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Generation of Mouse Thyroid Calcitonin-producing Cell Tumors from Primary Mouse Tumors
从原代鼠肿瘤中生成鼠甲状腺产降血钙素的肿瘤细胞   

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Abstract

Medullary thyroid cancers (MTCs) are derived from calcitonin-producing cells (C cells) of neuroendocrine origin. Rb heterozygous mice develop low-grade C cell adenocarcinoma following biallelic inactivation of the Rb tumor suppressor gene loci. Additional inactivation of another tumor suppressor gene such as Trp53, Arf or Cdkn1a allows Rb-deficient mice to generate more aggressive C cell adenocarcinoma (Takahashi et al., 2006; Shamma et al., 2009; Kitajima et al., 2015). To characterize C cell adenocarcinoma cells derived from Rb-deficient mice of different genetic backgrounds, we attempted to extract C cell adenocarcinoma cells from primary thyroid tumor tissue. Since primary mouse small cell lung cancer (SCLC) cells those originate in neuroendocrine cells that also stems C cells, can be established both as non-adhesive and adhesive cells (Calbo et al., 2011), we applied their method to MTCs. Here we describe our isolation technique for non-adhesive and adhesive cell cultures from primary medullary thyroid tumor tissue. We found that the molecular markers of C cell such as Calcitonin and Ascl1 are predominantly enriched in the non-adhesive population (Kitajima et al., 2015). This is in line with the fact that one of most commonly distributed human MTC cell line TT is non-adhesive.

Keywords: Retinoblastoma(视网膜母细胞瘤), Thyroid Medullary Cancer(甲状腺髓样癌), Calcitonin(降钙素), C cell(C细胞), Mouse(鼠标)

Materials and Reagents

  1. 12 well cell culture plate (Thermo Fisher Scientific, catalog number: 150628 )
  2. 100 mm cell culture dish (Thermo Fisher Scientific, catalog number: 172931 )
  3. 5 ml centrifuge tube (Thermo Fisher Scientific, catalog number: 339650 )
  4. Phosphate buffered saline (PBS) (pH 7.2) (Life Technologies, catalog number: 20012027 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 20012027”.
  5. 100x Antibiotic-Antimycotic (Life Technologies, catalog number: 15240062 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15240062”.
  6. Dulbecco’s modified Eagle's medium (DMEM) (Wako Pure Chemical Industries, catalog number: 04330085 )
  7. 3,000 U/ml Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C5138 )
  8. 1,000 U/ml Hyaluronidase (Wako Pure Chemical Industries, catalog number: 08006201 )
  9. 20 mg/ml Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
  10. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: SH3091003 )
    Note: Currently, it is “GE Healthcare, catalog number: SH3091003”.
  11. Penicillin-Streptomycin Mixed Solution (Nakarai tesque, catalog number: 2625384 )
  12. 0.5 g/l-Trypsin/0.53 mmol/l-EDTA Solution, with Phenol Red (Nakarai tesque, catalog number: 3277834 )
  13. 7-AAD (BD Pharmingen, catalog number: 5168981E )
  14. CELLBANKER1 (Nippon Zenyaku Kogyo Co., ZENOAQ, catalog number: CB011 )
  15. Wash buffer (see Recipes)
  16. Digestion solution (see Recipes)
  17. Growth medium (see Recipes)

Equipment

  1. Surgical scissors and forceps
  2. Pipettes
  3. Centrifuge
  4. Cell culture hood
  5. 37 °C, 5% CO2 cell culture incubator
  6. Microscope
  7. FACS Aria ll flow cytometer (BD Biosciences)

