Unbiased Screening of Activated Receptor Tyrosine Kinases (RTKs) in Tumor Extracts Using a Mouse Phospho-RTK Array Kit
利用小鼠Phospho-RTK Array试剂盒无偏筛选肿瘤提取物中激活受体酪氨酸激酶   

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PLOS Pathogens
Jul 2018

 

Abstract

Kaposi’s sarcoma (KS) herpesvirus (KSHV) is a virus that causes KS, an angiogenic AIDS-associated spindle-cell neoplasm, by activating host oncogenic signaling cascades through autocrine and paracrine mechanisms. Many host signaling cascades co-opted by KSHV including PI3K/AKT/mTORC, NFkB and Notch are critical for cell-specific mechanisms of transformation and their identification is paving the way to therapeutic target discovery. Analysis of the molecular KS signature common to human KS tumors and our mouse KS-like tumors showed consistent expression of KS markers VEGF and PDGF receptors with upregulation of other angiogenesis ligands and their receptors in vivo. This points to the autocrine and paracrine activation of various receptor tyrosine kinase (RTK) signaling axes. Hereby we describe a protocol to screen for activated receptor tyrosine kinase of KSHV-induced KS-like mouse tumors using a Mouse Phospho-RTK Array Kit and its validation by RTK western blots. We showed that this method can be successfully used to rank the tyrosine kinase receptors most activated in tumors in an unbiased manner. This allowed us to identify PDGFRA as an oncogenic driver and therapeutic target in AIDS-KS.

Keywords: PDGFRA (血小板源性生长因子受体α多肽), Tyrosine kinase (酪氨酸激酶), Proteomic array (蛋白质芯片), KSHV (KSHV), Kaposi’s sarcoma (卡波西肉瘤)

Background

Kaposi’s sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS) (Chang et al., 1994; Ganem, 2010; Mesri et al., 2010; Dittmer and Damania, 2016). KS is a major cancer associated with AIDS (AIDS-KS) and is the most prevalent type of cancer affecting men and children in Africa (Ganem, 2010; Mesri et al., 2010; Cavallin et al., 2014). Although the incidence of AIDS-KS in the western world has markedly declined since the wide-spread implementation of HAART (Highly Active Antiretroviral Therapy), a significant percentage of AIDS-KS patients never achieve total remission (Krown, 2003; Nguyen et al., 2008; Cavallin et al., 2014). Moreover, KSHV prevalence and KS incidence appear to be increasing, even in HAART treated HIV patients with controlled viremias (Maurer et al., 2007; Labo et al., 2015). Understanding the interplay of viral and host factors in KS oncogenesis is critical for the rational development of new therapies (Dittmer and Krown, 2007; Sullivan et al., 2009). Many host signaling cascades co-opted by KSHV including PI3K/AKT/mTORC, NFkB and Notch are critical for cell-specific mechanisms of transformation and their identification is paving the way to therapeutic target discovery (Sodhi et al., 2006; Emuss et al., 2009; Liu et al., 2010; Mesri et al., 2014; Dittmer and Damania, 2016). Analysis of the molecular KS signature common to human KS tumors and our mouse KS-like tumors, showed consistent expression of KS markers VEGFR 1, 2, 3, Podoplanin with upregulation of angiogenesis ligands and receptors in vivo, pointing to the upregulation of various receptor tyrosine kinase signaling axes (Mutlu et al., 2007; Mesri et al., 2010). Thus, we set out to rank the host tyrosine kinase signaling cascades activated by KSHV using a mouse model of KSHV-dependent tumorigenesis that helped us to identify PDGFRA as an oncogenic driver and therapeutic target in AIDS-KS (Cavallin et al., 2018).

Here we describe an assay protocol using a Mouse Phospho-RTK Array Kit in mouse KSHV-induced KS-like tumors, which can be used as a reliable method to rank the tyrosine kinase receptors most activated in tumors in an unbiased manner. The Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems) is a membrane-based sandwich immunoassay. Captured antibodies spotted in duplicate on nitrocellulose membranes bind to specific target proteins present in the sample. Tyrosine phosphorylation of the captured proteins is detected with an HRP-conjugated pan phospho-tyrosine antibody and then visualized using chemiluminescent detection reagents. The signal produced is proportional to the amount phosphorylation in the bound analyte.

