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Hypoxia Studies with Pimonidazole in vivo
体内哌莫硝唑缺氧研究   

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Abstract

Therapy-induced hypoxia drives changes in the tumor microenvironment that contribute to the poor response to therapy. Hypoxia is capable of driving the expression and/or activation of specific signaling cascades (e.g., c-Met, Axl, CTGF), the recruitment of tumor promoting immune cells, and the induction of cell survival pathways including autophagy (Phan et al., 2013; Hu et al., 2012; Ye et al., 2010). We have recently shown that anti-VEGF therapy-induced hypoxia can result in changes in the extracellular matrix that contribute to the aggressiveness of tumors post therapy (Aguilera et al., 2014). Importantly, therapies that induce hypoxia do not always increase epithelial plasticity and tumor aggressiveness (Ostapoff et al., 2013; Cenik et al., 2013). We have used pimonidazole to evaluate hypoxia in tumors and herein provide a detailed protocol for this useful tool to interrogate the levels of hypoxia in vivo.
The utility of the HypoxyprobeTM (pimonidazole hydrochloride) immunohistochemical analysis approach allows for the assessment of hypoxia in different tissues as well as cell types. Pimonidazole is a 2-nitroimidazole that is reductively activated specifically in hypoxic cells and forms stable adducts with thiol groups in proteins, peptides, and amino acids (Cenik et al., 2013; Arnold et al., 2010; Raleigh and Koch, 1990; Raleigh et al., 1998). Furthermore, the amount of pimonidazole that is detected is directly proportional to the level of hypoxia within tumors.

Materials and Methods

  1. HypoxyprobeTM-1 Plus Kit (HypoxyprobeTM, catalog number: HP1-1000Kit )
  2. A rabbit, FITC-conjugated, secondary reagent is included in the HypoxyprobeTM-1 Plus Kit (HypoxyprobeTM, catalog number: HP1-1000Kit)
  3. Sodium chloride ACS reagent (Sigma-Aldrich, catalog number: S9888 )
  4. Milli Q H2O
  5. Corning bottle-top vacuum filter system (Sigma-Aldrich, catalog number: CLS430769 )
  6. PBS (Sigma-Aldrich, catalog number: P-4417 )
  7. Anti-CD31 antibody (Dianova GmbH, catalog number: DIA-310 )
  8. Anti-Meca32 antibody (Abcam, catalog number: ab27853 )
  9. Anti-Endomucin antibody (Millipore, catalog number: MAB2624 )
  10. Cy3-conjugated secondary IgG (Jackson ImmunoResearch Laboratories)
  11. Tissue-Tek OCT compound (Ted Pella, catalog number: 27050 )
  12. Liquid nitrogen
  13. IsoFlo (isoflurane, USP) (Abbott Laboratories, catalog number: 05260-05 )
  14. Carbon dioxide (Sigma-Aldrich, catalog number: 295108 -227G)
  15. Superfrost plus; white microscope slides (Thermo Fisher Scientific, catalog number: 12-550-15 )
  16. Liquid scintillation vials, plastic (Sigma-Aldrich, catalog number: M2026 -1000EA)
  17. Prolong gold antifade mountant (Life Technologies, catalog number: P10144 )
  18. Aqua Block/EIA/WB (EastCoast Bio, catalog number: PP82 )
  19. Acetone (Sigma-Aldrich, catalog number: 320110 )
  20. Micro cover glasses, rectangular, No. 1 (VWR International, catalog number: 48393-106 )
  21. Tween 20 (Sigma-Aldrich, catalog number: P9416 -50ML)
  22. 0.9% saline (see Recipes)
  23. 30 mg/ml pimonidazole in 0.9% saline (see Recipes)
  24. Antibody diluent (see Recipes)
  25. 20% aqua block (see Recipes)
  26. PBS-t (see Recipes)

