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Obesity has been linked to breast cancer progression but the underlying mechanisms remain obscure. Being overweight or obese for a woman at the time she is diagnosed with breast cancer is linked to a high risk of recurrence regardless of treatment factors. In rodents, high body weight is also associated with increased incidence of spontaneous and chemically induced tumors. To study the complex interaction between the mammary epithelia and the microenvironment, with a focus on the mechanism underlying the role obesity plays in the regulation of the cancer stem cell traits and the development of mammary cancer in vivo, we have established a diet-induced obesity (DIO) rat model of breast cancer (Chang et al., 2015).

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High Fat Diet-induced Breast Cancer Model in Rat
高脂饮食诱导的大鼠乳腺癌模型

癌症生物学 > 通用技术 > 动物模型
作者: Meng Ju Wu
Meng Ju WuAffiliation 1: Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA
Affiliation 2: Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA
Bio-protocol author page: a3259
 and Chun Ju Chang
Chun Ju ChangAffiliation 1: Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA
Affiliation 2: Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA
For correspondence: chunjuchang@purdue.edu
Bio-protocol author page: a3260
Vol 6, Iss 13, 7/5/2016, 1460 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1852

[Abstract] Obesity has been linked to breast cancer progression but the underlying mechanisms remain obscure. Being overweight or obese for a woman at the time she is diagnosed with breast cancer is linked to a high risk of recurrence regardless of treatment factors. In rodents, high body weight is also associated with increased incidence of spontaneous and chemically induced tumors. To study the complex interaction between the mammary epithelia and the microenvironment, with a focus on the mechanism underlying the role obesity plays in the regulation of the cancer stem cell traits and the development of mammary cancer in vivo, we have established a diet-induced obesity (DIO) rat model of breast cancer (Chang et al., 2015).
Keywords: Obesity(肥胖), High-fat diet(高脂饮食), Breast cancer(乳腺癌)

[Abstract]

Materials and Reagents

  1. 40 µm cell strainer (STEMCELL Technologies, catalog number: 27305 )
  2. Female Sprague-Dawley rats (Envigo, Harlan Laboratories, catalog number: Sprague Dawley® SD®)
  3. 1 ml, 26 G x 3/8 in. BD Tuberculin syringe with detachable needle, slip tip (BD Biosciences, catalog number: 309625 )
  4. N-methylnitrosourea (Sigma-Aldrich, catalog number: N1517 )
  5. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
  6. 10% neutral formalin (Sigma-Aldrich, catalog number: HT501128 )
  7. EpiCultTM-B Mouse Medium Kit (STEMCELL Technologies, catalog number: 05610 )
  8. 10x Gentle Collagenase/Hyaluronidase (STEMCELL Technologies, catalog number: 07919 )
  9. Hanks’ balanced salt solution modified (HBSS) (STEMCELL Technologies, catalog number: 37150 )
  10. Trypsin-EDTA
  11. Ammonium chloride (NH4Cl) (STEMCELL Technologies, catalog number: 07800 )
  12. 40 µm cell strainer (STEMCELL Technologies, catalog number: 27305)
  13. Fetal bovine serum (FBS) (Corning, catalog number: 35-011-CV )
  14. Flow Cytometry Stain Buffer (BD Biosciences, catalog number: 554656 )
  15. FITC-conjugated anti-OBR antibody (aa956-986, polyclonal antibody) (Lifespan Biosciences, catalog number: LS-C261832 )
  16. Regular rat chow diet (Envigo, catalog number: 8604 ) (see Recipes)
  17. Western diet (Research Diets, catalog number: D12079B ) (see Recipes)
  18. 0.9% saline (see Recipes)
  19. N-methylnitrosourea solution (see Recipes)

Equipment

  1. Dual-energy x-ray absorptometry (Lunar Corporation, model: Lunar PIXImus II Densitometer )
  2. Cell analyzer (BD Biosciences, model: BD FACSCanto II )

Software

  1. BD FACSDiva and FCS Express Flow Cytometry Software

Procedure

  1. Obesity-induced breast cancer
    1. Obtain twenty-day-old outbred Sprague-Dawley rats from Harlan Laboratories.
    2. Wean pups 24 h after arrival and maintain in a temperature and light controlled animal facility.
    3. Give the animals ad libitum access to water and assign randomly to either a regular rat chow diet or a Western diet after grouped.
    4. At 54 days old, group the animals according to their body fat composition determined by dual-energy x-ray absorptiometry (DEXA). The average percent body fat mass of rats on the lean rat chow diet would be half of that on the obese Western diet. The example of body composition analysis from DEXA scans is shown in Table 1.