Surprisingly, analysis of the pattern of tumor RTK activation (Figures 1 and 2) showed a single prominently activated RTK spot that accounted for a significant portion of the total tyrosine kinase activation of the tumors. This signal corresponded to the PDGF receptor alpha-chain (PDGFRA) (Cavallin et al., 2018). Validation of the array by Western Blot analysis (Figure 3A) shows that PDGFRA is robustly expressed and much more phosphorylated in the tumors than in mouse normal skin. On the other hand, c-kit (Figure 3B), a related RTK, is similarly expressed in skin and tumors and displays low levels of activation in the tumors; that are similar to those in the skin control and correlates with the low activation of c-kit in our phospho-tyrosine kinase proteomic array (Cavallin et al., 2018).

Materials and Reagents

Common lab consumables

  1. Pipette tips 
  2. Gloves 
  3. Plastic containers with the capacity to hold 50 ml (for washing the arrays)
  4. Plastic wrap
  5. Absorbent lab wipes (KimWipes® or equivalent) 
  6. Paper towels
  7. Plastic transparent sheet protector (trimmed to 10 cm x 12 cm and open on three sides) 
  8. TissueRuptor Disposable Probes (QIAGEN, catalog number: 990890)

Reagent Preparation (Mouse Phospho-RTK Array Kit)
  1. Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems, catalog number: ARY014)
    Kit Contents:
    1. 4 Array Membranes
    2. 4-Well Multi-dish
    3. Array Buffers:
      1)
      Lysis Buffer 17 (see Recipes)
      2)
      Array Buffer 1
      3)
      1x Array Buffer 2 (see Recipes)
      4)
      1x Wash Buffer (see Recipes)
      5)
      Chemi Reagent Mix (see Recipes)
    4. Lysis Buffer
    5. Wash Buffer
    6. Anti-Phospho-Tyrosine-HRP Detection Antibody
    7. Chemiluminescent Detection Reagents
    8. Transparency Overlay Template
    9. Detailed Protocol
    Note: Store the unopened kit at 2 °C to 8 °C. Do not use past kit expiration date.
  2. Aprotinin (Sigma, catalog number: A6279)
  3. Leupeptin (Tocris, catalog number: 1167)
  4. Pepstatin (Tocris, catalog number: 1190)

Materials and reagents for Western blots
  1. PDVF membranes
  2. CL-XPosureTM Film, 5 x 7 in. (13 x 18 cm) (Thermo Fisher Scientific, catalog number: 34090)
  3. 4x Laemmli Sample Buffer (Bio-Rad Laboratories, catalog number: 1610747)
  4. 4%-20% Mini-PROTEAN® TGX Stain-FreeTM Protein Gels, 12-well, 20 µl (Bio-Rad Laboratories, catalog number: 4568095)
  5. 10x Tris/Glycine Buffer for Western Blots and Native Gels (Bio-Rad Laboratories, catalog number: 1610771)
  6. 10x Tris/Glycine/SDS (Bio-Rad Laboratories, catalog number: 1610772)
  7. Methanol ACS reagent, ≥ 99.8% (Sigma, catalog number: 179337)
  8. PierceTM BCA Protein Assay Kit (Thermo Scientific, catalog number: 23227)
  9. Blotting-Grade Blocker, nonfat dry milk (Bio-Rad Laboratories, catalog number: 1706404)
  10. Tween 20, 100% Nonionic Detergent (Bio-Rad Laboratories, catalog number: 1706531)
  11. TBS Buffer, 20x liquid (VWR, catalog number: 97064-338)
  12. RestoreTM PLUS Western Blot Stripping Buffer (Thermo Scientific, catalog number: 46430)
  13. SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, catalog number: 34577)
  14. Primary Antibodies:
    cKIT and p-cKIT (Cell Signaling Technology, catalog number: 9370)
    PDGFRA and p-PDGFRA (R&D Systems, catalog numbers: AF307 and AF2114)
  15. HRP-labeled secondary antibodies (Promega, catalog number: W4011)
  16. Deionized or distilled water
  17. 1x Tris/Glycine running buffer (see Recipes)
  18. Transfer buffer (see Recipes)
  19. 1x TBS (1 L) (see Recipes)
  20. TBS/Tween (see Recipes)
  21. 5% Blotting-Grade Blocker, nonfat dry milk/TBS/Tween (see Recipes)