Equipment

  1. Cryostat (Leica, catalog number: CM3050 S )
  2. Low profile microtome blades (Accu Edge, catalog number: 4689 )
  3. Surgical equipment (Braintree Scientific)
  4. Fluorescent or bright-field microscope (Nikon Eclipse, model: E600 )
  5. Fluorescent microscope camera (Photometrics, Cool Snap HQ)
  6. Nikon filters (FITC: 96170M F HQ, Cy3/TRITC: 96171M R HG, DAPI/nuclei: 96101M UV-2E/C)
  7. 30 G x 1/2 in. precision glide needles (BD Biosciences, catalog number: 305106 )
  8. Tuberculin syringe only (BD Biosciences, catalog number: 309659 )
  9. Tailveiner restrainer for mice (Braintree Scientific, catalog number: TV-150 STD )

Software

  1. NIS Elements software, ImageJ (open source software: http://imagej.nih.gov/ij/), or other image analysis software

Procedure

The following procedure measures hypoxia in tumor-bearing mice and can be used at any respective end point of an animal experiment.

  1. The HypoxyprobeTM (pimonidazole hydrochloride) is resuspended at a concentration of 30 mg/ml in 0.9% sterile saline.
  2. Multiple mice (n = 5 per group is optimal for statistical relevance) are injected intravenously (tail vein) with 60 mg/kg of the pimonidazole solution.


    Figure 1. Tail Vein Injection protocol. 1. Obtain a Tailveiner Restrainer. 2. Insert mouse and push the restrainer to set the mouse in position. 3. Rotate the tail 45 degrees in order to find and inject the tail vein with the needle. 4. Close representation of the tail vein injection. 5. The appearance of blood validates a successful tail vein injection.

  3. Let circulate in vivo for 90 min before the mice are euthanazied with isoflurane or carbon dioxide (CO2).
    Note: For animal euthanasia: In compliance with proper IACUC protocols, animals are placed in a drop jar or vaporizer in the presence of controlled concentrations of isoflurane or CO2 in order to ensure proper inhalation. Animals are then sacrificed by a secondary method with cervical dislocation.
  4. Organs are then removed, weighed, snap-frozen, and stored in plastic scintillation vials at -80 °C.
    Note: The pimonidazole stain can work on paraffin-embedded tissue, frozen tissue, and cell lines.
  5. Organs are then taken out of the scintillation vials, embedded in OCT, placed in a cryostat, and cut into 10 µm-thick sections. Sections are placed on microscope slides for staining.
    Notes:
    Cryostat settings
    1. Temperature used can range from -10 to -50 °C depending on tissue type, though -20 °C is recommended for the majority of tissues.
    2. Alternate between “Trimming mode” (cuts 5 - 150 µm) and “Sectioning mode” (0.5 - 50 µm) in order to adjust the tissue for the beginning of section collection.
    3. The speed of sectioning is dependent of the researcher’s own speed.
    4. As many sections as necessary can be cut per organ used.
    5. To achieve a proper representation of the sample, cut non-serial sections of the tissue.
  6. Frozen tissue sections are then interrogated with FITC-conjugated anti-pimonidazole primary antibody (included in kit) and endothelial cell markers (CD31, Meca-32, or endomucin) followed by respective Cy3-conjugated secondary IgG. Use a slide incubated with only antibody diluent as its primary antibody as a negative control.
    1. The Hypoxyprobe™-1 Plus Kit recommends a 1:50 - 100 dilution of the secondary rabbit anti-FITC horseradish peroxidase conjugated antibody.
    2. For other primary antibodies a 1:50 - 1:100 dilution is recommended, unless otherwise specified by respective primary antibody data sheets.
      Notes:
      1. Any suitably labeled, secondary anti-FITC antibody can be used which would bind to the pimonidazole adducts in hypoxic cells within the tissues.
      2. This staining should be done within a week of harvesting the organs as the signal will begin to fade.
      3. The interrogation of hypoxia within animal tissues is not limited to fluorescent staining as it can be done with brown stain (DAB) as well. Respective primary and secondary antibodies can be utilized to achieve both types of staining.
  7. Example of a fluorescent tissue-staining protocol for frozen sections:
    1. Air dry frozen slides.
    2. Fix the slides with Acetone for 30 sec - 2 min.
    3. Briefly air dry sections a few seconds (do not let tissue dry completely).
    4. Rehydrate sections with PBS-t 2 times, 5 min each.
      Note: Once the tissue is rehydrated, do not let the slides dry out.
    5. Block in your favorite blocking solution (typically 20% aqua block in PBS) 10 - 30 min.
    6. Pour off the blocking solution, add primary antibody and incubate 1 h at room temperature-overnight at 4 °C.
    7. Wash the slides with PBS-t 3 times, 5 min each.
    8. Incubate the slides with secondary antibody 1 h at room temperature.
    9. Wash with PBS-t 4 times, 5 min each.
    10. Mount the slides with prolong gold mounting solution and coverslip.
    11. Apply sealant (e.g. clear nail polish) along the edges of the coverslips to preserve the slides.
    12. Image the slides.
  8. Imaging of tissue sections should then be obtained with 20x or 40x microscopic magnification as desired and analyzed using NIS Elements software or ImageJ (open source software).
    Notes:
    1. The filters on the microscope should include the following wavelengths for fluorescent microscopy:
      1. FITC (green 470-520 nm)
      2. Cy3/TRITC (red 530-570 nm)
      3. DAPI/nuclei (blue 345-455 nm)
    2. Six to eight images per tissue area are recommended in order to capture the variable signal intensity in a wide representative sample of the respective tissue.
    3. The negative control slide from step 6 should be used as a background control. With the image analysis software, once the threshold of “background” is calculated use this threshold to image the remaining sections and ultimately normalize them to this threshold to eliminate the background.

