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Isolation of Primary Breast Cancer Cells from HER2 Transgenic Mice
从 HER2 转基因小鼠分离原代乳腺癌细胞   

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Abstract

HER2 is a tyrosine kinase receptor, which is overexpressed in about 30% of breast cancer patients. Its overexpression leads to mammary tumorigenesis and increased invasion and metastasis (Slamon et al., 1987). HER2 transgenic mouse (FVB/N-MMTVneu mouse) is a well-established model of mammary tumor in human (Fantozzi and Christofori, 2006). Although in vivo models are excellent for assessing the influence of various factors, especially microenvironment, on development of breast cancer, a convenient and less costly way to study the underlying molecular events is utilizing cells derived from the model under evaluation. In order to explore the molecular mechanism by which HOXB7 inhibits initiation, but promotes metastasis of breast tumors, we generated mouse breast cancer cell line from HER2 transgenic mouse (Liu et al., 2015). This protocol may be useful for the generation of breast cancer cell line from mice with other genetic backgrounds.

Keywords: HER2 Transgenic mice(HER2基因的转基因小鼠), Primary breast cancer cells(原发性乳腺癌细胞), Mouse breast cancer cell line(小鼠乳腺癌细胞株)

Materials and Reagents

  1. Cell strainer, sterile (100 μm) (Corning, Falcon®, catalog number: 352360 )
  2. 10 cm standard tissue culture dish (Corning, catalog number: 430293 )
  3. 50 ml conical tubes, sterile (Corning, Falcon®, catalog number: 352070 )
  4. Disposable scalpels, forceps and scissors (VWR International)
  5. HER2 transgenic mice (THE JACKSON LABORATORY, catalog number: 002376 )
  6. 0.25% trypsin-EDTA (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056 )
  7. Phosphate-buffered saline (PBS), sterile (self-preparation)
  8. HEPES (Sigma-Aldrich, catalog number: H3375 )
  9. DMEM/F12-with L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 11875-093 )
  10. Bovine serum albumin solution (BSA) (Sigma-Aldrich, catalog number: A9576 )
  11. Hydrocortisone (Sigma-Aldrich, catalog number: H0888 )
  12. Type IV collagenase (Sigma-Aldrich, catalog number: C5138 )
  13. Hyaluronidase (Sigma-Aldrich, catalog number: H3884 )
  14. Pen-Strep (GE Healthcare, HyCloneTM, catalog number: SV30010 )
  15. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434 )
  16. Tris (Sigma-Aldrich, catalog number: 93362 )
  17. Fetal bovine serum (FBS), heat inactivated (GE Healthcare, HyCloneTM, catalog number: SH30071.03 )
  18. Insulin (with transferrin/selenium) (Thermo Fisher Scientific, GibcoTM, catalog number: 51500-056 )
  19. Digestion buffer (see Recipes)
  20. Tris-buffered ammonium chloride (TAC buffer) (see Recipes)
  21. Complete media (see Recipes)

Equipment

  1. Shaking incubator (Thermo Fisher Scientific, Thermo Scientific, model: SHKA5000 )
  2. 37 °C, 5% CO2 cell culture incubator (VWR, symphonyTM, model: 5.3A )
  3. Pipette
  4. Refrigerated centrifuge (Eppendorf, model: 5418R )
  5. Water bath
  6. Hemocytometer
  7. Inverted microscope (Olympus Corporation, model: CKX41 )
  8. Tissue culture hood equipped with UV light source (Labconco, model: Purifier Logic+ Class II , Type A2 Biosafety Cabinet)

Procedure

  1. Processing solid tumor tissue into single cells
    1. Take out tumor from mouse mammary gland using forceps and scissors (Figure 1), and place it in a 10 cm Petri dish, pouring sterile DMEM/F12 medium on the tissue to keep it moist.


      Figure 1. Spontaneous breast tumor in MMTV-HER2 mice

    2. Cut the tumor into pieces using a scalpel, removing necrotic tissue if present. Necrotic tissue is normally darker and softer compared to the surrounding tumor.
    3. Mince the tissue using disposable scalpels until finely chopped (Figure 2A and 2B), and transfer the minced tissue to a 50 ml conical vial.


      Figure 2. Tumor tissue from spontaneous tumor. A. Before mincing; B. After mincing.

