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3D Gel Invasion Assay of Gastric Cancer Cells with Fibroblasts
用成纤维细胞对胃癌细胞进行三维凝胶侵袭试验   

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Abstract

Cancer tissue is composed of cancer cells and a large number of stromal cells including fibroblasts. In order to understand the relationship between fibroblasts and cancer cells during invasion of the stroma, 3D gel invasion assay is useful. Most tumors are associated with a biologically active type of fibroblasts known as cancer-associated fibroblasts (CAFs), which promote the invasion of cancer cells. Here, we describe the method of imaging the invasion by fluorescently labeled CAFs and gastric cancer cells in gels containing extracellular matrix. For two-color fluorescence labeling of living cells, long-chain dialkylcarbocyanines, DiO and DiI were used. This method is also applicable for studying invasion by other stromal cells and cancer cells, and for evaluation of drugs targeting cancer stromal cells.

Keywords: CAF(CAF), Scirrhous(硬癌), Invasion(入侵)

Materials and Reagents

  1. Collagen-coated dish (Sanyo, IWAKI, catalog number: 4010-010 )
  2. Transparent PET membrane 24 well 3.0 μm pore size (Corning, Falcon®, catalog number: 353096 )
  3. 24 well plate for use with cell culture inserts (Corning, Falcon®, catalog number: 353504 )
  4. Razor blades (Esbjerg, Feather, catalog number: FA-10 )
  5. Microslide glass (Matsunami Glass Ind, catalog number: TF0215M )
  6. Micro cover glass, 24 x 24 mm (Thickness NO.1: 0.12-0.17 mm) (Matsunami Glass Ind)
  7. Gastric cancer cells (44As3) (Yanagihara K et al., 2005)
    Note: It’s established from gastric cancer patient.
  8. Fibroblasts (CAF) (Fuyuhiro Y et al., 2011)
    Note: It’s isolated from surgical materials of gastric cancer patients.
  9. Type I-collagen (Nitta Gelatin Inc., Cellmatrix Type I-P)
  10. Matrigel matrix (Corning, catalog number: 356234 )
  11. 3, 3’-dioctadecycloxacarbo-cyanine perchlorate (DiO) (Thermo Fisher Scientific, Molecular Probes™, catalog number: D-275 )
  12. 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethyllindo-carbocyanine perchlorate (DiI) (Thermo Fisher Scientific, Molecular Probes™, catalog number: D-282 )
  13. Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T3924 )
  14. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P4333 )
  15. Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, catalog number: D6046 )
  16. RPMI-1640 medium (Sigma-Aldrich, catalog number: R8758 )
  17. 10x Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, catalog number: D2429 )
  18. 10x RPMI-1640 medium (Sigma-Aldrich, catalog number: R1145-500ML )
  19. Fetal bovine serum (heat inactivated) (Sigma life science, catalog number: 172012-500ML , batch: S13C490)
  20. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417 )
  21. 4% Paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, catalog number: 163-20145 )
  22. Polyvinyl alcohol mounting medium (Sigma-Aldrich, Fluka, catalog number: 10981 )
  23. Instant glue (Krazy Glue, catalog number: KG585 )
  24. NaHCO3 (Sigma-Aldrich, catalog number: S6014-500G )
  25. NaOH (Sigma-Aldrich, catalog number: S5881-500G )
  26. HEPES (Sigma-Aldrich, catalog number: H7006-25G )
  27. Gel (0.2 mg/ml type I-collagen and 2.5 mg/ml matrigel matrix) (see Recipes)
  28. Reconstitution buffer (see Recipes)

Equipment

  1. Tweezers (Electron Microscopy Sciences, Dumont, model: No.5 )
  2. 37 °C, 5% CO2 incubator (LabX, Sanyo, model: MCO-19AIC )
  3. Dissecting microscope (OLYMPUS CORPORATION, model: SZ61 )
  4. Light source (OLYMPUS CORPORATION, KL1600LED )
  5. Vibratome (DOSAKA EM, model: LinearSlicer PRO7 )
  6. Confocal laser scanning microscope (ZEISS, model: LSM 780 )