Representative data



Figure 2. Representative data (taken from Reference 1). 6-week-old KIC mice were treated with saline or mcr84 for 1 week. Animals were sacrificed 1 h after pimonidazole injection (i.v.). Tissue was stained for endothelial cells (MECA32, red) and pimonidazole adducts (Hypoxyprobe, green). Hypoxic area was quantified and is presented on the right. Error bars: *P<0.05, one-way ANOVA with Tukey multiple comparison test. Pancreata from 6 animals were sectioned per treatment group, an average intensity value was determined for each organ and the intensities were averaged and the SEM was calculated. Please view this reference for more information on the model and context of the representative data.


Figure 3. Representative data (taken from Aguilera et al., 2014). The amount of hypoxia within tumors was quantified using an antibody, FITC-conjugated mouse anti-pimonidazole, directed against an adduct that forms when Hypoxyprobe-1 enters hypoxic tissue. The percentage of threshold area was quantified and two separate experiments were combined by normalization to data from Sparc+/+ mice. Images display hypoxia (green) near the vasculature (red) stained with the rat antimouse endothelial cell (Meca-32) antibody within tumors. Nuclei are marked with DAPI (blue). Error bars: *P<0.05, student’s t-test. Please view this reference for more information on the model and context of the representative data.

  1. Representative data from our experiments are published in Aguilera et al. (2014), Arnold et al. (2010), Cenik et al. (2013) and Ostapoff et al. (2013).
  2. Other representative data in a tumor-bearing model can be found in Graves et al. (2010), and background information on the establishment of this assay can be found in Raleigh et al. (1998) defining the use of pimonidazole as a marker of hypoxia.

Recipes

  1. 0.9% saline
    Mix 0.9 grams of sodium chloride (NaCl) in 100 ml milli Q H2O
    Filter
  2. 30 mg/ml pimonidazole in 0.9% saline
    30 mg of pimonidazole in 1 ml of filtered 0.9% saline
  3. Antibody diluent (unless otherwise specified in the HypoxyprobeTM-1 Plus Kit)
    5% BSA in PBS
  4. 20% aqua block
    20 ml aqua block in 80 ml PBS
  5. PBS-t
    200 µl of Tween 20 in 99.8 ml PBS

Acknowledgments

Drs. Bercin Kutluk Cenik and Shanna Arnold for information on this assay, Dr. Miao Wang for the tail vein injection procedure, and Dr. Michael Dellinger for the frozen slide staining protocol. This work is supported in part by a sponsored research agreement from Affitech AS (RAB), the NIH (R01CA118240 to RAB; F31CA168350 to KYA; U01CA141576 and R01CA137181 to DHC), and the Effie Marie Cain Scholarship for Angiogenesis Research (RAB).