      Table 1. Body composition analysis from DEXA scans. Animals in the Western-obese group showed a significant increase in fat mass compared to animals in the Western-lean group. Values represent mean ± SEM. n = 9 animals per group. Asterisk indicates P < 0.05, double asterisk indicates P < 0.0001 (from Chang et al., 2015).


    5. According to body composition analysis from DEXA scans, place the animals on the Western diet into two groups with rats in the highest tertile being placed in the Western obese group and rats in the lowest tertile being placed in the Western lean group. Three groups would be designated: “Western obesity”, “Western lean” and “Regular chow/diet”.
    6. Dissolve N-methylnitrosourea immediately before use in 0.9% NaCl and adjust the pH with acetic acid to pH = 4. Prepare fresh solution prior to application.
    7. At 65 days of age, intraperitoneally inject half of the rats in each group with 50 mg/kg of the chemical carcinogen N-methylnitrosourea and the remaining animals receive 0.9% saline (the amount of the drug to is given per body weight in mg/kg). To reduce the likelihood of piercing an organ, use 26-gauge 3/8-inch-long needles.
    8. Scarface the rats by 168 days post-injection or when an animal reaches a terminal state as determined by established guidelines (Wallace, 2000).
    9. Collect intact mammary glands and tumors and flash-freeze in liquid nitrogen or fixed in 10% neutral formalin for histologic evaluation (shown in Figure 1).


      Figure 1. Tumor phenotypes in Western obese animals, Western lean and regular diet fed animals. H&E staining showing normal mammary gland in regular diet-fed rats, aggressive adenocarcinoma in Western-obese animals, and benign adenoma in Western-lean animals (from Chang et al., 2015).

  2. Isolation of mammary tumor cells for flow cytometry analysis
    1. Prepare dissociation solution by diluting 1 part 10x Gentle Collagenase/Hyaluronidase mixture with 9 parts Complete EpiCultTM-B Medium supplemented with 5% FBS and placed solution into a 15 ml centrifuge tube.
    2. Resect and transfer mammary glands or tumors to a sterile petri dish with the 3-5 ml dissociation solution. Mince the tissues with scalpels in a cross-wise pattern until tissues are rendered to a paste. Incubate the tissues overnight (for up to 15 h) at 37 °C.
    3. After dissociation, centrifuge the cells at 350 x g for 5 min. Discard the supernatant.
    4. Re-suspend the pellet with a 1:4 mixture of cold Hanks’ balanced salt solution modified (HBSS) supplemented with 2% FBS and ammonium chloride and centrifuged at 350 x g for 5 min.
    5. Discard the supernatant and add 1-5 ml of pre-warmed 1x Trypsin-EDTA to the partially-dissociated tissue and mix by pipetting.
    6. Gently pipette up and down with a P1000 micropipettor for 1-3 min.
    7. Add 10 ml of cold HBSS supplemented with 2% FBS and centrifuge at 350 x g for 5 min.
    8. Re-suspend the pellet with an additional 10 ml of cold HBSS with 2% FBS, and filter the cell suspension through a 40 µm cell strainer into a new 50 ml centrifuge tube. Centrifuge at 350 x g for 5 min and discard the supernatant.
    9. Re-suspend the pellet with Flow Cytometry Staining Buffer, and count the cells with hemocytometer or automatic cell counter.
    10. Aliquot 200 µl of cell suspension (105-106 cells) and stain the cells with FITC-conjugated anti-OBR antibody for 20 min.
    11. Wash the cells by adding PBS. Use 1 ml for tubes. Pellet the cells by centrifugation at 400-600 x g for 5 min at room temperature. Repeat with two washes and discard supernatant between washes.
    12. Re-suspend the stained cells in 500 µl Flow Cytometry Staining Buffer.
    13. Subject the stained cells to flow cytometry analysis and determine the percentage of the cancer stem cell-like OBRhi (high leptin receptor expression, FITC-high) population. Data can be analyzed by BD FACSDiva and FCS Express Flow Cytometry Software. All surgical procedures and animal manipulations are performed under regulations of Purdue Animal Care and Use Committee.