Procedure

  1. Euthanize the mouse when the thyroid tumor has reached appropriate size, approximately 50-500 mm3 (refer to Video 1 and Figure 1).
  2. Expose and remove the thyroid tumor from trachea using sterile scissors. Remove the surrounding tissues as carefully as possible.
  3. Place the thyroid tumor onto 12 well type plate, and wash it several times with 2 ml of cold wash buffer.
  4. Mince the thyroid tumor into small pieces as small as possible using sterile scissors on ice.
  5. Collect all of the tumor pieces into 15 ml centrifuge tube.
  6. Wash with 5 ml wash buffer by centrifugation at 300 rcf for 5 min at 4 °C.
  7. Resuspend the tumor pieces in 5 ml digestion solution.
  8. Incubate at 37 °C for 60 min. Meanwhile mix gently every 15 min.
  9. Centrifuge at 300 rcf for 5 min at room temperature, and remove the supernatant.
  10. Wash 2 times with 5 ml wash buffer by centrifugation at 300 rcf for 5 min at room temperature.
  11. Resuspend the tumor pieces in 10 ml growth medium, and plate them onto a 100 mm cell culture dish.
  12. Incubate at 37 °C in 5% CO2 for 4~5 days.
  13. You will see some cells attach to the dish and grow, while the major population of cells is floating.
  14. Collect all culture medium containing floating cells into 15 ml centrifuge tube.
  15. Add 1 ml growth medium to the 100 mm cell culture dish, wash and recollect it into 15 ml centrifuge tube. Repeat this step at least twice in order to recover all of the floating cells.
  16. Centrifuge at 300 rcf for 5 min at room temperature, discard the supernatant, resuspend the cells in 10 ml growth medium, and plate them onto a new 100 mm cell culture dish.
  17. Add 10 ml growth medium to the used 100 mm cell culture dish, and maintain the attached cells in the cell culture incubator as adhesive population.
  18. Repeat steps 13-17 after 3-4 day culture at least 3-4 times until attached cells never appear from floating cells in order to completely separate the non-adhesive population from the adhesive population.
  19. You will detect the markers of C cells such as Calcitonin and Ascl1 positive cells are highly enriched in the non-adhesive population.
  20. Dead cells should be removed by cell sorter using staining reagents such as 7-AAD for some applications.
  21. Non-adhesive and adhesive populations can be stored in liquid nitrogen tank with cell stock solution such as CELLBANKER1.

    Video 1. The video for how to remove the thyroid tumor


    Figure 1. The picture of mouse thyroid tumor



    Figure 2. Phase-contrast image of the non-adhesive cell population


    Figure 3. The non-adhesive population highly expressed C cell markers (A. Calcitonin, B. Ascl1) as compared to adhesive population and mouse embryonic fibroblast

Recipes

  1. Wash buffer
    2x antibiotic
    Antimycotic in PBS (pH 7.2)
  2. Digestion solution
    DMEM containing 300 units/ml collagenase, 100 units/ml hyaluronidase and 0.1 mg/ml DNase l
  3. Growth medium
    DMEM supplemented 10 % FBS, 100 U/ml Penicillin and 100 μg/ml Streptomycin

Acknowledgments

We thank S. Kohno, H. Muranaka and Y. Nishimoto for helping preparation of this manuscript. Also, we thank A. Berns’s group for stimulating us to create this protocol. This work was supported by Funding Program for Next Generation World-Leading Researchers (NEXT), Grant-in-Aid for Scientific Research (MEXT), Astellas Foundation for Research on Metabolic Disorders, Takeda Science Foundation, Naito Foundation, Daiichi-Sankyo Foundation for Life Science, NOVARTIS Foundation (Japan) for Promotion of Science and Hokkoku Foundation for Cancer Research.

References

  1. Calbo, J., van Montfort, E., Proost, N., van Drunen, E., Beverloo, H. B., Meuwissen, R. and Berns, A. (2011). A functional role for tumor cell heterogeneity in a mouse model of small cell lung cancer. Cancer Cell 19(2): 244-256.
  2. Kitajima, S., Kohno, S., Kondoh, A., Sasaki, N., Nishimoto, Y., Li, F., Abdallah Mohammed, M. S., Muranaka, H., Nagatani, N., Suzuki, M., Kido, Y. and Takahashi, C. (2015). Undifferentiated State induced by Rb-p53 double inactivation in mouse thyroid neuroendocrine cells and embryonic fibroblasts. Stem Cells 33(5): 1657-1669.
  3. Shamma, A., Takegami, Y., Miki, T., Kitajima, S., Noda, M., Obara, T., Okamoto, T. and Takahashi, C. (2009). Rb Regulates DNA damage response and cellular senescence through E2F-dependent suppression of N-ras isoprenylation. Cancer Cell 15(4): 255-269.
  4. Takahashi, C., Contreras, B., Iwanaga, T., Takegami, Y., Bakker, A., Bronson, R. T., Noda, M., Loda, M., Hunt, J. L. and Ewen, M. E. (2006). Nras loss induces metastatic conversion of Rb1-deficient neuroendocrine thyroid tumor. Nat Genet 38(1): 118-123.