    4. Add 10 ml digestion buffer (about 0.5 cm3 in 10 ml digestion buffer).
    5. Close the tube with a cap and wrap the cap with Parafilm. Place the tube in a 37 °C shaker at low to moderate speed (e.g., 100-200 rpm) for 1-2 h.
    6. Pipette up and down the digested tissue and centrifuge at 530 x g at room temperature for 5 min to pellet the cells.
    7. Aspirate the supernatant, which contains fat. If the pellet contains red blood cells (observed as a red layer on top of the pellet), resuspend the pellet in 5-10 ml of TAC buffer and incubate 3-10 min in a 37 °C water bath. Centrifuge at 530 x g at room temperature for 5 min. Repeat this step until the red blood cells are no longer visible.
    8. Resuspend the pellet in 10 ml of DMEM/F12 medium (room temperature, without supplements). Filter the digested tissue using cell strainer.
      Note: It is not necessary. Normally, I do it because I need to use it for FACS or sorting.
    9. Centrifuge at 530 x g at room temperature for 1 min. Resuspend the pellet in 10 ml of DMEM/F12 medium (room temperature, without supplements).
    10. Perform a trypan blue exclusion test using a hemocytometer to determine the number of viable cells per ml.
    11. Seed 4-6 x 106 viable cells into 10 cm dish with complete media. Add 1x Pen-Strp.

  2. Generating tumor cell line
    1. Split cells once it is confluent. Normally the cells grow very fast in the first 3 passages.
    2. Purify tumor cells via removing fibroblast. Fibroblasts are often more sensitive to trypsin and can therefore be removed from the plate while the tumor cells adhere for a longer time. So treating the cells with trypsin (normally 2-3 min at 37 °C), and constantly monitor it under a microscope. Once the fibroblast (blue arrowhead) detached from the plate, while the tumor cells (red arrowhead) still adhere on the plate (Figure 3A and 3B), gently suck trypsin and wash with PBS. Then trypsinize and transfer the tumor cells to a new plate. After repeating this step for several passages, the majority of cells are tumor cells.


      Figure 3. Purification of tumor cells. A. Before trypsinization; B. After trypsinization. Blue arrowhead: fibroblast; red arrowhead: tumor cells.

    3. Passaging cells every 5-7 days after 3-4 passages. In the course of passage, the cells grow slowly or even no growth. Over 90% of cells die.
    4. After passaging 8-9 times, the cells are immortalized, and grow faster and faster. Figure 4 shows the morphology of tumor cells at different passage.
    5. The morphology of generated tumor cell line is shown in Figure 5. And the expression of HER2 is examined by IHC (Figure 6A) and Western (Figure 6B).
    6. The tumorigenicity of generated tumor cell line is tested by inoculating cells into the mammary gland fat pad of HER2 transgenic mice (Figure 7).


      Figure 4. Morphology of tumor cells at different passages


      Figure 5. Representative tumor cell line


      Figure 6. HER2 expression in tumor cells. A. IHC staining for HER2 in tumor cells (blue arrowhead: fibroblast; red arrowhead: tumor cells). B. HER2 expression in tumor cell line (1: MCF10A-Vector; 2: MCF10A-HER2; 3: Representative cell line).


      Figure 7. Tumorigenicity of established tumor cell line. Tumors formed in HER2 transgenic mouse after inoculating representative cell line in 104/site for 4 weeks.

Notes

  1. Based on what I did, stable cell lines can be generated in about 30% of tumor tissues.
  2. The cells start to proliferate after passaging 8-9 times or more. I think it is a sign for immortalization. However, I do not know the underlying mechanism.

Recipes

  1. Digestion buffer
    DMEM-F/12 supplemented with:
    10 mM HEPES
    2% BSA
    0.5 μg/ml hydrocortisone
    1 mg/ml IV collagenase
    0.1 mg/ml hyaluronidase
    1x Pen-Strp
    Sterile filter and store at 4 °C.
  2. Tris-buffered ammonium chloride (TAC buffer)
    0.16 M NH4Cl
    0.17 M Tris
    Adjust pH to 7.65 with HCl
    To make the working solution:
    Mix 90 ml of 0.16 M NH4Cl and 10 ml of 0.17 M Tris
    Adjust to pH 7.2 with HCl
    Sterile filter and store at 4 °C.
  3. Complete media
    DMEM-F/12 supplemented with 10% FBS and 10 μg/ml insulin.

Acknowledgments

This work was supported by the American Cancer Society Research Award (RSG-10-067-01-TBE) to Hexin Chen.

References

  1. Fantozzi, A. and Christofori, G. (2006). Mouse models of breast cancer metastasis. Breast Cancer Res 8(4): 212.
  2. Liu, S., Jin, K., Hui, Y., Fu, J., Jie, C., Feng, S., Reisman, D., Wang, Q., Fan, D., Sukumar, S. and Chen, H. (2015). HOXB7 promotes malignant progression by activating the TGFβ signaling pathway. Cancer Res 75(4): 709-719.
  3. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A. and McGuire, W. L. (1987). Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235(4785): 177-182.

