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Mouse Mammary Intraductal (MIND) Method for Transplantation of Patient Derived Primary DCIS Cells and Cell Lines
小鼠乳腺管内(MIND)法移植病人来源的原代DCIS细胞和细胞系   

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参见作者原研究论文

本实验方案简略版
Breast Cancer Research
May 2009

Abstract

The MIND method involves intraductal injection of patient derived ductal carcinoma in situ (DCIS) cells and DCIS cell lines (MCF10DCIS.COM and SUM225) inside the mouse mammary ducts [Video 1 and Figure 1 in Behbod et al. (2009)]. This method mimics the normal environment of DCIS and facilitates study of the natural progression of human DCIS, i.e., their initial growth as carcinoma in situ within the ducts, followed by invasion into the stroma through the myoepithelial cell layer and basement membrane (Behbod et al., 2009; Valdez et al., 2011). In order to demonstrate that transplantation procedure is successful, the transplanted mammary glands may be excised as early as two weeks following intraductal injection of cells followed by Hematoxylin and Eosin (H&E) staining and/or immunofluorescence staining using human specific cytokeratin 5 and/or 19 [please see Figures 2-4 in Behbod et al. (2009)]. Additionally, the presence of trypan blue inside the mouse mammary ducts immediately following intraductal injection is the best indicator that the injection was successful (Video 1 starting at 4:33 sec).

Keywords: Intraductal, Mouse mammary, Mammary transplantation, Human DCIS

Materials and Reagents

  1. Hamilton syringe, 50 μl capacity, with a 30 gauge blunt-ended fixed 1/2-inch needle (Hamilton, catalog number: 80608 )
  2. 1 ml Tuberculin (TB) syringe (Benton Dickenson, catalog number: 309625 )
  3. Wound clips (Becton Dickinson, catalog number: 427631 )
  4. Animals: 8-12 week old female immunocompromised mice
    Note: NSG, NOD scid gamma [NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (HE JACKSON LABORATORY, catalog number: 005557 )]. Alternatively, SCID/beige mice (Fox Chase SCID® Beige CB17.Cg-PrkdcscidLystbg-J/Crl) may be used for the injection of DCIS cell lines only. While a formal comparison has not been made, patient derived DCIS epithelial cells seem to grow more efficiently in the NSG mice.
  5. Cells of interest [i.e., cell lines MCF10DCIS.COM and SUM225 (Behbod Lab, 2009 #329) or patient derived DCIS epithelial cells] in single cells
  6. PBS (pH 7.4, 1x), sterile (Life Technologies, catalog number: 10010023 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: 10010023”.
  7. Trypan blue 0.4%, sterile (Sigma-Aldrich, catalog number: T8154 )
  8. 70% ethanol
  9. Sterile water
  10. Nembutal Sodium Solution (Pentobarbital sodium injection, USP) (Akorn, Oak Pharmaceuticals, catalog number: NDC 7647850120 )
  11. KetofenTM (Ketoprofen) (Patterson Veterinary Supply, catalog number: 10004029 )

Equipment

  1. Scissors Small scale suitable for mice
    Sharp-Blunt (Fine Science Tool, catalog number: 14028-10 )
    Castroviejo Spring Scissors (Fine Science Tool, catalog number: 15017-10 )
    Balled Scissors (Fine Science Tool, catalog number: 14086-09 )
  2. Clippers (Fine Science Tool, catalog number: 1501710 )
  3. Rounded end flat handle tweezers (ROBOZ, model: RS5095 )
  4. Spring scissor (Fine Science Tool, catalog number: 1501710 )
  5. Balled scissor (Fine Science Tool, catalog number: 1408609 )
  6. Rounded end tweezers (Fine Science Tool, catalog number: 1105210 )
  7. Rat tooth tweezers (Fine Science Tool, catalog number: 1105310 )
  8. Clip applier (Becton Dickinson, catalog number: 427630 )
  9. Heat pad (Sunbeam Products, model: PN126985900 )
  10. Clip remover (Becton Dickinson, catalog number: 427638 )

Procedure

  1. Prepare the cells for injection
    1. Resuspend cells in PBS with 0.04% trypan blue. For cell lines, the final concentration should be 5,000-10,000 cells/μl. For primary cells the concentration should be 12,500-17,500 cells/μl. Trypan blue will indicate the presence of cells inside the duct after the injection procedure.
    2. Be sure all instruments have been sterilized and the Hamilton syringe washed by alternating pulling up sterile water and 70% alcohol, ending with sterile water.

  2. Prepare the mice for surgery
    1. Weigh the mouse.
    2. Using 1 ml TB syringe, administer Nembutal 40 μg/g body intraperitoneal (this will provide 1 h of sedation).
    3. Wait for mouse to fall asleep; confirm by checking hind limb reflex by pinching foot.
    4. Use clippers to shave the abdomen exposing the inguinal nipples and taking care not to cut them.
    5. Using 1 ml TB syringe, administer KetofenTM (ketoprofen) 5 mg/kg intraperitoneal for pain relief.
    6. Place the mouse on surgical board and secure limbs with surgical tape.
    7. Sterilize the abdomen with 70% alcohol.

