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In vivo Matrigel Plug Angiogenesis Assay
基质胶内血管生成实验   

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Abstract

The matrigel plug angiogenesis assay is a simple in vivo technique to detect the newly formed blood vessels in the transplanted gel plugs in nude mice. The matrigel matrix is derived from the engelbroth-holm-swarm (EHS) mouse sarcoma, and its composition is comparable to the basement membrane proteins. The matrigel can induce differentiation of a variety of cell types such as hepatocytes, mammary epithelial cells, and endothelial cells. In our case, tumor cells are mixed with the matrigel gel and are injected into the mice. The later immunohistochemistry (IHC) staining with the endothelial marker indicates the presence of the newly formed capillaries in the sectioned gel plugs.

Keywords: In vivo(在体内), Matrigel plug(Matrigel plug), Angiogenesis(血管生成), Nude mice(裸鼠), Endothelial cell(内皮细胞)

Materials and Reagents

  1. BALB/cAnN-nu (Nude) female mouse, 6 to 8 week-old
  2. Tumor cells
  3. Matrigel matrix (BD Biosciences, Falcon®, catalog number: 354234 )
  4. 10% formalin solution-neutral buffered (Sigma-Aldrich, catalog number: HT501128-4L )
  5. Anti-rat CD34 (Santa Cruz, catalog number: sc-18917 )
  6. Biotin goat Anti-Rat IgG (BD Biosciences, catalog number: 559286 )
  7. Streptavidin-peroxidase conjugate (Dako, catalog number: P0397 )
  8. Diaminobenzidine (DAB) (Dako, catalog number: K3467 )
  9. Paraffin
  10. Potassium chloride
  11. Potassium phosphate monobasic
  12. Dodium chloride (NaCl)
  13. Sodium phosphate dibasic anhydrous
  14. Hematoxylin and eosin (H&E)
  15. Trypsin
  16. Complete medium
  17. Plain medium
  18. Phosphate buffered saline (PBS) (see Recipes)

Software

  1. Aperio ScanScope System
  2. ImageScope v10 software (Aperio, Vista)

Equipment

  1. Aperio Scanscope CS-S microscopic slide scanning system
  2. Tumor cell culture set up
  3. Hemocytometer
  4. Centrifuges
  5. 24G syringe
  6. T175 flask

Procedure

  1. Remove the medium from a T175 flask containing an 80-90% confluent monolayer of tumor cells (e.g. the nasopharyngeal carcinoma HONE1 cells) and wash the cells with PBS.
  2. Detach the tumor cells by incubating with 3 ml trypsin for 5-10 min; stop trypsinization with 10 ml complete medium.
  3. Count the number of cells with a hemocytometer, spin down the cells at 1,200 rpm for 5 min and wash once with PBS.
  4. Mix a total of 5 × 106 to 1 × 107 cells (cell type-dependent) with 50 μl plain medium and 250 μl ice-cold matrigel (the matrigel maintains as liquid form at 2-8 °C and solidifies rapidly at 22 -37 °C).
  5. Subcutaneously inject the 300 μl cell matrigel mixture into a flank of five female athymic nude mice (one injection site per mouse) with an ice-cold syringe with a 24G one inch needle.
  6. Polymerize the matrigel mixed with the cells to form a solid gel plug, which allows cell growth and blood vessel formation.
  7. After inoculation for 7 days, excise the matrigel, fix with formalin overnight, embed in paraffin, and section onto slides.
  8. Stain the slides with hematoxylin and eosin (H&E) for histological observation.
  9. Stain the blood vessels formed with 150 μl endothelial cell marker CD34 monoclonal antibody (1:40) overnight at 4 °C and 150 μl secondary biotin-conjugated goat anti-Rat IgG antibody (1:100) for 1 h at room temperature (RT).
  10. Incubate the antibody with 150 μl streptavidin-peroxidase conjugate (1:200) for 1 h at RT.
  11. Add the substrate DAB for signal detection.
  12. Scan the slides by the Aperio ScanScope System and quantify the signal by ImageScope v10 software.


    Figure 1. Representative results from matrigel plug assay. The endothelial cells were stained with anti-CD34 antibody, as indicated by the brown stain (indicated by arrows). A. Plenty of blood capillaries are formed in the gel plug section with growing tumor cells. B. Representative image of cell necrosis observed in center region of tumor nodules formed with lack of blood capillaries (indicated by asterisks).

Recipes

  1. PBS (pH 7.4)
    2.67 mM potassium chloride
    1.47 mM potassium phosphate monobasic
    137.93 mM sodium chloride (NaCl)
    8.1 mM sodium phosphate dibasic anhydrous

Acknowledgments

The study was financed by the Research Grants Council of the Hong Kong Special Administrative Region, People’s Republic of China: Grant numbers HKU6617/08M and HKU6415/06M to MLL; the NIH award AR49930 to SSA; and the Swedish Cancer Society, the Swedish Research Council, the Swedish Institute, Cancer Research Institute in New York/Concern Foundation in Los Angeles and Karolinska Institute to ERZ.