Procedure

  1. 44As3 cells and gastric CAFs were cultured in RPMI-1640 medium or DMEM, respectively supplemented with 10% FBS and Penicillin (100 unit/ml)-Streptomycin (0.1 mg/ml) at 37 °C in a humidified atmosphere containing 5% CO2. CAFs were maintained in collagen-coated dishes.
  2. 44As3 cells and CAFs were labeled with DiO or DiI, respectively as follows. Stock solutions of DiO and DiI were prepared in dimethylformamide at 2.5 mg/ml. DiO or DiI was added in the medium at final concentration of 6 μg/ml, and cells were incubated for 1 h in a CO2 incubator. Cells were then washed by replacing with fresh medium containing 10% FBS and backed into a CO2 incubator for 20 min, repeat this step two cycles.
  3. During labeling the cells, prepare the gel. Serum-free DMEM/RPMI-1640 (1:1) medium containing 0.2 mg/ml type I-collagen and 2.5 mg/ml matrigel matrix (refer to the below recipe) was laid onto the upper chamber of transwells (150 μl/ well), and solidify in a CO2 incubator at 37 °C for 30 min.
  4. 44As3 cells and CAFs were detached by trypsin-EDTA (trypsin 0.5 g/L, incubate 3 min) and mixed (1.5 x 104 cells each) in 200 μl of medium composed by 1:1 mixture of DMEM and RPMI-1640 supplemented with 0.2% FBS, and placed on gels. The lower compartment of the transwell was filled with 700 μl of the same medium with 10% FBS. Cells were incubated for 5-9 days with replacing the upper and lower medium every other day (Figure 1).


    Figure 1. Cell-culture on the gel in transwell inserts. (Left). Mixture of cancer cells and fibroblasts, labeled with distinguishable fluorescence dyes were put on the gel, which was prepared in 3.0 μm pored transwell inserts. (Right). The cells invade into the gel according to serum gradient.

  5. Take the gels out from transwell inserts by cutting the insert membrane along the whole circumference (Video 1). The gels were fixed in 4% paraformaldehyde for 1 h at room temperature, or overnight at 4 °C. If necessary, take photos of the whole gel as activated fibroblasts contract the gel (Figure 2A).
  6. The fixed gel was once rinsed with PBS. Periphery of the gel was excised by a razor blade under dissecting microscope (Figure 2B). The edge of cell area, which is usually visible by labeled fluorescence is excised. The gel was attached to the stage of vibratome slicer by instant glue (Figure 3A, B), and cut into 200 μm thick slices in PBS (Figure 3C-D, Video 2).


    Figure 2. Appearance of the gel before setting on the vibratome. A. Appearance of the whole gel after taking out from the insert and fixation. B. After excision of peripheral part of the gel. The position of excision was shown at the bottom.


    Figure 3. Slice of the gel by a vibratome. A, B. Attach the gel face a section down to the metal stage of a vibratome. A. Superior view, B. Lateral view of the gel. C. The stage is filled with PBS. The gel is sectioned by a razor blade in PBS. D. Direction of the sectioning was shown.

  7. The slices were picked up from PBS, put on a glass slide, and fix the shape of the slices by straightening them with tweezers under dissecting microscope. Wipe excess PBS by a paper and mounted in polyvinyl alcohol mounting medium (Figure 4).


    Figure 4. Appearance of sliced gels. A. Illustrations of sliced gels. B. Sliced gels were arranged on a slide glass. Bar = 1 mm

  8. After solidified the mounting medium, the slices were visualized using a confocal microscope. The 3D-rendered images are obtained from the z-stack images (Figure 5).


    Figure 5. Representative confocal images of a gel section. Green: Cancer cells, Red: CAFs. Mixture of cancer (44As3) cells and CAFs protruded and invaded into the gel. Gels were fixed at day 7. Bar = 100 μm

Representative data

Video 1. Take the gels out from transwell inserts by cutting the insert membrane

Video 2. Gel sectioning by a vibratome. In this video, two gels are attached to the stage.

Notes

The optimum length of culture period should be determined empirically. Usually between 5-9 days. If solidified glue was attached to the sliced gel, it should be removed in PBS using tweezers under dissecting microscope before mounting.

Recipes

  1. Gel (0.2 mg/ml type I-collagen and 2.5 mg/ml matrigel matrix)
    Mix type-I collagen gel and matrigel matrix (3:1). For example, if you make total 800 μl mixed gel, at first prepare 600 μl of type-I collagen by mixing 10x conc. culture medium (60 μl), reconstitution buffer (60 μl) and type-I collagen (480 μl) in a tube on ice. Add 200 μl of matrigel matrix and mix thoroughly.
  2. Reconstitution buffer
    2.2 g NaHCO3 in 100 ml of 0.05 N NaOH and 200 mM HEPES

Acknowledgments

This protocol was adapted from the previously published studies, Satoyoshi et al. (2015a; 2015b) and Tsuji et al. (2015). This work was supported by JSPS KAKENHI (Grant Nos. 25290042, 26640068) and Research Grant of the Princess Takamatsu Cancer Research Fund (No. 14-24620).