References

  1. Aguilera, K. Y., Rivera, L. B., Hur, H., Carbon, J. G., Toombs, J. E., Goldstein, C. D., Dellinger, M. T., Castrillon, D. H. and Brekken, R. A. (2014). Collagen signaling enhances tumor progression after anti-VEGF therapy in a murine model of pancreatic ductal adenocarcinoma. Cancer Res 74(4): 1032-1044.
  2. Arnold, S. A., Rivera, L. B., Miller, A. F., Carbon, J. G., Dineen, S. P., Xie, Y., Castrillon, D. H., Sage, E. H., Puolakkainen, P., Bradshaw, A. D. and Brekken, R. A. (2010). Lack of host SPARC enhances vascular function and tumor spread in an orthotopic murine model of pancreatic carcinoma. Dis Model Mech 3(1-2): 57-72.
  3. Cenik, B. K., Ostapoff, K. T., Gerber, D. E. and Brekken, R. A. (2013). BIBF 1120 (nintedanib), a triple angiokinase inhibitor, induces hypoxia but not EMT and blocks progression of preclinical models of lung and pancreatic cancer. Mol Cancer Ther 12(6): 992-1001.
  4. Graves, E. E., Vilalta, M., Cecic, I. K., Erler, J. T., Tran, P. T., Felsher, D., Sayles, L., Sweet-Cordero, A., Le, Q. T. and Giaccia, A. J. (2010). Hypoxia in models of lung cancer: implications for targeted therapeutics. Clin Cancer Res 16(19): 4843-4852
  5. Hu, Y.-L., Jahangiri, A., DeLay, M. and Aghi, M. K. (2012). Tumor cell autophagy as an adaptive response mediating resistance to treatments such as antiangiogenic therapy. Cancer Res 72(17): 4294-4299.
  6. Ostapoff, K. T., Awasthi, N., Cenik, B. K., Hinz, S., Dredge, K., Schwarz, R. E. and Brekken, R. A. (2013). PG545, an angiogenesis and heparanase inhibitor, reduces primary tumor growth and metastasis in experimental pancreatic cancer. Mol Cancer Ther 12(7): 1190-1201.
  7. Phan, V. T., Wu, X., Cheng, J. H., Sheng, R. X., Chung, A. S., Zhuang, G., Tran, C., Song, Q., Kowanetz, M., Sambrone, A., Tan, M., Meng, Y. G., Jackson, E. L., Peale, F. V., Junttila, M. R. and Ferrara, N. (2013). Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF-induced neutrophil recruitment. Proc Natl Acad Sci U S A 110(15): 6079-6084.
  8. Raleigh, J. A. and Koch, C. J. (1990). Importance of thiols in the reductive binding of 2-nitroimidazoles to macromolecules. Biochem Pharmacol 40(11): 2457-2464.
  9. Raleigh, J. A., Calkins-Adams, D. P., Rinker, L. H., Ballenger, C. A., Weissler, M. C., Fowler, W. C., Novotny, D. B. and Varia, M. A. (1998). Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker. Cancer Res 58(17): 3765-3768.
  10. Ye, X., Li, Y., Stawicki, S., Couto, S., Eastham-Anderson, J., Kallop, D., Weimer, R., Wu, Y. and Pei, L. (2010). An anti-Axl monoclonal antibody attenuates xenograft tumor growth and enhances the effect of multiple anticancer therapies. Oncogene 29(38): 5254-5264.

