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Feb 2020

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A 3D Skin Melanoma Spheroid-Based Model to Assess Tumor-Immune Cell Interactions
基于球体的三维皮肤黑色素瘤模型评价肿瘤免疫细胞相互作用   

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Abstract

Three-dimensional (3D) tumor spheroids have the potential to bridge the gap between two-dimensional (2D) monolayer tumor cell cultures and solid tumors with which they share a significant degree of similarity. However, the progression of solid tumors is often influenced by the dynamic and reciprocal interactions between tumor and immune cells. Here we present a 3D tumor spheroid-based model that might shed new light on understanding the mechanisms of tumor and immune cell interactions. The model first utilizes the hanging drop assay, which serves as one of the simplest methods for generating 3D spheroids and requires no specialized equipment. Next, pre-established spheroids can be co-cultured either directly or indirectly with an immune cell population of interest. Using skin melanoma, we provide a detailed description of the model, which might hold a significant importance for the development of successful therapeutic strategies.

Keywords: Skin (皮肤), Cancer (癌症), Melanoma (黑素瘤), Spheroid (球体), 3D (3D), Hanging drop (悬滴), Innate lymphoid cells (固有淋巴细胞), ILC2 (ILC2)

Background

Three-dimensional (3D) tumor spheroids are spherical self-assembled tumor cell aggregates, which resemble micrometastases and replicate many features of solid tumors. As in the non-proliferating regions of avascular solid tumors, tumor cells within the inner regions of spheroids usually demonstrate perturbed gene and protein expression, altered metabolism, cell cycle arrest and necrotic death (Sant and Johnston, 2017). However, most currently available techniques for generating 3D spheroids are time-consuming, difficult and expensive. A simple, fast and easy method for generating 3D spheroids is through the use of the hanging drop assay (Foty, 2011; Berens et al., 2015). Within the hanging drop, cells have no direct contact with substratum and due to gravity accumulate at the free liquid-air interface to form a single spheroid. In light of the increasingly recognized role of interactions between tumor and immune cells, pre-established spheroids can be co-cultured either directly or indirectly with different populations of immune cells, as shown in Figure 1.

Direct co-culture can be performed by culturing pre-established spheroids with immune cells in close proximity in the same culture environment, for example by layering two components one on top of the other. In contrast, indirect co-culture employs cell culture inserts with porous membranes, to keep immune cells separated from spheroids. Co-culturing pre-established spheroids with immune cells directly can provide information on cell-cell interactions based on physical contact, whereas indirect co-culture can be used to study the effects of secreted signaling molecules.

In our recent work, we generated spheroids from B16 melanoma cells and demonstrated their potential to impair proliferation of recently identified innate immune cells, group 2 innate lymphoid cells (ILC2s), through the production of lactic acid (Moro et al., 2010 and 2015; Wagner and Koyasu, 2019; Wagner et al., 2020). Here we provide a detailed workflow necessary for the generation of 3D spheroids from B16 melanoma cells that can be co-cultured with an immune cell population of interest. Using a hanging drop assay, B16 spheroids were generated over a period of 7 days, as shown in Figure 2. The optimal number of uniformly shaped spherical tumor cell aggregates with a smooth surface was obtained using cultures initiated with 10,000 cells per 25 μl. At least 24 spheroids can be produced per dish. Pre-established spheroids can be co-cultured with an immune cell population of interest to assess an impact of tumor cells on immune cell effector functions and vice versa. In order to address specific research questions, alterations to this condition including cell transfection, transduction and cell culture media modification with different therapeutic agents can be easily performed.

The possibility to reproduce the complex interactions between tumor and immune cells represents an important area of unfulfilled need towards the design of efficient therapeutic strategies. This protocol describes a simple, fast, and easy method for generating 3D spheroids that can be used to study the mechanisms of tumor and immune cell interactions.


Figure 1. Scheme displaying the principle of the hanging drop assay. Tumor cells per drop of cell culture medium are placed on the lid of a PBS containing dish and incubated for 7 days. Pre-established spheroids are collected and co-cultured directly or indirectly with an immune cell population of interest.