简介

甲状腺髓样癌(MTC)源自神经内分泌起源的降钙素产生细胞(C细胞)。Rb 杂合小鼠在b 肿瘤抑制基因位点的双等位基因失活后发展为低级C细胞腺癌。另一种肿瘤抑制基因(例如Trp53 ,Arf 或 Cdkn1a)的额外失活使Rb缺陷型小鼠产生更具侵袭性的C细胞腺癌(Takahashi, 2006; Shamma等人,2009; Kitajima等人,2015年)。为了表征来自不同遗传背景的Rb缺陷小鼠的C细胞腺癌细胞,我们尝试从原发性甲状腺肿瘤组织中提取C细胞腺癌细胞。由于原代小鼠小细胞肺癌(SCLC)细胞起源于也干细胞C的神经内分泌细胞,因此可以建立为非粘附和粘附细胞(Calbo等人,2011),我们将其方法应用于MTC。在这里我们描述我们的隔离技术的非粘附和粘附细胞培养从原发性甲状腺髓样肿瘤组织。我们发现C细胞的分子标记物如降钙素和Ascl1主要富集在非粘附性群体中(Kitajima等人,2015)。这与以下事实一致:最常分布的人MTC细胞系TT之一是非粘附性的。

关键字:视网膜母细胞瘤, 甲状腺髓样癌, 降钙素, C细胞, 鼠标

材料和试剂

  1. 12孔细胞培养板(Thermo Fisher Scientific,目录号:150628)
  2. 100mm细胞培养皿(Thermo Fisher Scientific,目录号:172931)
  3. 5ml离心管(Thermo Fisher Scientific,目录号:339650)
  4. 磷酸盐缓冲盐水(PBS)(pH7.2)(Life Technologies,目录号:20012027)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:20012027" />
  5. 100x抗生素 - 抗真菌剂(Life Technologies,目录号:15240062)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:15240062" />
  6. Dulbecco改良的Eagle培养基(DMEM)(Wako Pure Chemical Industries,目录号:04330085)
  7. 来自溶组织梭菌(Clostridium histolyticum)(Sigma-Aldrich,目录号:C5138)的3,000U/ml胶原酶
  8. 1000 U/ml透明质酸酶(Wako Pure Chemical Industries,目录号:08006201)
  9. 20mg/ml来自牛胰腺的脱氧核糖核酸酶I(Sigma-Aldrich,目录号:DN25)
  10. 胎牛血清(FBS)(Thermo Fisher Scientific,目录号:SH3091003)
    注意:目前,它是"GE Healthcare,目录号:SH3091003"。
  11. 青霉素 - 链霉素混合溶液(Nakarai tesque,目录号:2625384)
  12. 0.5g/l-胰蛋白酶/0.53mmol/l-EDTA溶液,具有酚红(Nakarai tesque,目录号:3277834)
  13. 7-AAD(BD Pharmingen,目录号:5168981E)
  14. CELLBANKER1(Nippon Zenyaku Kogyo Co.,ZENOAQ,目录号:CB011)
  15. 洗涤缓冲液(见配方)
  16. 消解解决方案(参见配方)
  17. 生长培养基(参见食谱)

设备

  1. 外科剪刀和镊子
  2. 移液器
  3. 离心机
  4. 细胞培养罩
  5. 37℃,5%CO 2细胞培养箱中培养
  6. 显微镜
  7. FACS Aria 11流式细胞仪(BD Biosciences)