References

  1. Fuyuhiro, Y., Yashiro, M., Noda, S., Kashiwagi, S., Matsuoka, J., Doi, Y., Kato, Y., Hasegawa, T., Sawada, T. and Hirakawa, K. (2011). Upregulation of cancer-associated myofibroblasts by TGF-beta from scirrhous gastric carcinoma cells. Br J Cancer 105(7): 996-1001.
  2. Satoyoshi, R., Kuriyama, S., Aiba, N., Yashiro, M. and Tanaka, M. (2015a). Asporin activates coordinated invasion of scirrhous gastric cancer and cancer-associated fibroblasts. Oncogene 34(5): 650-660.
  3. Satoyoshi, R., Aiba, N., Yanagihara, K., Yashiro, M. and Tanaka, M. (2015b). Tks5 activation in mesothelial cells creates invasion front of peritoneal carcinomatosis. Oncogene 34(24): 3176-3187.
  4. Tsuji, T., Satoyoshi, R., Aiba, N., Kubo, T., Yanagihara, K., Maeda, D., Goto, A., Ishikawa, K., Yashiro, M. and Tanaka, M. (2015). Agr2 mediates paracrine effects on stromal fibroblasts that promote invasion by gastric signet-ring carcinoma cells. Cancer Res 75(2): 356-366.
  5. Yanagihara, K., Takigahira, M., Tanaka, H., Komatsu, T., Fukumoto, H., Koizumi, F., Nishio, K., Ochiya, T., Ino, Y. and Hirohashi, S. (2005). Development and biological analysis of peritoneal metastasis mouse models for human scirrhous stomach cancer. Cancer Sci 96(6): 323-332.

简介

癌组织由癌细胞和大量包括成纤维细胞的基质细胞组成。 为了理解在基质侵入期间成纤维细胞和癌细胞之间的关系,3D凝胶入侵测定是有用的。 大多数肿瘤与称为癌症相关成纤维细胞(CAF)的生物活性类型的成纤维细胞相关,其促进癌细胞的侵入。 在这里,我们描述的成像入侵的荧光标记CAF和胃癌细胞在含有细胞外基质的凝胶中的方法。 对于活细胞的双色荧光标记,使用长链二烷基碳菁,DiO和DiI。 该方法也适用于研究其他基质细胞和癌细胞的侵袭,以及用于评价靶向癌症基质细胞的药物。

关键字:CAF, 硬癌, 入侵

材料和试剂

  1. 胶原包被的皿(Sanyo,IWAKI,目录号:4010-010)
  2. 透明PET膜24孔3.0μm孔径(Corning,Falcon ,目录号:353096)
  3. 24孔板用于细胞培养插入物(Corning,Falcon ,目录号:353504)。
  4. 剃刀刀片(Esbjerg,Feather,目录号:FA-10)
  5. 微滑玻璃(Matsunami Glass Ind,目录号:TF0215M)
  6. 微型玻璃,24×24mm(厚度NO.1:0.12-0.17mm)(Matsunami Glass Ind)
  7. 胃癌细胞(44As3)(Yanagihara K et al。,2005)
    注意:它是由胃癌患者建立的。
  8. 成纤维细胞(CAF)(Fuyuhiro Y et al。,2011)
    注意:它与胃癌患者的手术材料隔离。
  9. I型胶原(Nitta Gelatin Inc.,Cellmatrix Type I-P)
  10. Matrigel矩阵(Corning,目录号:356234)
  11. (DiO)(Thermo Fisher Scientific,Molecular Probes TM,目录号:D-275)
  12. 1,1'-二(十八烷基)-3,3,3',3'-四甲基吲哚 - 羰花青高氯酸盐(DiI)(Thermo Fisher Scientific,Molecular Probes TM,目录号:D-282)
  13. 胰蛋白酶-EDTA溶液(Sigma-Aldrich,目录号:T3924)
  14. 青霉素 - 链霉素(Sigma-Aldrich,目录号:P4333)
  15. Dulbecco改良的Eagle培养基(Sigma-Aldrich,目录号:D6046)
  16. RPMI-1640培养基(Sigma-Aldrich,目录号:R8758)
  17. 10x Dulbecco改良的Eagle培养基(Sigma-Aldrich,目录号:D2429)
  18. 10x RPMI-1640培养基(Sigma-Aldrich,目录号:R1145-500ML)
  19. 胎牛血清(热灭活)(Sigma life science,目录号:172012-500ML,批次:S13C490)
  20. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417)
  21. 4%多聚甲醛磷酸盐缓冲液(Wako Pure Chemical Industries,目录号:163-20145)
  22. 聚乙烯醇封固剂(Sigma-Aldrich,Fluka,目录号:10981)
  23. 速溶胶(Krazy Glue,目录号:KG585)
  24. NaHCO 3(Sigma-Aldrich,目录号:S6014-500G)
  25. NaOH(Sigma-Aldrich,目录号:S5881-500G)
  26. HEPES(Sigma-Aldrich,目录号:H7006-25G)
  27. 凝胶(0.2mg/ml I型胶原和2.5mg/ml基质胶基质)(参见Recipes)
  28. 重建缓冲区(请参阅配方)

