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This experiment is used to test the efficacies of chemo treatments or gene therapy in an in vivo system. In this protocol, the mouse xenograft model is used.

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[Bio101] Murine xenograft model
[Bio101] 小鼠异种移植模型

癌症生物学 > 通用技术 > 动物模型 > 细胞侵袭
作者: FengZhi Liu
FengZhi LiuAffiliation: School of Biomedical Sciences, Thomas Jefferson University, Philadelphia, USA
For correspondence: fengzhi6@yahoo.com
Bio-protocol author page: a51
6/5/2012, 10118 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.221

[Abstract] This experiment is used to test the efficacies of chemo treatments or gene therapy in an in vivo system. In this protocol, the mouse xenograft model is used.

[Abstract] 这个实验方法通常是用来检测在体内环境下化疗或基因治疗的效果。

Materials and Reagents

  1. Severe combined immunodeficiency (SCID) mice
  2. NaCl
  3. Pluronic acid
  4. Phosphate buffered saline (PBS)
  5. HBSS solution

Equipment

  1. 1 ml syringe with 22-24 gauge of needls
  2. Bioluminescent imaging instrument (in university core facility)

Procedure

  1. For single tumor cell (or any kind of tumor cells)
    1. Six-week-old female in-bred Fox Chase SCID mice were obtained from Charles River Laboratories (Hartford, CT, USA). Animals were handled according to a protocol approved by the Institutional Animal Care and Use Committee at our university.
    2. Mice were allowed to acclimate to animal housing, and xenografts were developed by subcutaneously injecting 5 x 106 cancer cells in murine flank bilaterally. Twice weekly, tumor volume was determined using digital caliper measurements and the formula:
      large diameter2 x small diameter
                         2
    3. After 14 days, all mice had measurable tumours and were sorted into treatment and control groups with equal number of animals (n=5). Treatment group mice received some dose of chemodrugs dissolved in vehicle (0.1 M NaCl, 0.05% pluronic acid in PBS) per treatment while control mice received vehicle only.
    4. All mice received 10 intraperitoneal injections over a 14-day period (cycle: Five treatment days followed by two non-treatment days). After 14 days (2 cycles), mice were killed. So the whole experiment lasts 28 days.
    5. The tumor sample can be used either for extract RNA, protein or immunohistochemistry staining.

  2. For interaction of tumor and Stella cells (for cancers containing big volume of stella cells)
    --using pancreatic cancer model.
    1. Bioluminescent imaging was done weekly to follow the luciferase signal from BxPC3 cells.
    2. Mice were sacrificed and tumors were harvested and measured.
      1. BxPC3-FL alone, either 0.5 x 106 or 1 x 106 per mouse.
      2. BxPC3-FL with immortalized HPSCs (human pancreatic stellate cells), in varying tumor-to-stroma ratios (1:1, 1:1, or 1:5).
      3. Immortalized HPSCs alone (0.5 x 106 or 1 x 106). All cell suspensions, including the mixture of BxPC3 and HPSCs, were injected in a 50 μl volume of HBSS.
    3. Statistical analysis was done using GraphPad Prism (GraphPad). Comparisons were made using two-tailed Student’s t test and significant difference was defined as P < 0.05. Data are shown as mean ±SE.

Acknowledgments

This protocol was adapted from Hwang et al. (2008).

References

  1. Hwang, R. F., Moore, T., Arumugam, T., Ramachandran, V., Amos, K. D., Rivera, A., Ji, B., Evans, D. B. and Logsdon, C. D. (2008). Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res 68(3): 918-926.

材料和试剂

  1. 重症联合免疫缺陷(SCID)小鼠
  2. NaCl
  3. 聚丙烯酸
  4. 磷酸盐缓冲盐水(PBS)
  5. HBSS解决方案

设备

  1. 1 ml注射器,带22-24号针头
  2. 生物发光成像仪器(在大学核心机构)

程序

  1. 对于单个肿瘤细胞(或任何种类的肿瘤细胞)
    1. 从Charles River Laboratories(Hartford,CT,USA)获得6周龄雌性原种Fox Chase SCID小鼠。根据我们大学的机构动物护理和使用委员会批准的方案处理动物
    2. 使小鼠适应动物住房,并通过在鼠侧腹部皮下注射5×10 6个癌细胞来开发异种移植物。每周两次,使用数字卡尺测量和公式:
      测定肿瘤体积 大直径 2 x小直径
                           2
    3. 14天后,所有小鼠具有可测量的肿瘤,并且用相等数量的动物(n = 5)分入治疗组和对照组。治疗组小鼠每次治疗接受溶于载体(0.1M NaCl,0.05%泊洛沙姆酸的PBS)中的一些剂量的化学药物,而对照小鼠仅接受载体。
    4. 所有小鼠在14天的时间段内接受10次腹膜内注射(周期:五个治疗天,随后两个非治疗天)。 14天(2个周期)后,杀死小鼠。因此,整个实验持续28天
    5. 肿瘤样品可用于提取RNA,蛋白质或免疫组织化学染色

  2. 对于肿瘤和斯特拉细胞(对于含有大体积的斯特拉细胞的癌症)的相互作用 - 使用胰腺癌模型。
    1. 每周进行生物发光成像以跟踪来自BxPC3细胞的荧光素酶信号。
    2. 处死小鼠并收获并测量肿瘤。
      1. BxPC3-FL,每只小鼠0.5×10 6个/1cm或1×10 6个。
      2. BxPC3-FL与永生化HPSCs(人胰腺星形细胞)以不同的肿瘤 - 基质比例(1:1,1:1或1:5)接触。
      3. 单独的永生化HPSC(0.5×10 6个或1×10 6个)。 将所有细胞悬浮液(包括BxPC3和HPSCs的混合物)注射到50μl体积的HBSS中。
    3. 使用GraphPad Prism(GraphPad)进行统计分析。 使用双尾学生t检验进行比较,显着差异定义为P < 0.05。 数据显示为平均值±SE。

致谢

该方案改编自Hwang等人(2008)。

参考文献

  1. Hwang,R.F.,Moore,T.,Arumugam,T.,Ramachandran,V.,Amos,K.D.,Rivera,A.,Ji,B.,Evans,D.B。和Logsdon,C.D。(2008)。 癌症相关基质成纤维细胞促进胰腺肿瘤进展 癌症研究< (3):918-926。
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How to cite this protocol: Liu, F. (2012). Murine xenograft model. Bio-protocol Bio101: e221. DOI: 10.21769/BioProtoc.221; Full Text



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7/8/2012 12:30:51 PM  

Q1. Which part in mouse body do you expect to see the tumors? Does it depend on types of injected tumor cells or injection location?

Q2. Can you elaborate in more details how you visualize the tumors in step 1-(2)?

7/8/2012 9:24:55 PM  

FengZhi Liu (Author)
School of Biomedical Sciences, Thomas Jefferson University, USA

The mouse usually is SCID mouse. Any tumor can grow if you use enough amount. The side is subcutaneous on the back of neck. You just hod the skin with thumb and index and inject the tumor cells.

The tumors take some time to grow to visible. Some tumors grow fast, and may takes one week, some not, it depends. you just measure the sizes in different groups with a gauze.

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