References

  1. Cheung, A. K., Ko, J. M., Lung, H. L., Chan, K. W., Stanbridge, E. J., Zabarovsky, E., Tokino, T., Kashima, L., Suzuki, T., Kwong, D. L., Chua, D., Tsao, S. W. and Lung, M. L. (2011). Cysteine-rich intestinal protein 2 (CRIP2) acts as a repressor of NF-kappaB-mediated proangiogenic cytokine transcription to suppress tumorigenesis and angiogenesis. Proc Natl Acad Sci U S A 108(20): 8390-8395.
  2. Law, E. W., Cheung, A. K., Kashuba, V. I., Pavlova, T. V., Zabarovsky, E. R., Lung, H. L., Cheng, Y., Chua, D., Lai-Wan Kwong, D., Tsao, S. W., Sasaki, T., Stanbridge, E. J. and Lung, M. L. (2012). Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma. Oncogene 31(6): 728-738.
  3. Lo, P. H., Lung, H. L., Cheung, A. K., Apte, S. S., Chan, K. W., Kwong, F. M., Ko, J. M., Cheng, Y., Law, S., Srivastava, G., Zabarovsky, E. R., Tsao, S. W., Tang, J. C., Stanbridge, E. J. and Lung, M. L. (2010). Extracellular protease ADAMTS9 suppresses esophageal and nasopharyngeal carcinoma tumor formation by inhibiting angiogenesis. Cancer Res 70(13): 5567-5576.

简介

基质胶塞血管生成测定是在裸鼠中检测移植的凝胶栓中新形成的血管的简单的体内技术。 基质胶基质来源于engelbroth-holm-swarm(EHS)小鼠肉瘤,其组成与基底膜蛋白相当。 基质胶可诱导多种细胞类型的分化,例如肝细胞,乳腺上皮细胞和内皮细胞。 在我们的情况下,将肿瘤细胞与基质胶凝胶混合并注射到小鼠中。 用内皮标记物的后期免疫组织化学(IHC)染色表明在切片的凝胶塞中存在新形成的毛细血管。

关键字:在体内, Matrigel plug, 血管生成, 裸鼠, 内皮细胞

材料和试剂

  1. BALB/cAnN-nu(裸鼠)6到8周龄的雌性小鼠
  2. 肿瘤细胞
  3. Matrigel矩阵(BD Biosciences,Falcon ,目录号:354234)
  4. 10%福尔马林溶液中性缓冲液(Sigma-Aldrich,目录号:HT501128-4L)
  5. 抗大鼠CD34(Santa Cruz,目录号:sc-18917)
  6. 生物素山羊抗大鼠IgG(BD Biosciences,目录号:559286)
  7. 链霉亲和素 - 过氧化物酶缀合物(Dako,目录号:P0397)
  8. 二氨基联苯胺(DAB)(Dako,目录号:K3467)
  9. 石蜡
  10. 氯化钾
  11. 磷酸二氢钾
  12. 氯化钠(NaCl)
  13. 磷酸氢二钠
  14. 苏木精和曙红(H& E)
  15. 胰蛋白酶
  16. 完成媒介
  17. 普通介质
  18. 磷酸盐缓冲盐水(PBS)(见Recipes)

软件

  1. Aperio ScanScope系统
  2. ImageScope v10软件(Aperio,Vista)

设备

  1. Aperio Scanscope CS-S显微镜载玻片扫描系统
  2. 肿瘤细胞培养设置
  3. 血细胞计数器
  4. 离心机
  5. 24G注射器
  6. T175烧瓶

程序

  1. 从含有80-90%铺满的单层肿瘤细胞(例如鼻咽癌HONE1细胞)的T175烧瓶中取出培养基,并用PBS洗涤细胞。
  2. 通过与3ml胰蛋白酶孵育5-10分钟来分离肿瘤细胞; 用10ml完全培养基停止胰蛋白酶消化
  3. 用血细胞计数器计数细胞数,以1,200rpm旋转细胞5分钟,并用PBS洗涤一次。
  4. 用50μl普通培养基和250μl冰冷的基质胶混合总共5×10 6个/〜1×10 7个细胞(细胞类型依赖性)(基质胶维持 在2-8℃下为液体形式,并在22-37℃下快速固化)
  5. 使用带有24G一英寸针的冰冷注射器将300μl细胞基质胶混合物皮下注射到5只雌性无胸腺裸鼠的小鼠腹部(每只小鼠一个注射部位)。
  6. 聚合与细胞混合的基质胶以形成固体凝胶塞,其允许细胞生长和血管形成。
  7. 接种7天后,切除基质胶,用福尔马林固定过夜,包埋在石蜡中,并切片到载玻片上。
  8. 用苏木精和伊红(H& E)染色载玻片用于组织学观察
  9. 将在150μl内皮细胞标记CD34单克隆抗体(1:40)形成的血管在4℃和150μl第二生物素偶联的山羊抗大鼠IgG抗体(1:100)在室温下1小时(RT )。
  10. 将抗体与150μl链霉抗生物素蛋白 - 过氧化物酶缀合物(1:200)在室温下孵育1小时。
  11. 添加基材DAB用于信号检测。
  12. 通过Aperio ScanScope系统扫描幻灯片,并通过ImageScope v10软件对信号进行量化