Materials and Reagents

  1. 75 cm2 cell culture flasks (Thermo Fisher, catalog number: 156499)

  2. 10 cm dishes (Thermo Fisher, catalog number: 150464)

  3. 15 ml conical tubes (Corning, catalog number: 352196)

  4. FalconTM permeable support for 24-well plate with 0.4 µm transparent PET membrane (Corning, catalog number: 353095)

  5. FalconTM 24-well TC-treated cell polystyrene permeable support companion plate (Corning, catalog number: 353504)

  6. Mus musculus B16F10 skin melanoma cells (ATCC, catalog number: CRL-6475)

  7. PBS, pH 7.4 (Gibco, catalog number: 10010023)

  8. 0.25% Trypsin-EDTA, phenol red (Gibco, catalog number: 25200072)

  9. Trypan Blue solution (0.4%) (Thermo Fisher, catalog number: 15250061)

  10. RPMI-1640 medium with L-glutamine and sodium bicarbonate, liquid, sterile-filtered (Sigma-Aldrich, catalog number: R8758)

  11. Heat inactivated Fetal Bovine Serum (Japan Bioserum, catalog number: S1820-500) (Testing of different batches is recommended)

  12. MEM Non-essential Amino Acid solution (100×) (Sigma-Aldrich, catalog number: M7145)

  13. Sodium Pyruvate (100 mM) (Gibco, catalog number: 11360070)

  14. HEPES solution (1 M) (Sigma-Aldrich, catalog number: H3537)

  15. Penicillin-Streptomycin (10,000 U/ml) (Gibco, catalog number: 15140122)

  16. 2-Mercaptoethanol (Gibco, catalog number: 21985023)

  17. L-Glutamine (200 mM) (Lonza, catalog number: 17-605E)

  18. Complete cell culture medium (see Recipes)

Equipment

  1. Pipettes

  2. Hemocytometer

  3. Incubator (37 °C, 5% CO2)

  4. Water bath (37 °C)

  5. Refrigerated benchtop centrifuge for spinning conical tubes

  6. Inverted microscope

Procedure

  1. Single Cell Suspension Preparation

    1. Plate early (3-4) passage of B16F10 cells in a 75 cm2 cell culture flask in complete cell culture medium. Incubate at 37 °C until cells reach 80-90% confluency.

    2. Remove and discard cell culture medium.

    3. Briefly rinse the monolayer with PBS to remove all traces of serum since it contains the trypsin inhibitor.

    4. Detach adherent cells from the surface of a cell culture flask by adding 1.0 to 2.0 ml of 0.25% Trypsin-EDTA solution to the flask. Incubate at 37 °C until the monolayer is dispersed (usually within 5-10 min). Monitor cells under an inverted microscope.

    5. As soon as cells have detached, neutralize the Trypsin-EDTA solution by adding 8.0 to 9.0 ml of complete cell culture medium (shown below). Gently pipette up and down the mixture until cells are in suspension.

    6. After spinning down, count cells using a hemocytometer and perform a dilution to allow for the seeding of 10,000 cells per 25 μl of complete cell culture medium.


  2. Generation of Spheroids

    1. Remove the lid from the 10 cm dish and carefully deposit 25 μl drops on the inner side of the lid.

    2. Fill the bottom of the dish with 10 ml PBS to humidify the culture chamber after distribution of the drops.

    3. Flip the lid in a confident, yet controlled manner. Cells under the force of gravity will be aggregated at the bottom of the hanging drop. Make sure to place the drops sufficiently apart from each other and not too close to the dish edges.

    4. Carefully place the dish in the incubator and preferably avoid touching or moving for the first 2 days.

    5. Change cell culture medium on the 3rd and 6th day in each drop. To this end, carefully remove the lid from the dish and replace approximately 10 μl of used media with 10-15 μl of fresh media in each drop, in order to maintain constant osmolality and nutrient supply. Replace the medium from the edge of the drop. Avoid touching spheroids. Flip the lid in a confident, yet controlled manner.

    6. Monitor the drops under an inverted microscope. Pay attention not to disturb the droplets while moving the dish.



      Figure 2. Representative photographs of skin melanoma spheroids. B16F10 melanoma cells (10,000 cells per 25 ul drop of cell culture medium) placed on the lid of a PBS containing dish and incubated for 3 and 7 days. Objective magnification: 3.2x (lower panel).


  3. Co-cultures with Immune Cells

    1. Collect spheroids by the wide end of the tip (100 or 1,000 μl pipette tip cut widely 5-6 mm from the end) and transfer to an appropriate cell culture plate.

    2. Co-culture spheroids with immune cells in complete cell culture medium by either layering two components one on top of the other in the same well of the cell culture plate or by employing cell culture inserts with porous membranes, to keep immune cells separated from spheroids.

Notes

  1. Antibiotics (and antimycotics) may be added to PBS in order to minimize bacterial (and fungal) contamination during the period of spheroid generation.

