-231 cells that are sandwiched between two collagen-fibronectin gels, depicting the cell morphology changes during its 3D migration. Scale bar: 40 µm. For the exemplary depiction of the 3D migration video, refer to Visweshwaran et al., 2018, Movie EV2.

Data analysis

  1. Once the video acquisition is finished, assign the image properties. Open the video/image stack file in Fiji and assign the image properties by selecting ‘Image’ → ‘Properties…’ and fill in ‘Unit of length’ (example-µm), ‘Pixel width’ (pixel size in µm), ‘Pixel height’ (pixel size in µm) and ‘Frame interval’ (20 min). Save the video/image stack. A similar assignment can also be done by selecting ‘Analyze’ → ‘Set scale’ option.
  2. Manual tracking and analysis of cell trajectories are done as described in Dang and Gautreau, 2018, and Gorelik and Gautreau, 2014. Briefly, manual tracking performed with the help of Fiji software plugin 'MTrackJ', which gives the trajectory data. These data are then copied to MS Excel file containing DiPer suite of custom-made macros for quantifying migration parameters. By running the DiPer macros, various migration parameters like cell speed, directional persistence (direction autocorrelation), and mean squared displacement (MSD) are calculated and plotted, which are used to make publication-quality figures (Figure 2).
  3. Automated tracking and analysis of cell trajectories are also possible for this set-up. Visweshwaran and Maritzen, 2019 describes a suitable procedure to quantify 3D cell migration automatically with minimal input by the experimenter.
    Note: MS Excel file loaded with DiPer suite of macros are available as supplementary file: Excel_loaded_with_DiPer. Detailed description about DiPer and the migration parameters like cell speed, directional persistence (direction autocorrelation) and mean squared displacement (MSD) available in Gorelik and Gautreau, 2014.


    Figure 2. Representative 3D migration quantification of HSBP1 depleted MDA-MB-231 cells. Please refer to Visweshwaran et al. (2018) for details. Briefly, top panels display single-cell trajectories. Mean Square Displacement (MSD) analysis shows that HSBP1 depleted cells explore a smaller territory than control cells. This effect might be accounted for by decreased speed and directional persistence. At least 10 cells were tracked per condition, in 3 independent experiments; data are mean ± S.E.M; ANOVA **P < 0.01; ****P  <  0.0001.

Notes

  1. When MDA-MB-231 cells are cultured in Leibovitz’s L-15 medium, no CO2 should be used for pH control. The L-15 medium formulation was devised for use with atmospheric air. Incubation in an atmosphere enriched with CO2 would be detrimental to cells.
  2. For running macros to analyze the cell trajectories, ‘Developer’ tab in the MS Excel must be enabled. The ‘Developer’ tab is enabled by going to ‘Excel Options’ → ‘Customize Ribbon’ → ‘Customize the Ribbon’ → ‘Main tab’ → checking ‘Developer’.
  3. The effects of drugs/inhibitors on 3D migration of the cells could be investigated in this set-up. For a long-term effect analysis, the drug could be added while making the collagen-fibronectin gels and in the medium that is added at the final stage to prevent the gel drying. In this method, cells are exposed constantly to the drug for a full period of observation. On the other end, the addition of drugs/inhibitors during 3D migration is also possible. In this method, a high concentration of drug should be added (1-10 µl) gently to the medium that is present on the top of the gels to prevent the gel drying. Here, the drug will diffuse into the gels. In this case, the experimenter initially would record a 3D cell migration for a period where there is no drug treatment and after addition of the drug, a period of 3D cell migration under drug treatment. If the drugs/inhibitors are toxic, a brief pre-treatment of the cells with the drug and sandwiching them between gels for migration analysis are also an alternative method.

Recipes

  1. MDA-MB-231 culture medium
    L-15 medium
    10% FBS
    1x Penicillin-Streptomycin
  2. Collagen-Fibronectin gel mix
    2 mg/ml Collagen I
    10 µg/ml FN
    25 mM HEPES
    10% FBS
    1x MEM completed with sterile water
    For 1 ml gel volume:
    10x MEM
    100 µl
    HEPES (1 M)
    25 µl
    Collagen I (4.41 mg/ml)
    452 µl
    FBS
    100 µl
    FN
    10 µl
    Sterile water
    313 µl

Acknowledgments

This protocol was adapted from Dang et al. (2013) with modifications. The establishment of this protocol was funded by AG’s group grant: the Agence Nationale de la Recherche (ANR ANR-15-CE13-0016-01), the fondation ARC pour la Recherche sur le Cancer (PGA120140200831), and Institut National du Cancer (INCA_6521). SPV was supported by PhD fellowships from Ministère de l'Enseignement Supérieur et de la Recherche for the first 3 years and from Ligue Nationale contre le Cancer for the 4th year.