设备

  1. 镊子(Electron Microscopy Sciences,Dumont,型号:5)
  2. 37℃,5%CO 2培养箱(LabX,Sanyo,型号:MCO-19AIC)中。
  3. 解剖显微镜(OLYMPUS CORPORATION,型号:SZ61)
  4. 光源(OLYMPUS CORPORATION,KL1600LED)
  5. Vibratome(DOSAKA EM,型号:LinearSlicer PRO7)
  6. 共聚焦激光扫描显微镜(ZEISS,型号:LSM 780)

程序

  1. 44As3细胞和胃CAF在37℃下在含有5%CO 2的湿润气氛中在分别补充有10%FBS和青霉素(100单位/ml) - 链霉素(0.1mg/ml)的RPMI-1640培养基或DMEM中培养, sub> 2 。 CAF保持在胶原包被的皿中
  2. 44As3细胞和CAF分别用DiO或DiI标记,如下。在2.5mg/ml的二甲基甲酰胺中制备DiO和DiI的储备溶液。将DiO或DiI以6μg/ml的终浓度加入培养基中,并将细胞在CO 2培养箱中孵育1小时。然后通过用含有10%FBS的新鲜培养基替换来洗涤细胞,并且放回CO 2培养箱中20分钟,重复该步骤两个循环。
  3. 在标记细胞期间,制备凝胶。将含有0.2mg/ml I型胶原和2.5mg/ml基质胶基质的无血清DMEM/RPMI-1640(1:1)培养基置于转座上室(150μl/孔),并在CO 2培养箱中在37℃下固化30分钟。
  4. 通过胰蛋白酶-EDTA(胰蛋白酶0.5g/L,孵育3分钟)分离44As3细胞和CAF,并在200μl由1:1混合物的混合物组成的培养基中混合(各1.5×10 4个细胞) DMEM和补充有0.2%FBS的RPMI-1640,并置于凝胶上。 Transwell的下部室填充有700μl具有10%FBS的相同培养基。将细胞孵育5-9天,每隔一天更换上和下培养基(图1)

    图1. transwell插入物中凝胶上的细胞培养(左)。将用可区分的荧光染料标记的癌细胞和成纤维细胞的混合物放在凝胶上,所述凝胶在3.0μm多孔的transwell插入物中制备。 (对)。细胞根据血清梯度侵入凝胶
  5. 通过沿整个圆周切割插入膜片(视频1),从transwell插入物中取出凝胶。将凝胶在4%多聚甲醛中在室温下固定1小时,或在4℃下过夜。如果需要,拍摄整个凝胶作为活化的成纤维细胞收缩凝胶(图2A)。
  6. 固定的凝胶一次用PBS漂洗。在解剖显微镜下用剃刀刀片切下凝胶的周边(图2B)。切除通常通过标记的荧光可见的细胞区域的边缘。通过瞬间胶将凝胶附着在振动切片机的台上(图3A,B),并在PBS中切成200μm厚的切片(图3C-D,视频2)。


    图2.在置于振动器上之前凝胶的外观。 A.从插入物取出并固定后的整个凝胶的外观。 B.切除凝胶的周边部分。切除的位置显示在底部。


    图3.通过vibratome切片的凝胶。 A,B.将凝胶面朝下贴在vibratome的金属台上。 A.高级视图,B.凝胶的侧视图。该阶段充满PBS。将凝胶用PBS中的剃刀刀片切片。 D.显示切片的方向。