  2. Always prepare a surplus number of spheroids in case some of the drops detach from the lid or slide on the surface of the lid during the change of the medium process.

  3. The longer time of incubation is possible, however the cell viability should be assessed. We have observed around 90% cell viability following 7 days of incubation.

  4. For the use of other types of cancer cells, the number of cells and the volume of the drops should be adjusted.

Recipes

  1. Complete cell culture medium

    RPMI-1640 medium

    10% Fetal Calf Serum

    100 μM MEM Non-essential Amino Acid

    10 mM HEPES

    100 U/ml Penicillin-Streptomycin

    1 mM Sodium Pyruvate

    50 μM 2-mercaptoethanol

    400 μM L-Glutamine

Acknowledgments

This work was supported by the FRIPRO Mobility Grant Fellowship from the Research Council of Norway (302241) to M.W., and by a Grant-in-Aid for Scientific Research (A) (20H00511) from the Japan Society for the Promotion of Science, to S.K. This protocol was adapted from the original research paper published in Wagner et al. (2020).

Competing interests

The authors declare no competing interests.

References

  1. Berens, E. B., Holy, J. M., Riegel, A. T. and Wellstein, A. (2015). A cancer cell spheroid assay to assess invasion in a 3D setting. J Vis Exp (105): 53409.
  2. Foty, R. (2011). A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp (51): 2720.
  3. Moro, K., Yamada, T., Tanabe, M., Takeuchi, T., Ikawa, T., Kawamoto, H., Furusawa, J., Ohtani, M., Fujii, H. and Koyasu, S. (2010). Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. Nature 463(7280): 540-544.
  4. Moro, K., Ealey, K. N., Kabata, H. and Koyasu, S. (2015). Isolation and analysis of group 2 innate lymphoid cells in mice. Nat Protoc 10(5): 792-806.
  5. Sant, S. and Johnston, P. A. (2017). The production of 3D tumor spheroids for cancer drug discovery. Drug Discov Today Technol 23: 27-36.
  6. Wagner, M., Ealey, K. N., Tetsu, H., Kiniwa, T., Motomura, Y., Moro, K. and Koyasu, S. (2020). Tumor-derived lactic acid contributes to the paucity of intratumoral ILC2s. Cell Rep 30(8): 2743-2757 e2745.
  7. Wagner, M. and Koyasu, S. (2019). Cancer immunoediting by innate lymphoid cells. Trends Immunol 40(5): 415-430.

简介

[摘要]三维(3D)肿瘤球体具有弥合二维(2D)单层肿瘤细胞培养物与实体瘤之间的差距的潜力,它们之间有着显着的相似性。然而,实体瘤的进展通常受肿瘤与免疫细胞之间的动态相互作用和相互影响的影响。在这里,我们提出了一个基于3D肿瘤球体的模型,该模型可能会为了解肿瘤与免疫细胞相互作用的机制提供新的思路。的该模型首先利用了悬滴法,这是生成3D球体的最简单方法之一,不需要专门的设备。接下来,可以将预先建立的球体与目标免疫细胞群体直接或间接共培养。使用皮肤黑色素瘤,我们提供了该模型的详细说明,这可能对成功治疗策略的开发具有重要意义。

[背景]三维(3D)肿瘤球体是球形的自组装肿瘤细胞聚集体,类似于微转移瘤并复制实体瘤的许多特征。就像在无血管实体瘤的非增生区域一样,球体内部区域的肿瘤细胞通常表现出扰动的基因和蛋白质表达,新陈代谢改变,细胞周期停滞和坏死(Sant and Johnston ,2017)。但是,用于生成3D椭球体的大多数当前可用技术是耗时,困难和昂贵的。一种简单,快速,简便的生成3D球体的方法是使用悬滴法(Foty ,2011;Berens等,2015)。在悬滴内,细胞不直接与基质接触,并且由于重力而聚集在自由的液-气界面形成单个球体。在肿瘤和免疫细胞之间的相互作用的日益认识到的作用的光,预先建立的球状体可以是共培养任一直接或间接地与免疫细胞的不同群体,如图1中所示。

直接共培养可以通过在相同的培养环境中将预先建立的球状体与免疫细胞紧密相邻地进行来进行,例如通过将两个组件一个接一个地层叠。相反,间接共培养采用带有多孔膜的细胞培养插入物,以使免疫细胞与球体分离。直接将预先建立的球体与免疫细胞共培养可提供基于物理接触的细胞间相互作用信息,而间接共培养可用于研究分泌的信号分子的作用。