Competing interests

The authors declare that they have no conflict of interests.

References

  1. Biswenger, V., Baumann, N., Jurschick, J., Hackl, M., Battle, C., Schwarz, J., Horn, E. and Zantl, R. (2018). Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system. PLoS One 13(9): e0203040.
  2. Dang, I. and Gautreau, A. (2018). Random migration assays of mammalian cells and quantitative analyses of single cell trajectories. Methods Mol Biol 1749: 1-9.
  3. Dang, I., Gorelik, R., Sousa-Blin, C., Derivery, E., Guerin, C., Linkner, J., Nemethova, M., Dumortier, J. G., Giger, F. A., Chipysheva, T. A., Ermilova, V. D., Vacher, S., Campanacci, V., Herrada, I., Planson, A. G., Fetics, S., Henriot, V., David, V., Oguievetskaia, K., Lakisic, G., Pierre, F., Steffen, A., Boyreau, A., Peyrieras, N., Rottner, K., Zinn-Justin, S., Cherfils, J., Bieche, I., Alexandrova, A. Y., David, N. B., Small, J. V., Faix, J., Blanchoin, L. and Gautreau, A. (2013). Inhibitory signalling to the Arp2/3 complex steers cell migration. Nature 503(7475): 281-284.
  4. Friedl, P. and Weigelin, B. (2008). Interstitial leukocyte migration and immune function. Nat Immunol 9(9): 960-969.
  5. Friedl, P. and Wolf, K. (2010). Plasticity of cell migration: a multiscale tuning model. J Cell Biol 188(1): 11-19.
  6. Friedl, P., Sahai, E., Weiss, S. and Yamada, K. M. (2012). New dimensions in cell migration. Nat Rev Mol Cell Biol 13(11): 743-747.
  7. Gorelik, R. and Gautreau, A. (2014). Quantitative and unbiased analysis of directional persistence in cell migration. Nat Protoc 9(8): 1931-1943.
  8. Kunwar, P. S., Siekhaus, D. E. and Lehmann, R. (2006). In vivo migration: a germ cell perspective. Annu Rev Cell Dev Biol 22: 237-265.
  9. Lämmermann, T. and Sixt, M. (2009). Mechanical modes of 'amoeboid' cell migration. Curr Opin Cell Biol 21(5): 636-644.
  10. Molinie, N. and Gautreau, A. (2018). Directional collective migration in wound healing assays. Methods Mol Biol 1749: 11-19.
  11. Petrie, R. J. and Yamada, K. M. (2016). Multiple mechanisms of 3D migration: the origins of plasticity. Curr Opin Cell Biol 42: 7-12.
  12. Petrie, R. J., Doyle, A. D. and Yamada, K. M. (2009). Random versus directionally persistent cell migration. Nat Rev Mol Cell Biol 10(8): 538-549.
  13. Reig, G., Pulgar, E. and Concha, M. L. (2014). Cell migration: from tissue culture to embryos. Development 141(10): 1999-2013.
  14. Rommerswinkel, N., Niggemann, B., Keil, S., Zanker, K. S. and Dittmar, T. (2014). Analysis of cell migration within a three-dimensional collagen matrix. J Vis Exp (92): e51963.
  15. Thiery, J. P. (2002). Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2(6): 442-454.
  16. Visweshwaran, S. P., Thomason, P. A., Guerois, R., Vacher, S., Denisov, E. V., Tashireva, L. A., Lomakina, M. E., Lazennec-Schurdevin, C., Lakisic, G., Lilla, S., Molinie, N., Henriot, V., Mechulam, Y., Alexandrova, A. Y., Cherdyntseva, N. V., Bieche, I., Schmitt, E., Insall, R. H. and Gautreau, A. (2018). The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes. EMBO J 37(13): pii: e97706.
  17. Visweshwaran, S. P. and Maritzen, T. (2019). A simple 3D cellular chemotaxis assay and analysis workflow suitable for a wide range of migrating cells. MethodsX 6: 2807-2821.