Recipes

  1. Regular rat chow diet
    3.30 kcal/g with 18% protein
    5% fat, mainly from corn oil and 57% carbohydrates
  2. Western diet
    4.47 kcal/g with 20% protein
    21% fat in the form of anhydrous milk fat, and 50% carbohydrates, 34% sucrose
  3. 0.9% saline
    Add 0.9 g of NaCl to 100 ml of ddH2O in a sterilized glass container and pass through 0.2 µm sterile filter
  4. N-methylnitrosourea solution
    Dissolve immediately before use in 0.9% saline and adjust with acetic acid to pH value 4. Prepare fresh solutions prior to application

Acknowledgments

This protocol was used in Chang et al., (2015). This work was supported by Walther Cancer Foundation-Obesity and Cancer Pilot Grant (I.G. Camarillo and C-J. Chang) and P30 CA023168 to the Purdue University Center for Cancer Research in support of the use of facilities.

References

  1. Chang, C. C., Wu, M. J., Yang, J. Y., Camarillo, I. G. and Chang, C. J. (2015). Leptin-STAT3-G9a signaling promotes obesity-mediated breast cancer progression. Cancer Res 75(11): 2375-2386.
  2. Wallace, J. (2000). Humane endpoints and cancer research.ILAR J 41(2): 87-93.

材料和试剂

  1. 40μm细胞过滤器(STEMCELL Technologies,目录号:27305)
  2. 雌性Sprague-Dawley大鼠(Envigo,Harlan Laboratories,目录号:Sprague Dawley SD )
  3. 1ml,26G×3/8in。BD具有可分离针头的结核菌素注射器(slip tip)(BD Biosciences,目录号:309625)
  4. (Sigma-Aldrich,目录号:N1517)
  5. 氯化钠(Sigma-Aldrich,目录号:S7653)
  6. 10%中性福尔马林(Sigma-Aldrich,目录号:HT501128)
  7. EpiCult TM -B小鼠培养基试剂盒(STEMCELL Technologies,目录号:05610)
  8. 10x温和胶原酶/透明质酸酶(STEMCELL Technologies,目录号:07919)
  9. Hanks平衡盐溶液改性(HBSS)(STEMCELL Technologies,目录号:37150)
  10. 胰蛋白酶-EDTA
  11. 氯化铵(NH 4 Cl)(STEMCELL Technologies,目录号:07800)
  12. 40μm细胞过滤器(STEMCELL Technologies,目录号:27305)
  13. 胎牛血清(FBS)(Corning,目录号:35-011-CV)
  14. 流式细胞染色缓冲液(BD Biosciences,目录号:554656)
  15. FITC缀合的抗OBR抗体(aa956-986,多克隆抗体)(Lifespan Biosciences,目录号:LS-C261832)
  16. 常规大鼠食物饮食(Envigo,目录号:8604)(参见食谱)
  17. 西方饮食(Research Diets,目录号:D12079B)(参见食谱)
  18. 0.9%盐水(见配方)
  19. N - 甲基亚硝基脲溶液(参见配方)

设备

  1. 双能X射线吸收测定法(Lunar Corporation,型号:Lunar PIXImus II密度计)
  2. 细胞分析仪(BD Biosciences,型号:BD FACSCanto II)

软件

  1. BD FACSDiva和FCS Express流式细胞术软件

程序

  1. 肥胖诱导的乳腺癌
    1. 从Harlan实验室获得二十日龄的远交的Sprague-Dawley大鼠。
    2. Wean小狗在到达后24小时保持在温度和光控制的动物设施中。
    3. 给动物随意获取水,并在分组后随机分配给正常大鼠食物饮食或西方饮食。
    4. 在54天龄时,根据通过双能量x射线吸收测定法(DEXA)测定的它们的身体脂肪组成对动物进行分组。瘦大鼠食物饮食中大鼠的平均体脂肪百分比将是肥胖西方饮食的一半。来自DEXA扫描的身体组成分析的实例显示在表1中
      表1.来自DEXA扫描的身体组成分析。与西方贫困组中的动物相比,西方 - 肥胖组中的动物显示脂肪量显着增加。值表示平均值±SEM。 n =每组9只动物。星号表示P < 0.05,双星号表示P < 0.0001(来自Chang&et al。,2015)。


    5. 根据来自DEXA扫描的身体组成分析,将动物对西方饮食放置成两组,将最高三分位数的大鼠置于西方肥胖组中,将最低三分位数的大鼠置于西方瘦弱组中。三组将被指定:"西方肥胖","西方瘦"和"常规食物/饮食"
    6. 在使用前立即溶解于0.9%NaCl中,并用乙酸将pH调节至pH = 4。在使用前制备新鲜溶液。
    7. 在65天龄时,用50mg/kg的化学致癌物N 1 - 甲基亚硝基脲腹膜内注射每组中的一半大鼠,剩余的动物接受0.9%盐水(药物的量为给予每单位体重,以mg/kg计)。为了减少刺穿器官的可能性,使用26号3/8英寸针头
    8. 注射后168天或当动物达到终末状态时,通过确定的指南(Wallace,2000)确定大鼠的表面。
    9. 收集完整的乳腺和肿瘤,在液氮中快速冷冻或固定在10%中性福尔马林中用于组织学评估(如图1所示)。