在我们最近的工作中,我们从B16黑色素瘤细胞中产生了球状体,并证明了它们通过产生乳酸来破坏最近鉴定的第2组先天性免疫细胞(ILC2s)增殖的潜力(Moro等人,2010年和2015年) ; Wagner和Koyasu ,2019; Wagner等人,2020)。在这里,我们提供了从B16黑色素瘤细胞生成3D球状体所需的详细工作流程,该球状体可与感兴趣的免疫细胞群体共培养。使用悬滴试验中,在7天期间产生B16球状体,如示于图2的unifor最佳数目毫升ý圆球状的肿瘤细胞聚集物与使用具有每25万个细胞发起培养物中获得的平滑表面微升。每道菜至少可以生产24个sph类固醇。可以将预先建立的球体与感兴趣的免疫细胞群体共培养,以评估肿瘤细胞对免疫细胞效应子功能的影响,反之亦然。为了解决具体的研究问题,可以容易地进行这种条件的改变,包括用不同的治疗剂进行细胞转染,转导和细胞培养基修饰。

再现肿瘤与免疫细胞之间复杂相互作用的可能性代表了对有效治疗策略设计未满足需求的重要领域。该协议描述了一种用于生成3D球体的简单,快速,简便的方法,该方法可用于研究肿瘤与免疫细胞相互作用的机制。





图1.显示悬滴法原理的方案。将每滴细胞培养基的肿瘤细胞置于含有PBS的皿的盖子上,并孵育7天。收集预先建立的球体,并与感兴趣的免疫细胞群体直接或间接共培养。

关键字:皮肤, 癌症, 黑素瘤, 球体, 3D, 悬滴, 固有淋巴细胞, ILC2

材料和试剂
 
75 cm 2细胞培养瓶(Thermo Fisher,目录号:156499)
10厘米长的盘子(Thermo Fisher,货号:150464)
15 ml锥形管(Corning,目录号:352196)
Falcon TM渗透性支持物,用于带有0.4 µm透明PET膜的24孔板(Corning,目录号:353095)
Falcon TM 24孔TC处理过的细胞聚苯乙烯可渗透支撑伴侣板(Corning,目录号:353504)
小家鼠B16F10皮肤黑色素瘤细胞(ATCC,目录号:CRL-6475)
PBS,pH 7.4(Gibco,目录号:10010023)
0.25%胰蛋白酶-EDTA,酚红(Gibco,目录号:25200072)
台盼蓝溶液(0.4%)(Thermo Fisher,目录号:15250061)
带有L-谷氨酰胺和碳酸氢钠的RPMI-1640培养基,液体,无菌过滤(Sigma-Aldrich,目录号:R8758)
热灭活胎牛血清(日本生物血清,目录号:S1820-500)(建议测试不同批次)
MEM非必需氨基酸溶液(100×)(Sigma-Aldrich,目录号:M7145)
丙酮酸钠(100 mM)(Gibco,目录号:11360070)
HEPES溶液(1 M)(Sigma-Aldrich,目录号:H3537)
青霉素-链霉素(10,000 U / ml )(Gibco,目录号:15140122)
2-巯基乙醇(Gibco,目录号:21985023)
L-谷氨酰胺(200毫米)(Lonza,目录号:17-605E)
完整的细胞培养基(请参阅食谱)
 
设备
 
移液器
血细胞计数器
保温箱(37 °C,5%CO 2 )
水浴(37 °C)
锥形台式冷冻台式离心机
倒置显微镜
 
程序
 
单细胞悬液制备
在完全细胞培养基中的75 cm 2细胞培养瓶中接种B16F10细胞的早期(3-4)传代。在37°C下孵育,直到细胞达到80-90%融合为止。
除去并丢弃细胞培养基。
由于它包含胰蛋白酶抑制剂,因此用PBS短暂冲洗单层以除去所有痕量的血清。
通过向烧瓶中添加1.0到2.0 ml的0.25%胰蛋白酶-EDTA溶液,从细胞培养瓶的表面上分离粘附的细胞。在37 °C下孵育直至单层分散(通常在5-10分钟内)。在倒置显微镜下监控细胞。
细胞脱离后,通过向8.0 ml完整细胞培养基中添加8.0 (如下所示)来中和胰蛋白酶-EDTA溶液。轻轻地上下吸移混合物,直到细胞悬浮。
旋转下来后,使用血细胞计数器对细胞计数,并进行稀释以使每25μl完整细胞培养基可接种10,000个细胞。
 