简介

癌细胞在复杂的三维(3D)环境中迁移的能力是癌症转移的标志性事件。因此,用于评估3D环境中癌细胞迁移的体外迁移分析对于检查癌症进展具有重要意义。在这里,我们描述了一种在3D胶原纤连蛋白凝胶中的简单迁移试验,用于观察细胞形态并比较癌细胞的迁移能力。我们将在下面介绍如何准备胶原纤维连接蛋白凝胶铸件,如何设置延时记录,如何从电影中绘制单细胞轨迹以及如何提取表征细胞运动性的关键参数,例如细胞速度,方向性,均方位移以及定向持久性。在我们的设置中,细胞被夹在两个胶原-纤连蛋白凝胶之间的单个平面中。此技巧有助于对单元轨迹进行分析,至少在开始时,但在3D环境中,大多数情况下是2D的。该协议先前已在Visweshwaran et al。(2018)中发布,并在此进行了更详细的描述。
【背景】细胞在我们体内的迁移是一个必不可少的过程,并由复杂的基础细胞功能驱动。它在许多生理过程中都起着重要作用,包括发育,免疫应答和组织再生(Kunwar等,2006; Friedl和Weigelin,2008; Reig等。) >,2014年)。此外,某些病理情况,例如肿瘤的侵袭和转移依赖于细胞运动性(Thiery,2002)。因此,在基础研究和转化研究的背景下,细胞迁移已成为研究的主要领域。尽管在过去的十年中,我们已经看到了对高度可塑性细胞迁移过程的机制的理解的巨大进步,但仍有许多问题尚待解决。特别是,在由细胞和细胞外基质拥挤的不同组成的微环境中,迁移的复杂调控机制仍不清楚(Petrie et al。,2009; Friedl和Wolf,2010)。

为了分析细胞迁移,这些年来已经开发了几种体外和体内细胞迁移测定法。体内细胞迁移测定最接近地模拟了真实的生理状况,并通过可变的细胞外基质(ECM)组成,几何形状,形貌和孔径大小的复杂性来观察自然环境中的细胞。然而,进行这样的实验是劳动和成本密集,费时,难以控制的,并且需要先进的成像技术和动物实验。由于此类实际挑战,传统上已在伤口愈合检测的背景下(Molinie和Gautreau,2018)研究了二维(2D)表面上的细胞迁移(Dang和Gautreau,2018) eg )。尽管这在一定程度上对诸如乳腺上皮癌细胞之类的贴壁细胞有效,但2D迁移分析的生理相关性很小,因此预测价值也很小。此外,根据最近对细胞运动性的研究,可以理解2D和3D设置之间的显着差异(Lämmermann等,2009; Friedl等, 2012; Petrie和Yamada,2016)。这些最新研究表明,细胞迁移是一个非常可塑性的过程,嵌入到由胶原蛋白或基质胶组成的3D矩阵中的细胞与2D表面上的细胞具有非常不同的运动机制。因此,研究嵌入在类似生理3D环境中的细胞的迁移可能会导致结果具有更大的意义和更好的预测价值。

除了比真正的体内迁移实验更容易执行之外,具有更简单基质组成的3D迁移测定法还提供了受控,易于操作的环境的优势。因此,它促进了分子机理的简单分解和实验结果的解释。3D迁移研究通常是在博伊登会议厅的协助下进行的( eg ,transwell分析[Visweshwaran et al。,2018])。然而,这些测定法通常仅提供细胞迁移效率的终点读数,因此没有太多信息可用于深入分析。相反,基于实时显微镜的3D分析可以观察和跟踪单个细胞,因此可以分析各种参数,例如迁移过程中的细胞形态,细胞速度和方向性。
据我们所知,没有很多可用于3D细胞迁移分析的最佳方法。所有现有方法都有其局限性。它们或者允许实验者使用一种设置,即胶原蛋白凝胶中嵌入的细胞易于在包括Z轴在内的所有方向上随机迁移,从而使其难以跟踪和量化迁移参数,或者迫使实验者使用复杂的结构。 ,难以处理且通常昂贵的设置(Rommerswinkel等,2014; Biswenger等,2018)来执行3D迁移分析。为了克服这些局限性,我们开发了一种装置,其中细胞被夹在两个胶原纤连蛋白凝胶之间的单个平面中。此过程有助于对单元轨迹进行分析,至少在开始时,但在3D环境中,大多数情况下是2D的。实质上,在这里,我们描述了一种基于自制的设置和简便的分析流程执行和分析3D迁移测定的简便方法。