  3. Surgery
    1. Cut off the tip of the nipples of the inguinal glands so that the syringe can be directly inserted through the nipple. Use the rounded end flat handle tweezers to hold the skin under the nipple and force it to protrude. Use the spring scissor to cut off about the top 0.5 mm of the nipple. Take care not to remove too much or the syringe will slide under the skin. A properly cut nipple will have a volcano cone shape.
    2. Using the balled scissors, make an inverted Y-incision on the abdomen beginning on the midline slightly below the inguinal nipple and extending up about 1 inch. The other two cuts of the incision should extend between and fourth and fifth nipples on either side.
    3. Using the rounded end tweezers and rat tooth tweezers, separate the skin covering the inguinal mammary fat pads to expose the inguinal gland.
    4. Using the Hamilton syringe, draw up two microliters of PBS (with 0.04% trypan blue) containing cells at the proper concentration; holding the skin flap up, insert the tip of the syringe into the nipple at a 90 degree angle to the skin flap and carefully inject the cells. Trypan blue will make the injected liquid visible in the duct.
    5. Reposition the skin flaps and suture with wound clips.

      Video 1. Intraductal injection
      Note: The video contains material that can be disturbing to some viewers.

  4. Post-surgery
    1. Allow mice to recover on heat pad set on the lowest setting.
    2. Administer KetofenTM 5 mg/kg intraperitoneal 24 h later.
    3. Remove clips after 7-10 days.

Acknowledgments

This protocol was developed in the Department of Molecular and Cellular Biology, Baylor College of Medicine and Department of Pathology and Laboratory Medicine, University of Kansas Medical School. We thank David Edwards for assistance in the production of the procedure video. The work was supported by NCI (1K99/R00 CA127462) to FB and NCI (1K22 CA160587-01A1) to KV.

References

  1. Behbod, F., Kittrell, F. S., LaMarca, H., Edwards, D., Kerbawy, S., Heestand, J. C., Young, E., Mukhopadhyay, P., Yeh, H. W., Allred, D. C., Hu, M., Polyak, K., Rosen, J. M. and Medina, D. (2009). An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ. Breast Cancer Res 11(5): R66.
  2. Valdez, K. E., Fan, F., Smith, W., Allred, D. C., Medina, D. and Behbod, F. (2011). Human primary ductal carcinoma in situ (DCIS) subtype-specific pathology is preserved in a mouse intraductal (MIND) xenograft model. J Pathol 225(4): 565-573.

简介

MIND方法涉及管内注射患者来源的导管原位(DCIS)细胞和DCIS细胞系(MCF10DCIS.COM和SUM225)(在小鼠乳腺导管内)[Video 1和FIG 1 in Behbod et al。 >(2009)]。该方法模拟DCIS的正常环境,并且有助于研究人DCIS的自然进展,即它们在导管内原位癌的初始生长,随后是侵入通过肌上皮细胞层和基底膜的基质(Behbod等人,2009; Valdez等人,2011)。为了证明移植手术是成功的,可以早在导管内注射细胞后两周,随后使用人特异性细胞角蛋白5和/或19的苏木精和曙红(H&E)染色和/或免疫荧光染色,切除移植的乳腺[请参见Behbod等人(2009)中的图2-4]。此外,在管内注射后立即在小鼠乳腺导管内存在台盼蓝是注射成功的最佳指示(视频1从4:33秒开始)。

关键字

材料和试剂

  1. Hamilton注射器,50μl容量,用30号钝端固定的1/2英寸针(Hamilton,目录号:80608),
  2. 1ml结核菌素(TB)注射器(Benton Dickenson,目录号:309625)
  3. 伤口夹(Becton Dickinson,目录号:427631)
  4. 动物:8-12周龄雌性免疫受损小鼠
    注意:NSG,NOD scidγ[NOD.Cg-Prkdcscid II2rgtm1Wj1/SzJ(HE JACKSON LABORATORY,目录号:005557)]。或者,SCID /米色小鼠(Fox Chase SCID Beige CB17.Cg-PrkdcscidLystbg-J/Crl)可以仅用于注射DCIS细胞系。虽然尚未进行形式比较,但患者衍生的DCIS上皮细胞在NSG小鼠中似乎更有效地生长。
  5. 在单个细胞中感兴趣的细胞[即EM细胞系MCF10DCIS.COM和SUM225(Behbod Lab,2009#329)或患者来源的DCIS上皮细胞]
  6. PBS(pH 7.4,1×),无菌(Life technologies,目录号:10010023)
    注意:目前,它是"Thermo Fisher Scientific,目录号:10010023"。
  7. 无菌的台盼蓝(Sigma-Aldrich,目录号:T8154)
  8. 70%乙醇
  9. 无菌水
  10. Nembutal钠溶液(戊巴比妥钠注射液,USP)(Akorn,Oak Pharmaceuticals,目录号:NDC 7647850120)
  11. Ketofen TM (Ketoprofen)(Patterson Veterinary Supply,目录号:10004029)