简介

治疗诱导的缺氧驱动肿瘤微环境中的变化,其导致对治疗的差的反应。缺氧能够驱动特异性信号级联(例如,c-Met,Axl,CTGF)的表达和/或活化,肿瘤促进免疫细胞的募集和细胞存活途径的诱导,包括自噬(Phan等人,2013; Hu等人,2012; Ye等人,2010)。我们最近已经显示,抗VEGF治疗诱导的缺氧可以导致细胞外基质的变化,其有助于治疗后肿瘤的侵袭性(Aguilera等人,2014)。重要的是,诱导缺氧的治疗不总是增加上皮可塑性和肿瘤侵袭性(Ostapoff等人,2013; Cenik等人,2013)。我们已经使用哌莫硝唑来评价肿瘤中的缺氧,并且在本文中为这种有用的工具提供详细的方案来询问体内缺氧水平。
Hypoxyprobe TM (哌莫硝唑盐酸盐)免疫组织化学分析方法的实用性允许评估不同组织以及细胞类型中的缺氧。哌莫硝唑是2-硝基咪唑,其在缺氧细胞中特异性地被还原活化,并与蛋白质,肽和氨基酸中的巯基形成稳定的加合物(Cenik等人,2013; Arnold等人。,2010; Raleigh和Koch,1990; Raleigh等人,1998)。此外,检测到的吡莫硝唑的量与肿瘤内的缺氧水平成正比

材料和方法

  1. Hypoxyprobe TM -1 Plus Kit(Hypoxyprobe TM ,目录号:HP1-1000Kit)
  2. 在Hypoxyprobe TM Plus Plus试剂盒(Hypoxyprobe TM ,目录号:HP1-1000Kit)中包含兔,FITC缀合的第二试剂。
  3. 氯化钠ACS试剂(Sigma-Aldrich,目录号:S9888)
  4. Milli Q H sub 2 O 2 /
  5. Corning瓶顶真空过滤系统(Sigma-Aldrich,目录号:CLS430769)
  6. PBS(Sigma-Aldrich,目录号:P-4417)
  7. 抗CD31抗体(Dianova GmbH,目录号:DIA-310)
  8. 抗Meca32抗体(Abcam,目录号:ab27853)
  9. 抗Endomucin抗体(Millipore,目录号:MAB2624)
  10. Cy3缀合的第二IgG(Jackson ImmunoResearch Laboratories)
  11. Tissue-Tek OCT化合物(Ted Pella,目录号:27050)
  12. 液氮
  13. IsoFlo(异氟烷,USP)(Abbott Laboratories,目录号:05260-05)
  14. 二氧化碳(Sigma-Aldrich,目录号:295108-227G)
  15. 超级霜加; 白色显微镜载玻片(Thermo Fisher Scientific,目录号:12-550-15)
  16. 液体闪烁小瓶,塑料(Sigma-Aldrich,目录号:M2026-1000EA)
  17. 长寿黄金防褪色填充剂(Life Technologies,目录号:P10144)
  18. Aqua Block/EIA/WB(EastCoast Bio,目录号:PP82)
  19. 丙酮(Sigma-Aldrich,目录号:320110)
  20. 微型保护玻璃,矩形,1号(VWR国际,目录号:48393-106)
  21. 吐温20(Sigma-Aldrich,目录号:P9416-50ML)
  22. 0.9%盐水(见配方)
  23. 30mg/ml哌莫硝唑在0.9%盐水中(见Recipes)
  24. 抗体稀释剂(参见配方)
  25. 20%水块(见配方)
  26. PBS-t(参见配方)

设备

  1. 低温恒温器(Leica,目录号:CM3050S)
  2. 薄型切片机刀片(Accu Edge,目录号:4689)
  3. 手术设备(Braintree Scientific)
  4. 荧光或明视场显微镜(Nikon Eclipse,型号:E600)
  5. 荧光显微镜相机(Photometrics,Cool Snap HQ)
  6. 尼康过滤器(FITC:96170M F HQ,Cy3/TRITC:96171M R HG,DAPI /核:96101M UV-2E/C)
  7. 30G×1/2英寸精密滑动针(BD Biosciences,目录号:305106)
  8. 仅结核菌素注射器(BD Biosciences,目录号:309659)
  9. 小鼠尾部限制器(Braintree Scientific,目录号:TV-150STD)