关键字:胶原凝胶, 三维迁移, 细胞运动, MDA-MB-231, 细胞示踪, MSD, 方向性, 细胞速度

材料和试剂

  1. 带玻璃底部的µ-slide 8孔(Ibidi,目录号:80827)
  2. 细胞系:MDA-MB-231(ATCC,目录号:HTB-26)
  3. L-15培养基(Thermo Fishers Scientific,目录号:11415049)
  4. 胎牛血清(FBS)(Thermo Fishers Scientific,目录号:A31605)
  5. 青霉素-链霉素(100x)(Thermo Fishers Scientific,目录号:15140122)
  6. 胰蛋白酶(0.25%)(Thermo Fishers Scientific,目录号:15050065)
  7. DPBS(Thermo Fishers Scientific,目录号:14190250)
  8. 10x MEM(Thermo Fishers Scientific,目录号:11430030)
  9. HEPES(Thermo Fishers Scientific,目录号:15630080)
  10. 胶原I(尾巴,4.41 mg / ml)(BD Biosciences,目录号354236)
  11. 纤连蛋白(5 mg)(FN)(Sigma,目录号:F1141)
  12. MDA-MB-231培养基(请参阅食谱)
  13. 胶原-纤连蛋白凝胶混合物(请参阅食谱)

设备

  1. 相衬显微镜,具有20倍空气物镜,温度模块,CO 2 模块和自动聚焦模块(Zeiss Axio Observer显微镜)
  2. 37°C细胞培养培养箱
  3. 层流通风罩

软件

  1. 斐济:带有MTrackJ插件的ImageJ
    注意:斐济:ImageJ,可以从 https:// imagej 下载软件。 net / Fiji / Downloads ,有关MTrackJ插件的信息,请访问 https://imagescience.org/meijering/software/mtrackj/ 。
  2. Microsoft(MS)Excel 2007或更高版本

程序

  1. 按照食谱2中所述准备适当体积的胶原蛋白-纤连蛋白混合物。
  2. 每孔µ-Slide 8孔中加入100 µl凝胶混合物,使其在37°C下聚合1小时(图1A)。
    注意:胶凝后,胶原纤连蛋白凝胶变成淡乳白色,并且胶原纤维均匀可见。
  3. 在此凝胶化期间,用胰蛋白酶消化细胞并计数。将细胞浓度设置为每毫升10 4 个细胞。
  4. 凝胶聚合后,将200 µl含细胞的培养基( ie ,2,000个细胞)放在凝胶顶部。
  5. 将µ-slide 8放入37°C的培养箱中,并使细胞粘附在凝胶表面约2小时(图1A)。
  6. 细胞粘附后,轻轻除去培养基。这可以通过从µ-slide孔的角落缓慢吸出培养基来完成。
    注意:此步骤很微妙。除去培养基时,必须格外小心,以免干扰凝胶和细胞。如果凝胶受到干扰/损坏,则不利于成像。
  7. 除去培养基后,将μ玻片放在层流通风橱中,并迅速重新制备胶原蛋白-纤连蛋白混合物。
  8. 将100 µl凝胶混合物放在附着于先前浇铸的凝胶的细胞顶部(图1A)。
  9. 在培养箱中于37°C孵育1小时,以使凝胶聚合。
    注意:第二层凝胶凝胶化后,可以观察到在单个z平面中夹在两个凝胶之间的细胞。
  10. 同时,打开显微镜和显微镜载物台加热器。将温度调节至37°C。
  11. 第二层凝胶凝胶化后,在第二个凝胶的顶部添加150 µl完全培养基,以确保凝胶不会变干(图1A)。
  12. 小心地将µ-玻片放入设计用于固定玻片的显微镜插入物中,并进行温度,湿度和CO 2 控制。
  13. 将插入物放在显微镜载物台上。将焦点放在孔中心的单元格上。使用20倍物镜。使用具有相位对比的物镜(图1B)。
    注意:µ-slide孔的中心最适合成像,以避免靠近孔侧面的像差。
  14. 以20分钟的间隔拍摄60小时的延时图像。


    图1. 3D迁移设置中的步骤。 A.示意图概述了协议中涉及的步骤。B.夹在两个胶原-纤连蛋白凝胶之间的MDA-MB-231细胞的示例延时图像,描绘了其3D迁移过程中的细胞形态变化。比例尺:40 µm。有关3D迁移视频的示例性描述,请参阅Visweshwaran et al。,2018年,Movie EV2。