球体的产生
从10厘米的培养皿上取下盖子,并小心地将25μl液滴滴在盖子的内侧。
在滴液分布后,用10 ml PBS填充培养皿底部,以润湿培养室。
以自信但可控的方式翻转盖子。在重力作用下的细胞将聚集在悬滴的底部。确保液滴彼此之间充分分开,并且不要太靠近培养皿边缘。
小心地将培养皿放在培养箱中,最好在开始的2天避免触摸或移动。
改变细胞培养mediu米上的3次和6次的一天中每个液滴。为此,小心地从盘移除盖子并更换大约10微升与10-15使用的介质的微升在每个液滴的新鲜培养基中,为了保持恒定的重量克分子渗透压浓度和营养供应。从液滴边缘更换介质。避免触摸球体。以自信但可控的方式翻转盖子。
在倒置显微镜下监控液滴。注意在移动培养皿时不要干扰液滴。
 


图2.皮肤黑色素瘤球体的代表性照片。将B16F10黑色素瘤细胞(每25 ul细胞培养基滴入10,000个细胞)装在含PBS的培养皿盖上,孵育3天和7天。物镜放大倍率:3.2倍(下图)。
 
与免疫细胞共培养
收集球状体由尖端(100或1的宽端,000微升移液管尖端从广泛5-6毫米切割的端部),并转移到合适的细胞培养板中。
通过在细胞培养板的同一孔中将两个组件一个接一个地层叠在一起,或者通过使用带有多孔膜的细胞培养插入物,将球状体与免疫细胞共培养,以保持免疫细胞与球状体分离。 
 
笔记
 
可以将抗生素(和抗真菌药)添加到PBS中,以最大程度减少球体生成期间细菌(和真菌)的污染。
在更换介质的过程中,一定要准备多余数量的球体,以防某些液滴从盖子上脱落或在盖子表面上滑动。
可能需要更长的孵育时间,但是应评估细胞活力。孵育7天后,我们观察到大约90%的细胞活力。
为了使用其他类型的癌细胞,应调整细胞数量和液滴体积。
 
菜谱
 
完整的细胞培养基
RPMI-1640中
10%胎牛血清
100μM MEM非必需氨基酸
10毫米HEPES
100 U / ml青霉素-链霉素
1毫米丙酮酸钠
50 μM2-巯基乙醇
400 μM L-谷氨酰胺
 
致谢
 
这项工作得到了挪威研究理事会(302241)向MW的FRIPRO流动性研究金以及日本科学促进会的科学研究补助金(A)(20H00511)的支持。 SK该方案改编自Wagner等人发表的原始研究论文。(2020)。
 
利益争夺
 
作者宣称没有利益冲突。
 
参考文献
 
Berens,EB,Holy,JM,Riegel,AT和Wellstein ,A.(2015)。癌细胞球体分析,用于评估3D环境中的入侵。Ĵ显示精通(105):53409。
Foty ,R。(2011)。一个简单的悬滴细胞培养方案,用于生成3D球体。对外教育杂志(51):2720。
莫罗(K.),山田(T.),田边(M.),竹内(T.),井川(T.),河本(H.),藤泽(J.),大谷(M.),藤井(H.)和小安(S.) 2010)。脂肪组织相关的c-Kit + Sca-1 +淋巴样细胞先天产生T H 2细胞因子。自然463(7280):540-544。
Moro,K.,Ealey,KN,Kabata ,H.和Koyasu,S.(2015年)小鼠第2组天然淋巴样细胞的分离和分析。纳特Protoc 10(5):792-806。              
桑特(Sant)和宾夕法尼亚州约翰斯顿(2017)。用于癌症药物发现的3D肿瘤球体的生产。Drug Discov Today Technol 23:27-36。
瓦格纳,M.,Ealey,KN,哲,H.,Kiniwa ,T.,本村,Y.,摩洛,K。和子安,S。(2020)。肿瘤来源的乳酸导致肿瘤内ILC2缺乏。细胞代表30(8):2743-2757 e2745。              
Wagner,M.和Koyasu,S.(2019年)。先天性淋巴样细胞对癌症进行免疫编辑。趋势免疫学40(5):415-430。        
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引用:Wagner, M. and Koyasu, S. (2020). A 3D Skin Melanoma Spheroid-Based Model to Assess Tumor-Immune Cell Interactions. Bio-protocol 10(23): e3839. DOI: 10.21769/BioProtoc.3839.
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