简介

HER2是酪氨酸激酶受体,其在约30%的乳腺癌患者中过表达。 其过表达导致乳腺肿瘤发生和增加的侵袭和转移(Slamon等人,1987)。 HER2转基因小鼠(FVB/N-MMTVneu小鼠)是人类乳腺肿瘤的良好建立的模型(Fantozzi和Christofori,2006)。 虽然体内模型对于评估各种因素,特别是微环境对乳腺癌发展的影响是极好的,但是一种方便且成本较低的研究基础分子事件的方法是利用来自模型的细胞 评价。 为了探索HOXB7抑制起始,但促进乳腺肿瘤转移的分子机制,我们从HER2转基因小鼠产生小鼠乳腺癌细胞系(Liu等人,2015)。 该方案可用于从具有其他遗传背景的小鼠产生乳腺癌细胞系。

关键字:HER2基因的转基因小鼠, 原发性乳腺癌细胞, 小鼠乳腺癌细胞株

材料和试剂

  1. 细胞过滤器,无菌(100μm)(Corning,Falcon ,目录号:352360)
  2. 10cm标准组织培养皿(Corning,目录号:430293)
  3. 50ml锥形管,无菌(Corning,Falcon ,目录号:352070)
  4. 一次性手术刀,镊子和剪刀(VWR International)
  5. HER2转基因小鼠(THE JACKSON LABORATORY,目录号:002376)
  6. 0.25%胰蛋白酶-EDTA(1x)(Thermo Fisher Scientific,Gibco TM ,目录号:25200-056)
  7. 磷酸盐缓冲盐水(PBS),无菌(自制)
  8. HEPES(Sigma-Aldrich,目录号:H3375)
  9. DMEM/F12-与L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM ,目录号:11875-093)
  10. 牛血清白蛋白溶液(BSA)(Sigma-Aldrich,目录号:A9576)
  11. 氢化可的松(Sigma-Aldrich,目录号:H0888)
  12. IV型胶原酶(Sigma-Aldrich,目录号:C5138)
  13. 透明质酸酶(Sigma-Aldrich,目录号:H3884)
  14. Pen-Strep(GE Healthcare,HyClone ,目录号:SV30010)
  15. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A9434)
  16. Tris(Sigma-Aldrich,目录号:93362)
  17. 胎牛血清(FBS),热灭活(GE Healthcare,HyClone ,目录号:SH30071.03)
  18. 胰岛素(用转铁蛋白/硒)(Thermo Fisher Scientific,Gibco TM ,目录号:51500-056)
  19. 消化缓冲液(参见配方)
  20. Tris缓冲氯化铵(TAC缓冲液)(参见配方)
  21. 完成媒体(见配方)

设备

  1. 振荡培养箱(Thermo Fisher Scientific,Thermo Scientific,型号:SHKA5000)
  2. 37℃,5%CO 2细胞培养箱(VWR,symphony TM,型号:5.3A)中,
  3. 移液器
  4. 冷冻离心机(Eppendorf,型号:5418R)
  5. 水浴
  6. 血细胞计数器
  7. 倒置显微镜(Olympus Corporation,型号:CKX41)
  8. 装配有UV光源的组织培养罩(Labconco,型号:Purifier Logic + Class II,A2型生物安全柜)

程序

  1. 将实体肿瘤组织处理成单个细胞
    1. 使用镊子和剪刀从小鼠乳腺取出肿瘤(图1),并将其放置在10厘米培养皿,倾吐无菌DMEM/F12培养基上保持湿润。

      图1. MMTV-HER2小鼠中的自发性乳腺肿瘤
    2. 使用手术刀切割肿瘤块,去除坏死组织(如果存在)。坏死组织通常比周围肿瘤更暗更软。
    3. 使用一次性手术刀切碎组织直至切碎(图2A和2B),并将切碎的组织转移到50ml锥形小瓶中。


      图2.来自自发性肿瘤的肿瘤组织 A.切碎前; B.绞肉后。

    4. 加入10ml消化缓冲液(在10ml消化缓冲液中约0.5cm 3)。
    5. 用盖子关闭管子,用Parafilm包裹帽子。将管在低至中等速度(例如,100-200rpm)下在37℃摇床中放置1-2小时。
    6. 吸取上下消化的组织,并在室温下以530×g离心5分钟以沉淀细胞。
    7. 吸出上清液,其中含有脂肪。如果沉淀含有红细胞(在沉淀顶部观察为红色层),将沉淀重悬于5-10ml TAC缓冲液中,并在37℃水浴中孵育3-10分钟。在530×g下在室温下离心5分钟。重复此步骤,直到红细胞不再可见。
    8. 将沉淀重悬在10ml DMEM/F12培养基(室温,无补充)中。使用细胞过滤器过滤消化的组织。
      注意:没有必要。通常,我这样做,因为我需要使用它进行FACS或排序。
    9. 在530×g下在室温下离心1分钟。将沉淀重悬在10ml DMEM/F12培养基(室温,无补充)中
    10. 使用血细胞计数器进行台盼蓝排除试验以确定每ml的活细胞数
    11. 种子4-6×10 6个存活细胞进入具有完全培养基的10cm培养皿中。添加1x Pen-Strp。