  7. 从PBS中取出切片,放在载玻片上,并且通过在解剖显微镜下用镊子矫正切片的形状来固定切片的形状。用纸擦拭多余的PBS,并安装在聚乙烯醇固定介质中(图4)

    图4.切片凝胶的外观。 A.切片凝胶的图示。 B.将切片凝胶布置在载玻片上。 Bar = 1 mm

  8. 在固化封固介质后,使用共焦显微镜观察切片。 3D渲染的图像从z-stack图像获得(图5)。


    图5.凝胶切片的代表性共聚焦图像。绿色:癌细胞,红色:CAF。癌症(44As3)细胞和CAF的混合物突出和侵入凝胶。凝胶在第7天固定。条=100μm

笔记

培养期的最佳长度应根据经验确定。通常在5-9天之间。如果固化的胶粘在切片的凝胶上,应该在解剖显微镜下安装前使用镊子在PBS中除去。

食谱

  1. 凝胶(0.2mg/mlI型胶原和2.5mg/ml基质胶基质) 混合I型胶原凝胶和基质胶基质(3:1)。例如,如果你使总共800μl混合凝胶,首先通过混合10x浓度准备600μl的I型胶原。培养基(60μl),重建缓冲液(60μl)和I型胶原(480μl)。加入200μl基质胶基质并彻底混合
  2. 重建缓冲区
    2.2g NaHCO 3在100ml 0.05N NaOH和200mM HEPES中的溶液

致谢

该方案改编自先前公开的研究,Satoyoshi等人(2015a; 2015b)和Tsuji等人(2015)。这项工作由JSPS KAKENHI(批准号25290042,26640068)和高松公主癌症研究基金研究基金(No.14-24620)支持。

参考文献

  1. Fuyuhiro,Y.,Yashiro,M.,Noda,S.,Kashiwagi,S.,Matsuoka,J.,Doi,Y.,Kato,Y.,Hasegawa,T.,Sawada,T.and Hirakawa, 2011)。 通过TGF-β从胃癌胃癌细胞上调癌相关肌成纤维细胞。 Br J Cancer 105(7):996-1001。
  2. Satoyoshi,R.,Kuriyama,S.,Aiba,N.,Yashiro,M.and Tanaka,M.(2015a)。 Asporin激活scirrhous胃癌和癌相关成纤维细胞的协调入侵。癌基因 34(5):650-660。
  3. Satoyoshi,R.,Aiba,N.,Yanagihara,K.,Yashiro,M.and Tanaka,M.(2015b)。 在间皮细胞中的Tks5激活产生腹膜癌发生的侵袭。 癌基因 34(24):3176-3187。
  4. Tsuji,T.,Satoyoshi,R.,Aiba,N.,Kubo,T.,Yanagihara,K.,Maeda,D.,Goto,A.,Ishikawa,K.,Yashiro,M.and Tanaka, 2015)。 Agr2介导旁分泌对基质成纤维细胞的作用,促进胃癌细胞癌侵袭 Cancer Res 75(2):356-366。
  5. Yanagihara,K.,Takigahira,M.,Tanaka,H.,Komatsu,T.,Fukumoto,H.,Koizumi,F.,Nishio,K.,Ochiya,T.,Ino,Y.and Hirohashi, 2005)。 人类胃癌的腹膜转移小鼠模型的开发和生物分析 Cancer Sci 96(6):323-332。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tanaka, M. (2016). 3D Gel Invasion Assay of Gastric Cancer Cells with Fibroblasts. Bio-protocol 6(9): e1798. DOI: 10.21769/BioProtoc.1798.
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kyungHwa Cho
college of Medicine, pharmacology
Gel does not harden. Why? Gel mixture =culture medium (60 μl)+reconstitution buffer (60 μl) +type-I collagen (480 μl) + matrigel (200 μl). In addition, I did not understand this part. What are the ‘at first prepare 600 μl of type-I collagen by mixing 10x conc' Please ask your answer.
9/23/2017 8:30:39 AM Reply
Masamitsu Tanaka
Department of Molecular Medicine and Biochemistry, Akita University Graduate School of Medicine, Japan

Please put in a tube as following order on ice. Culture medium (10x conc, 60 μl) Reconstitution buffer (60 μl) Type-I collagen (480 μl) . Mix well. Then, add 200 μl of matrigel matrix and mix thoroughly.

9/23/2017 10:01:55 PM