软件

  1. NIS Elements软件,ImageJ(开源软件: http://imagej.nih.gov/ij/ )或其他图像分析软件

程序

以下程序测量荷瘤小鼠中的缺氧,并且可以在动物实验的任何相应终点使用。

  1. 将Hypoxyprobe TM sup(盐酸哌莫硝唑)以30mg/ml的浓度在0.9%无菌盐水中重悬浮。
  2. 多个小鼠(每组n = 5对于统计学相关性是最佳的)用60mg/kg的咪蒙唑溶液静脉内(尾静脉)注射。


    图1.尾静脉注射方案。1.获得Tailveiner Restrainer。 2。插入鼠标并按下限制器,将鼠标置于位置。 < u> 3。将尾部旋转45度,以找到并用针注射尾静脉。 4。尾静脉注射的近端表示。 5。血液的出现验证了成功的尾静脉注射。

  3. 在小鼠用异氟烷或二氧化碳安乐死前(CO 2),在体内循环90分钟。
    注意:对于动物安乐死:根据适当的IACUC方案,将动物置于滴剂罐或蒸发器中,在控制浓度的异氟烷或CO 2气体存在下, ,以确保正确吸入。然后通过颈部脱臼的第二种方法处死动物。
  4. 然后取出器官,称重,快速冷冻,并在-80℃下储存在塑料闪烁瓶中。
    注意:哌莫硝唑染色可用于石蜡包埋的组织,冷冻组织和细胞系。
  5. 然后将器官从闪烁瓶中取出,包埋在OCT中,置于低温恒温器中,切成10μm厚的切片。将切片置于显微镜载玻片上用于染色。
    注意:
    低温恒温器设置
    1. 使用的温度范围可以是-10至-50℃,取决于组织类型, 虽然-20°C推荐用于大多数组织。
    2. 在"修剪模式"(切割5 - 150μm)和"切割"之间切换 模式"(0.5-50μm),以便调整组织的开始 节集合。
    3. 切片的速度取决于研究者自己的速度。

    4. 为了实现样品的适当表示,切割组织的非连续部分。
  6. 然后用FITC缀合的抗咪莫唑一抗(包含在试剂盒中)和内皮细胞标记(CD31,Meca-32或内皮抑制素),然后用各自的Cy3-缀合的第二IgG来探查冷冻的组织切片。 使用仅用抗体稀释剂作为其一抗作为阴性对照孵育的载玻片。
    1. Hypoxyprobe™-1 Plus Kit建议稀释1:50 - 100 二抗兔抗FITC辣根过氧化物酶偶联抗体。
    2. 对于其他一抗,推荐1:50 - 1:100稀释,   除非各自的一抗数据表另有规定 注意:
      1. 可以使用任何适当标记的二抗FITC抗体 将结合于咪达唑加合物中的缺氧细胞内 组织。
      2. 这种染色应在收获器官的一周内完成,因为信号将开始褪色。
      3. 动物组织内缺氧的询问不限于 荧光染色,因为它可以用棕色染色(DAB)进行。 可以利用相应的一级和二级抗体来实现 两种类型的染色。
  7. 冷冻切片的荧光组织染色方案的实施例:
    1. 风干冻结的滑梯。
    2. 用丙酮固定幻灯片30秒-2分钟。
    3. 短暂空气干燥几秒钟(不要让组织完全干燥)。
    4. 用PBS-t进行两次水合切片,每次5分钟 注意:一旦组织再水化,不要让幻灯片变干。
    5. 阻止在您最喜欢的封锁解决方案(通常20%的水溶性块在PBS)10 - 30分钟
    6. 倒出封闭溶液,加入一抗,在室温下孵育1小时,在4℃过夜
    7. 用PBS-t洗涤载玻片3次,每次5分钟
    8. 在室温下用二抗孵育载玻片1小时
    9. 用PBS-t洗涤4次,每次5分钟
    10. 用延长的金色安装溶液和盖玻片安装载玻片
    11. 沿着盖玻片的边缘涂抹密封剂(例如清除指甲油),以保留幻灯片。
    12. 对幻灯片进行映像。
  8. 然后根据需要使用20x或40x显微镜放大获得组织切片的成像,并使用NIS Elements软件或ImageJ(开源软件)进行分析。
    注意:
    1. 显微镜上的滤光片应包括用于荧光显微镜的以下波长:
      1. FITC(绿色470-520 nm)
      2. Cy3/TRITC(红色530-570 nm)
      3. DAPI/nuclei(蓝色345-455 nm)
    2. 为了每个组织区域建议六到八个图像 在广泛的代表性样本中捕获可变信号强度   相应的组织。
    3. 阴性对照从步骤6滑动   应该用作后台控件。 用图像分析 软件,一旦"背景"的阈值被计算使用这个 阈值以对剩余部分成像,并最终将它们标准化 到此阈值以消除背景。