设备

  1. 剪刀小号适合小鼠
    Sharp-Blunt(Fine Science Tool,目录号:14028-10)
    Castroviejo弹簧剪刀(Fine Science Tool,目录号:15017-10)
    球形剪刀(Fine Science Tool,目录号:14086-09)
  2. 快船(Fine Science Tool,目录号:1501710)
  3. 圆头平头手柄镊子(ROBOZ,型号:RS5095)
  4. 弹簧剪(Fine Science Tool,目录号:1501710)
  5. 球形剪刀(Fine Science Tool,目录号:1408609)
  6. 圆头镊子(Fine Science Tool,目录号:1105210)
  7. 大鼠牙镊(Fine Science Tool,目录号:1105310)
  8. 施夹器(Becton Dickinson,目录号:427630)
  9. 热垫(Sunbeam Products,型号:PN126985900)
  10. 夹子去除剂(Becton Dickinson,目录号:427638)

程序

  1. 准备注射用细胞
    1. 重悬细胞在PBS与0.04%台盼蓝。对于细胞系,最终 ?浓度应为5,000-10,000个细胞/μl。对于原代细胞 浓度应为12,500-17,500个细胞/μl。台盼蓝会 表示注射后管道内存在细胞 程序。
    2. 确保所有仪器已灭菌, Hamilton注射器通过交替地提起无菌水和70% 酒精,以无菌水结束。

  2. 准备手术小鼠
    1. 称重鼠标。
    2. 使用1 ml TB注射器,给予Nembutal 40μg/g腹膜内(这将提供1小时的镇静)。
    3. 等待鼠标入睡;通过捏脚检查后肢反射来确认。
    4. 使用剪刀刮削腹部暴露腹股沟乳头,并注意不要割伤他们
    5. 使用1ml TB注射器,施用5mg/kg的Ketofen TM(酮洛芬)腹膜内用于缓解疼痛。
    6. 将鼠标放在手术板上,用手术胶带固定四肢。
    7. 用70%酒精消毒腹部。

  3. 手术
    1. 切断腹股沟腺的乳头的尖端,使 注射器可以直接插入通过乳头。使用圆角端 ?平面手柄镊子,以保持皮肤下的乳头,并强迫它 突出。使用弹簧剪刀剪掉顶部0.5mm处 乳头。注意不要取出太多,否则注射器会滑下 ?皮。正确切割的乳头将具有火山锥形状
    2. 使用球形剪刀,在腹部做一个倒Y形切口 开始于腹股沟乳头下方的中线 向上延伸约1英寸。切口的另外两个切口应该 在两侧和第四和第五接头之间延伸。
    3. 使用圆端镊子和大鼠牙镊,分离皮肤 ?覆盖腹股沟乳腺脂肪垫以暴露腹股沟
    4. 使用Hamilton注射器,吸取两微升PBS(含有 0.04%台盼蓝),含有适当浓度的细胞;保持 ?皮肤瓣向上,将注射器的尖端插入乳头90° ?角度到皮瓣,仔细注射细胞。台盼 蓝色将使注入的液体在管道中可见。
    5. 重新放置皮瓣和缝合用伤口夹。

      <! - flashid1744v17开始 - >
      视频1.导管内注射
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      获取Adobe Flash Player

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      <! - <![endif] - >
      <! - flashid1744v17结束 - > 注意:视频包含可能对某些观看者造成干扰的内容。

  4. 手术后
    1. 允许小鼠在最低设置的热垫上恢复。
    2. 24小时后腹膜内给予酮托芬 TM 5mg/kg
    3. 7-10天后移除剪辑。

致谢

该协议是在贝勒医学院分子和细胞生物学系以及堪萨斯大学医学院病理和实验室医学系发展起来的。我们感谢David Edwards在制作过程视频方面的帮助。该工作由NCI(1K99/R00CA127462)到FB和NCI(1K22CA160587-01A1)到KV支持。

参考文献

  1. Behbod,F.,Kittrell,FS,LaMarca,H.,Edwards,D.,Kerbawy,S.,Heestand,JC,Young,E.,Mukhopadhyay,P.,Yeh,HW,Allred,DC, ,Polyak,K.,Rosen,JM和Medina,D。(2009)。 导管内人类小鼠移植模型模拟导管原位癌的亚型,/em>。乳腺癌研究 11(5):R66
  2. Valdez,K.E.,Fan,F.,Smith,W.,Allred,D.C.,Medina,D.and Behbod,F。(2011)。 人类原发性导管原位癌(DCIS)亚型特异性病理是保存在小鼠管内(MIND)异种移植模型中。 Pathol 225(4):565-573。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kittrell, F., Valdez, K., Elsarraj, H., Hong, Y., Medina, D. and Behbod, F. (2016). Mouse Mammary Intraductal (MIND) Method for Transplantation of Patient Derived Primary DCIS Cells and Cell Lines. Bio-protocol 6(5): e1744. DOI: 10.21769/BioProtoc.1744.
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