      图1.在西方肥胖动物,瘦瘦和正常饮食饲养动物中的肿瘤表型。在正常饮食喂养大鼠中显示正常乳腺的H& E染色,在西方肥胖动物中显示侵袭性腺癌,在西方贫困动物中的腺瘤(来自Chang等人,2015)。

  2. 分离乳腺肿瘤细胞用于流式细胞术分析
    1. 通过用9份补充有5%FBS的完整EpiCult -B培养基稀释1份10x温和胶原酶/透明质酸酶混合物并将溶液置于15ml离心管中来制备解离溶液。
    2. 切除和转移乳腺或肿瘤到3-5毫升解离溶液的无菌培养皿。用交叉图案的手术刀切碎组织,直到组织变成糊状。在37℃下孵育组织过夜(达15小时)。
    3. 解离后,将细胞在350×g离心5分钟。弃去上清液。
    4. 用补充有2%FBS和氯化铵的冷Hanks平衡盐溶液修饰(HBSS)的1:4混合物重悬浮沉淀,并以350xg离心5分钟。
    5. 弃去上清液,加入1-5 ml预热的1×胰蛋白酶-EDTA至部分解离的组织,用移液器混匀。
    6. 用P1000微量移液管轻轻吸上1-3分钟。
    7. 加入10ml补充有2%FBS的冷HBSS,并在350×g离心5分钟。
    8. 用另外10ml含有2%FBS的冷HBSS重悬浮沉淀,并通过40μm细胞过滤器将细胞悬浮液过滤到新的50ml离心管中。以350×g离心5分钟,弃去上清液
    9. 用流式细胞仪染色缓冲液重悬细胞沉淀,并用血细胞计数器或自动细胞计数器计数细胞。
    10. 等分200μl细胞悬浮液(10×10 6 -10 6个细胞),并用FITC缀合的抗OBR抗体将细胞染色20分钟。
    11. 通过加入PBS洗涤细胞。使用1毫升管。通过在室温下以400-600×g离心5分钟来沉淀细胞。重复两次洗涤,并在洗涤之间弃去上清液
    12. 将染色的细胞重悬在500μl流式细胞染色缓冲液中
    13. 对染色的细胞进行流式细胞术分析,并测定癌干细胞样OBR hi(高瘦素受体表达,FITC高)群体的百分比。数据可以通过BD FACSDiva和FCS Express流式细胞仪软件分析。所有外科手术和动物操作在Purdue动物护理和使用委员会的规定下进行。

食谱

  1. 常规大鼠饮食
    3.30 kcal/g,含18%的蛋白质 5%脂肪,主要来自玉米油和57%碳水化合物
  2. 西方饮食
    4.47 kcal/g,含20%的蛋白质 21%的无脂乳脂形式的脂肪和50%的碳水化合物,34%的蔗糖
  3. 0.9%盐水
    在灭菌的玻璃容器中将0.9g NaCl加入100ml ddH 2 O中,并通过0.2μm无菌过滤器
  4. N - 甲基亚硝基脲溶液
    在使用前立即溶解于0.9%盐水中,并用乙酸调节至pH值4.在使用前制备新鲜溶液

致谢

该方案用于Chang等人。,(2015)。这项工作是由沃尔特癌症基金会 - 肥胖和癌症试点格兰特(I.G。卡马里洛和CJ。张)和P30 CA023168支持普渡大学癌症研究中心支持使用的设施。

参考文献

  1. Chang,CC,Wu,MJ,Yang,JY,Camarillo,IG and Chang,CJ(2015)。  Leptin-STAT3-G9a信号传导促进肥胖介导的乳腺癌进展。癌症研究75(11):2375-2386。
  2. Wallace,J。(2000)。 人道终点和癌症研究。ILAR J(4):87-93。
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How to cite this protocol: Wu, M. J. and Chang, C. J. (2016). High Fat Diet-induced Breast Cancer Model in Rat. Bio-protocol 6(13): e1852. DOI: 10.21769/BioProtoc.1852; Full Text



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