代表数据



图2.代表性数据(取自参考文献1)。将6周龄的KIC小鼠用盐水或mcr84处理1周。哌莫硝唑注射后1小时处死动物(i.v.)。组织对内皮细胞(MECA32,红色)和哌莫硝唑加合物(Hypoxyprobe,绿色)染色。缺氧区域被量化并且呈现在右边。误差棒:* P <0.05,使用Tukey多重比较检验的单因素方差分析。每个处理组切割6只动物的胰腺,测定每个器官的平均强度值,并且平均强度并计算SEM。有关代表性数据的模型和上下文的更多信息,请查看此参考

图3.代表性数据(取自Aguilera等人,2014)。使用抗体,FITC缀合的小鼠抗哌莫硝唑对肿瘤内缺氧的量进行定量,针对当Hypoxyprobe-1进入缺氧组织时形成的加合物。定量阈面积的百分比,并通过归一化到来自Sparc +/+小鼠的数据来组合两个单独的实验。图像显示在肿瘤内用鼠抗鼠内皮细胞(Meca-32)抗体染色的脉管系统附近的缺氧(绿色)(红色)。用DAPI(蓝色)标记核。误差条:* P <0.05,学生t检验。有关代表数据的模型和上下文的更多信息,请查看此参考。

  1. 来自我们实验的代表性数据公开在Aguilera等人(2014),Arnold等人中。 (2010),Cenik等人。 (2013)和Ostapoff等人。 (2013)。
  2. 荷瘤模型中的其他代表性数据可以在Graves等人中找到。 (2010),以及关于建立该测定的背景信息可以在Raleigh等人中找到。 (1998) 使用哌莫硝唑作为缺氧的标志物。

食谱

  1. 0.9%盐水
    将0.9克氯化钠(NaCl)在100ml milli Q H 2 O中混合 过滤
  2. 30mg/ml哌莫硝唑的0.9%盐水溶液
    30mg哌莫硝唑在1ml过滤的0.9%盐水中
  3. 抗体稀释剂(除非在Hypoxyprobe TM -1 Plus试剂盒中另有规定)
    5%BSA的PBS溶液
  4. 20%水性块
    在80ml PBS中的20ml水性块
  5. PBS-t
    200μl吐温20在99.8ml PBS中的溶液

致谢

博士。 Bercin Kutluk Cenik和Shanna Arnold提供关于该测定的信息,Miao Wang博士用于尾静脉注射方法,以及Michael Dellinger博士用于冷冻玻片染色方案。 这项工作部分由Affitech AS(RAB),NIH(R01CA118240至RAB; F31CA168350至KYA; U01CA141576和R01CA137181至DHC)和Effie Marie Cain血管生成研究奖学金(RAB)的赞助研究协议部分支持。