  2. 产生肿瘤细胞系
    1. 一旦融合就分裂细胞。通常,细胞在前3次传代中生长非常快。
    2. 通过去除成纤维细胞来纯化肿瘤细胞。成纤维细胞通常对胰蛋白酶更敏感,因此可以从板上除去,而肿瘤细胞粘附更长的时间。因此用胰蛋白酶处理细胞(通常在37℃下2-3分钟),并在显微镜下连续监测。一旦成纤维细胞(蓝色箭头)从板上分离,而肿瘤细胞(红色箭头)仍粘附在板上(图3A和3B),轻轻吸入胰蛋白酶并用PBS洗涤。然后胰蛋白酶消化并将肿瘤细胞转移至新平板。重复该步骤几次后,大多数细胞是肿瘤细胞

      图3.肿瘤细胞的纯化 A.在胰蛋白酶消化前; B.胰蛋白酶消化后。蓝色箭头:成纤维细胞;红色箭头:肿瘤细胞
    3. 3-4代后每5-7天传代一次细胞。在传代过程中,细胞生长缓慢或甚至没有生长。超过90%的细胞死亡。
    4. 传代8-9次后,细胞永生化,生长更快更快。图4显示了不同传代的肿瘤细胞的形态
    5. 所产生的肿瘤细胞系的形态如图5所示。通过IHC(图6A)和Western(图6B)检测HER2的表达。
    6. 通过将细胞接种到HER2转基因小鼠的乳腺脂肪垫中来测试所产生的肿瘤细胞系的致瘤性(图7)。


      图4.不同传代时肿瘤细胞的形态学


      图5.代表性肿瘤细胞系


      图6.肿瘤细胞中的HER2表达。A.肿瘤细胞中HER2的IHC染色(蓝色箭头:成纤维细胞;红色箭头:肿瘤细胞)。 B.在肿瘤细胞系(1:MCF10A-Vector; 2:MCF10A-HER2; 3:代表性细胞系)中的HER2表达。


      图7.已建立的肿瘤细胞系的致瘤性。在10天4次/位点接种代表性细胞系4周后,在HER2转基因小鼠中形成肿瘤。

笔记

  1. 基于我所做的,可在约30%的肿瘤组织中产生稳定的细胞系。
  2. 细胞在传代8-9次或更多次后开始增殖。我认为这是永生化的标志。但是,我不知道底层机制。

食谱

  1. 消化缓冲区
    DMEM-F/12补充:
    10 mM HEPES
    2%BSA
    0.5μg/ml氢化可的松
    1mg/ml IV胶原酶
    0.1mg/ml透明质酸酶
    1x Pen-Strp
    无菌过滤并在4°C下保存
  2. Tris缓冲氯化铵(TAC缓冲液)
    0.16 M NH 4 Cl
    0.17M Tris
    用HCl调节pH至7.65
    要制作工作解决方案:
    将90ml 0.16M NH 4 Cl和10ml 0.17M Tris混合 用HCl
    调节至pH 7.2 无菌过滤并在4°C下保存
  3. 填写媒体
    补充有10%FBS和10μg/ml胰岛素的DMEM-F/12

致谢

这项工作得到了美国癌症协会研究奖(RSG-10-067-01-TBE)给陈和明的支持。

参考文献

  1. Fantozzi,A.和Christofori,G.(2006)。  乳腺癌转移的小鼠模型 乳腺癌研究 8(4):212.
  2. Liu,S.,Jin,K.,Hui,Y.,Fu,J.,Jie,C.,Feng,S.,Reisman,D.,Wang,Q.,Fan,D.,Sukumar, Chen,H。(2015)。  HOXB7促进恶性进展通过激活TGFβ信号通路。 Cancer Res 75(4):709-719。
  3. Slamon,DJ,Clark,GM,Wong,SG,Levin,WJ,Ullrich,A.and McGuire,WL(1987)。  人乳腺癌:复发和存活与HER-2/neu癌基因扩增的相关性 Science 235 4785):177-182
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, S. and Chen, H. (2016). Isolation of Primary Breast Cancer Cells from HER2 Transgenic Mice. Bio-protocol 6(19): e1956. DOI: 10.21769/BioProtoc.1956.
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