程序

  1. 当甲状腺肿瘤达到合适的大小(约50-500mm <3cm)时,安乐死小鼠(参见视频1和图1)。
  2. 使用无菌剪刀暴露和从气管删除甲状腺肿瘤。尽可能小心地除去周围的组织
  3. 将甲状腺肿瘤放置在12孔板上,并用2ml冷洗涤缓冲液洗涤几次
  4. 使用无菌剪刀在冰上将甲状腺肿瘤切碎成尽可能小的碎片。
  5. 将所有肿瘤块收集到15ml离心管中
  6. 用5ml洗涤缓冲液通过在4℃下以300rcf离心5分钟洗涤
  7. 将肿瘤块重悬在5ml消化溶液中
  8. 在37℃孵育60分钟。同时每15分钟轻轻混匀。
  9. 在室温下以300rcf离心5分钟,除去上清液
  10. 在室温下以300rcf离心5分钟,用5ml洗涤缓冲液洗涤2次
  11. 将肿瘤块重悬在10ml生长培养基中,并将其平板在100mm细胞培养皿上
  12. 在37℃在5%CO 2中孵育4?5天
  13. 你会看到一些细胞附着在盘子上并生长,而主要的细胞群是漂浮的
  14. 收集所有含有浮动细胞的培养基到15ml离心管中
  15. 加入1毫升生长培养基到100毫米细胞培养皿,洗涤,并回收到15毫升离心管。重复此步骤至少两次,以恢复所有浮动单元格。
  16. 在室温下以300rcf离心5分钟,弃去上清液,将细胞重悬于10ml生长培养基中,并将它们平板接种到新的100mm细胞培养皿上。
  17. 加入10毫升生长培养基到使用的100毫米细胞培养皿,并保持附着的细胞在细胞培养孵化器作为粘合剂种群。
  18. 在3-4天培养后重复步骤13-17至少3-4次,直到附着的细胞从浮游细胞中不出现,以便完全分离非粘附群体与粘附群体。
  19. 您将检测C细胞的标记物,如降钙素和Ascl1阳性细胞在非粘附性人群中高度富集。
  20. 死细胞应通过细胞分选仪使用染色试剂如7-AAD在某些应用中除去
  21. 非粘合剂和粘合剂群可以储存在具有细胞储存溶液例如CELLBANKER1的液氮罐中。

    视频1.如何删除甲状腺肿瘤的视频
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    图1.小鼠甲状腺肿瘤的图片


    图2.非粘附细胞群体的相差图像


    图3.与粘附群体和小鼠胚胎成纤维细胞相比,非粘附群体高度表达C细胞标记物(A.降钙素,B.Ascl1)

食谱

  1. 洗涤缓冲液
    2x抗生素
    在PBS(pH 7.2)中抗真菌的
  2. 消化解决方案
    含有300单位/ml胶原酶,100单位/ml透明质酸酶和0.1mg/ml DNase I的DMEM
  3. 生长培养基
    DMEM,补充10%FBS,100U/ml青霉素和100μg/ml链霉素

致谢

我们感谢S. Kohno,H. Muranaka和Y.Nishimoto帮助准备这份手稿。此外,我们感谢A.伯恩斯的小组激励我们创建这个协议。这项工作得到下一代世界领先研究人员(NEXT)资助计划,科学研究资助(MEXT),阿斯特拉斯代谢疾病研究基金会,武田科学基金会,内藤基金会,第一三共基金会生命科学,NOVARTIS基金会(日本)促进科学和Hokkoku癌症研究基金会。

参考文献

  1. Calbo,J.,van Montfort,E.,Proost,N.,van Drunen,E.,Beverloo,H.B.,Meuwissen,R。和Berns,A。 小细胞肺癌小鼠模型中肿瘤细胞异质性的功能作用。 Cancer Cell 19(2):244-256。
  2. Kitajima,S.,Kohno,S.,Kondoh,A.,Sasaki,N.,Nishimoto,Y.,Li,F.,Abdallah Mohammed,MS,Muranaka,H.,Nagatani,N.,Suzuki, Kido,Y.和Takahashi,C。(2015)。 由小鼠甲状腺神经内分泌细胞和胚胎成纤维细胞中Rb-p53双重失活诱导的未分化状态。 Stem Cells 33(5):1657-1669。
  3. Shamma,A.,Takegami,Y.,Miki,T.,Kitajima,S.,Noda,M.,Obara,T.,Okamoto,T.and Takahashi,C。(2009)。 Rb通过E2F依赖性抑制N-ras异戊二烯化,调节DNA损伤反应和细胞衰老。 a> Cancer Cell 15(4):255-269
  4. Takahashi,C.,Contreras,B.,Iwanaga,T.,Takegami,Y.,Bakker,A.,Bronson,R.T.,Noda,M.,Loda,M.,Hunt,J.L.and Ewen,M.E。 Nras损失诱导Rb1缺陷的神经内分泌性甲状腺肿瘤的转移性转化。 Genet 38(1):118-123。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kitajima, S., Li, F. and Takahashi, C. (2015). Generation of Mouse Thyroid Calcitonin-producing Cell Tumors from Primary Mouse Tumors. Bio-protocol 5(24): e1681. DOI: 10.21769/BioProtoc.1681.
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