参考文献

  1. Aguilera,K.Y.,Rivera,L.B.,Hur,H.,Carbon,J.G.,Toombs,J.E.,Goldstein,C.D.,Dellinger,M.T.,Castrillon,D.H。和Brekken, 胶原信号在胰腺导管腺癌的鼠模型中在抗VEGF治疗后增强肿瘤进展。 a> Cancer Res 74(4):1032-1044。
  2. Arnold,S.A.,Rivera,L.B.,Miller,A.F.,Carbon,J.G.,Dineen,S.P.,Xie,Y.,Castrillon,D.H.,Sage,E.H.,Puolakkainen,P.,Bradshaw,A.D.and Brekken, 缺乏宿主SPARC在胰腺癌的原位小鼠模型中增强血管功能和肿瘤扩散。 a> Dis Model Mech 3(1-2):57-72
  3. Cenik,B.K.,Ostapoff,K.T.,Gerber,D.E.and Brekken,R.A。(2013)。 BIBF 1120(nintedanib),一种三重血管激酶抑制剂,诱导缺氧,但不诱导EMT并阻断进展肺癌和胰腺癌的临床前模型。 Mol Cancer Ther 12(6):992-1001。
  4. Graves,E.E.,Vilalta,M.,Cecic,I.K.,Erler,J.T.,Tran,P.T.,Felsher,D.,Sayles,L.,Sweet- Cordero,A.,Le,Q.T.and Giaccia,A.J。 肺癌模型中的缺氧:对靶向治疗的影响临床癌症Res 16(19):4843-4852
  5. Hu,Y.-L.,Jahangiri,A.,DeLay,M.and Aghi,M.K。(2012)。 肿瘤细胞自噬作为一种适应性反应介导对抗血管生成疗法等治疗的耐药性。 Cancer Res 72(17):4294-4299
  6. Ostapoff,K.T.,Awasthi,N.,Cenik,B.K.,Hinz,S.,Dredge,K.,Schwarz,R.E.and Brekken,R.A。(2013)。 PG545是一种血管生成和肝素酶抑制剂,可减少实验性胰腺癌的原发性肿瘤生长和转移。 a> Mol Cancer Ther 12(7):1190-1201
  7. Phan,VT,Wu,X.,Cheng,JH,Sheng,RX,Chung,AS,Zhuang,G.,Tran,C.,Song,Q.,Kowanetz,M.,Sambrone, ,Meng,YG,Jackson,EL,Peale,FV,Junttila,MR和Ferrara,N。(2013)。 致癌性RAS途径活化通过G-CSF诱导的嗜中性粒细胞募集促进对抗VEGF治疗的抗性。/a> Proc Natl Acad Sci USA 110(15):6079-6084。
  8. Raleigh,J.A。和Koch,C.J。(1990)。 硫醇在2-硝基咪唑还原结合大分子中的重要性。 Biochem Pharmacol 40(11):2457-2464。
  9. Raleigh,J.A.,Calkins-Adams,D.P.,Rinker,L.H.,Ballenger,C.A.,Weissler,M.C.,Fowler,W.C.,Novotny,D.B.and Varia,M.A。(1998)。 使用哌莫硝唑作为缺氧标记物的人鳞状细胞癌中的缺氧和血管内皮生长因子表达。 Cancer Res 58(17):3765-
  10. Ye,X.,Li,Y.,Stawicki,S.,Couto,S.,Eastham-Anderson,J.,Kallop,D.,Weimer,R.,Wu,Y.and Pei, 抗Axl单克隆抗体可减弱异种移植肿瘤生长,并增强多种抗癌治疗的效果。 a> Oncogene 29(38):5254-5264。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Aguilera, K. Y. and Brekken, R. A. (2014). Hypoxia Studies with Pimonidazole in vivo. Bio-protocol 4(19): e1254. DOI: 10.21769/BioProtoc.1254.
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