数据分析

  1. 视频采集完成后,分配图像属性。在斐济打开视频/图像堆栈文件,然后选择“图像”→“属性…”来分配图像属性,然后填写“长度单位”(示例-µm),“像素宽度”(像素大小以µm为单位),“像素高度”(像素大小,单位为µm)和“帧间隔”(20分钟)。保存视频/图像堆栈。也可以通过选择“分析”→“设置比例”选项来完成类似的分配。
  2. 如Dang和Gautreau,2018年以及Gorelik和Gautreau,2014年所述,完成了对细胞轨迹的手动跟踪和分析。简而言之,借助斐济软件插件“ MTrackJ”执行了手动跟踪,从而提供了轨迹数据。然后,将这些数据复制到包含DiPer定制宏套件的MS Excel文件中,以量化迁移参数。通过运行DiPer宏,可以计算并绘制各种迁移参数,如单元速度,方向持久性(方向自相关)和均方根位移(MSD),这些参数可用于制作出版物质量的数字(图2)。
  3. 对于这种设置,细胞轨迹的自动跟踪和分析也是可能的。Visweshwaran和Maritzen,2019年描述了一种合适的程序,该方法可以在实验人员只需最少的输入的情况下自动量化3D细胞迁移。
    注意:加载了DiPer宏套件的MS Excel文件可以通过 辅助文件:Excel_loaded_with_DiPer 。GoPerlik和Gautreau,2014年提供了有关DiPer以及迁移参数(如单元速度,方向持久性(方向自相关)和均方根位移(MSD))的详细说明。


    图2。HSBP1耗尽的MDA-MB-231细胞的代表性3D迁移量化。请参阅Visweshwaran 等。(2018年)以获取详细信息。简而言之,顶部面板显示单细胞轨迹。均方位移(MSD)分析显示,与对照细胞相比,HSBP1耗竭的细胞探索的区域更小。这种影响可能是由于速度降低和方向持久性降低所致。在3个独立的实验中,每种条件下至少跟踪了10个细胞;数据是平均值±SEM;方差分析** P &lt; 0.01; **** P &lt; 0.0001。

笔记

  1. 当在Leibovitz的L-15培养基中培养MDA-MB-231细胞时,不应使用CO 2 进行pH控制。L-15培养基配方设计用于大气。在富含CO 2 的气氛中温育将对细胞有害。
  2. 为了运行宏来分析单元格轨迹,必须启用MS Excel中的“开发人员”选项卡。通过转到“ Excel选项”→“自定义功能区”→“自定义功能区”→“主选项卡”→“开发人员”,可以启用“开发人员”选项卡。
  3. 可以在此设置中研究药物/抑制剂对细胞3D迁移的影响。对于长期效果分析,可以在制造胶原纤连蛋白凝胶的同时添加药物,并在最后添加的介质中添加以防止凝胶干燥。在这种方法中,细胞会不断暴露于药物中,进行完整的观察。另一方面,还可以在3D迁移过程中添加药物/抑制剂。在这种方法中,应将高浓度的药物(1-10 µl)轻轻加入到凝胶顶部的培养基中,以防止凝胶干燥。在这里,药物会扩散到凝胶中。在这种情况下,实验人员最初会在没有药物治疗的期间记录3D细胞迁移,而在添加药物之后,则是在药物处理下3D细胞迁移的时期。如果药物/抑制剂有毒,用药物对细胞进行简短的预处理并将其夹在凝胶之间进行迁移分析也是一种替代方法。

菜谱

  1. MDA-MB-231培养基
    L-15培养基
    10%FBS
    1x青霉素-链霉素
  2. 胶原-纤连蛋白凝胶混合物
    2 mg / ml胶原I
    10 µg / ml FN
    25 mM HEPES
    10%FBS
    1x MEM用无菌水完成
    对于1毫升凝胶体积:
    <身体>
    10个MEM
    100 µl
    HEPES(1 M)
    25 µl
    胶原蛋白I(4.41 mg / ml)
    452微升
    FBS
    100 µl
    FN
    10 µl
    无菌水
    313微升

致谢

该协议改编自Dang et al。(2013),并进行了修改。该协议的建立是由AG的团体资助资助的:国家研究机构(ANR ANR-15-CE13-0016-01),基金会癌症研究基金会(PGA120140200831)和国家癌症研究所( INCA_6521)。SPV在最初的3年中获得了来自Ministèrede l'EnseignementSupérieuret de la Recherche的博士奖学金的支持,并在第四年获得了来自国立控制癌症中心的博士学位。

利益争夺

作者宣称他们没有利益冲突。

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引用:Visweshwaran, S. P. and Gautreau, A. (2020). Analysis of Random Migration of Cancer Cells in 3D. Bio-protocol 10(1): e3482. DOI: 10.21769/BioProtoc.3482.
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