    图1.来自基质胶栓测定的代表性结果。如棕色染色所指示的,用抗CD34抗体染色内皮细胞(由箭头指示)。 在具有生长的肿瘤细胞的凝胶塞部分中形成大量的毛细血管。 B.在缺乏毛细血管形成的肿瘤结节的中心区域中观察到的细胞坏死的代表性图像(由星号表示)。

食谱

  1. PBS(pH 7.4)
    2.67mM氯化钾
    1.47mM磷酸二氢钾 137.93mM氯化钠(NaCl) 8.1mM二水基无水磷酸钠

致谢

研究资助由中华人民共和国香港特别行政区研究资助委员会拨款:拨款HKU6617/08M及HKU6415/06M至MLL; 美国国家卫生研究院AR49930授予SSA; 和瑞典癌症协会,瑞典研究委员会,瑞典研究所,洛杉矶纽约/关注基金会的癌症研究所和爱沙尼亚的卡罗林斯卡研究所。  

参考文献

  1. Cheung,A.K.,Ko,J.M.,Lung,H.L.,Chan,K.W。,Stanbridge, Zabarovsky,E.,Tokino,T.,Kashima,L.,Suzuki,T.,Kwong,D.L.,Chua,   D.,Tsao,S.W.and Lung,M.L。(2011)。 富含半胱氨酸   肠蛋白2(CRIP2)作为NF-κB介导的阻遏物 促血管生成细胞因子转录以抑制肿瘤发生 angiogenesis。 Proc Natl Acad Sci U S A 108(20):8390-8395。
  2. 结果显示,与正常对照组相比,对照组患者的生存率明显高于对照组,差异有统计学意义(P <0.05)。 ,Stanbridge,EJ和Lung,ML(2012)。 候选肿瘤抑制基因(Fibulin-2)在鼻咽部的抗血管生成和肿瘤抑制作用癌。癌基因 31(6):728-738
  3. HL,Cheung,AK,Apte,SS,Chan,KW,Kwong,FM,Ko,JM,Cheng,Y.,Law,S.,Srivastava,G.,Zabarovsky,ER,Tsao,SW ,Tang,JC,Stanbridge,EJand Lung,ML(2010)。 细胞外蛋白酶ADAMTS9通过抑制血管生成抑制食道和鼻咽癌肿瘤形成。 Cancer Res 70(13):5567-5576
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lung, H. and Lung, M. L. (2012). In vivo Matrigel Plug Angiogenesis Assay. Bio-protocol 2(18): e261. DOI: 10.21769/BioProtoc.261.
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cookie jie
IOZ
Dear teacher: Thank you for the protocol you have provided.When I have done the In Vivo Matrigel Plug Angiogenesis Assay following your protocol. After remove the matrix glue form the mouse, fixed with formalin overnight, and then embed in paraffin.And this step is what I do not understanding. So I want to know should I directly embed the Matrigel in paraffin or after a gradient of ethanol dehydration and transparent in xylene. and then embed in paraffin? Thank you very much!
9/19/2017 9:29:07 AM Reply
HongLok Lung
University of Hong Kong

You are right, after formalin fixation, the gel plug has to go through the serial ETOH dehydration and xylene treatment, just like preparation of other normal/tumor tissues.

9/28/2017 7:38:16 PM


Andrés Vargas
UCR
In some cases the whole implante becomes in a haematoma and in other cases the implant disappear around the fourth day post injection... some idea of what i am doing wrong?
3/7/2015 12:24:05 AM Reply
HongLok Lung
University of Hong Kong

Thanks for your message, Andres. How many cells did you inject?

Make sure you see the tumor cells bubble after injection, if the bubble is not formed, it is most likely that the implant will disappear. Similarily, if there is no tumor cells or not enough cells in the gel plug, the impalnt will be absorbed. You can try increasing the volume of matrigel (eg. up to 450ul gel and 50ul cells) or increasing the cell numbers (up to 1 x 10e7).

For the case of haematoma, have you done the tissue sectioning of the "haematoma" and subsequent H&E/CD34 staining? that happens sometimes since the tumor blood vessels are leaky.

Good Luck!

3/9/2015 8:07:16 AM


Ramon Messeguer
LEITAT Technological Centre
Why an injection of matrigel latero-abdominal instead of dorsal?
2/19/2015 2:43:26 AM Reply
HongLok Lung
University of Hong Kong

Thanks a lot for your question, actually either latero or dorsal injection is acceptable.

2/20/2015 6:41:13 AM