Equipment

  1. Pipettes (Gilson, Pipetman Classic)
  2. Flat-tipped tweezers
  3. Autoradiography cassette
  4. Rocking platform shaker
  5. TissueRuptor II (QIAGEN, catalog number: 9002755)
  6. Centrifuge 5810R (Eppendorf, catalog number: 5811000010)
  7. Film developer Alphatek AX-390-SE (Alphatek)
  8. Rocking platform shaker

Software

  1. Protein Array Analyzer for ImageJ (Author: Gilles Carpentier, Faculte des Sciences et Technologies, Universite Paris Est Creteil Val de Marne, France, https://imagej.nih.gov/ij/macros/toolsets/Protein%20Array%20Analyzer.txt)
  2. GraphPad Prism 7.0a (GraphPad Software, Inc., www.graphpad.com)

Procedure

  1. Reagent Preparation (Mouse Phospho-RTK Array Kit)
    Note: Bring all reagents to room temperature before use.
    1. Mouse Phospho-RTK Array
      Four nitrocellulose membranes each containing 39 different anti-RTK antibodies printed in duplicate. Handle arrays only with gloved hands and flat-tipped tweezers. 
    2. Anti-Phospho-Tyrosine-HRP Detection Antibody: 50 μl of mouse anti-phospho-tyrosine antibody conjugated to HRP
      Immediately before use, dilute the Detection Antibody to the working concentration specified on the vial label using 1x Array Buffer 2.
      Note: Prepare only as much Detection Antibody as needed to run each experiment.
    3. Lysis Buffer 17 (see Recipe 5)
    4. 1x Array Buffer 2 (see Recipe 6)
    5. 1x Wash Buffer (see Recipe 7)
    6. Chemi Reagent Mix (see Recipe 8)

  2. Sample Preparation
    1. Excise by dissection and disrupt 1-5 mg of tumor tissue (approx 0.2-0.3 cm3) using TissueRuptor II in 1 ml Lysis Buffer 17 prepared with protease inhibitors. 
    2. Centrifuge samples at 10,621 x g for 10 min at 4 °C in an Eppendorf Centrifuge 5810R. 
    3. Collect supernatant and quantified protein concentrations in tumor lysates using the PierceTM BCA Protein Assay Kit.
    Notes:
    1. The suggested starting range for tissue lysates is 100-300 μg and the maximum allowable lysate volume is 250 μl/array. 
    2. Tissue lysates should be used immediately or aliquoted and stored at ≤ -70 °C.
    3. Thawed lysates should be kept on ice prior to use.

  3. Array Procedure
    Note: Bring all reagents to room temperature before use. Keep samples on ice. To avoid contamination, wear gloves while performing the procedures.
    1. Prepare all reagents and samples as directed in the previous sections. 
    2. Pipette 2.0 ml of Array Buffer 1 into each well of the 4-well Multi-dish that will be used. Array Buffer 1 is used as a block buffer. 
    3. Using flat-tip tweezers, remove each array to be used from between the protective sheets. 
    4. Place one array into each well of the 4-well Multi-dish. The array number should be facing upward.
      Note: Upon contact with Array Buffer 1 the blue dye will disappear from the spots. The capture antibodies are retained in their specific locations. 
    5. Incubate for 1 h at room temperature on a rocking platform shaker. Orient the tray so that each array rocks from end to end in its well. 
    6. While the arrays are blocking, prepare samples by diluting the desired quantity of lysate in 1.25 ml of Array Buffer 1. Adjust to a final volume of 1.5 ml with Lysis Buffer 17 as necessary. The maximum allowable lysate volume is 250 μl/array. 
    7. Aspirate Array Buffer 1 from the 4-well Multi-dish. Add the prepared samples and place the lid on the 4-well Multi-dish. 
    8. Incubate overnight at 2-8 °C on a rocking platform shaker.
      Note: A shorter incubation time may be used if optimal sensitivity is not required. 
    9. Carefully remove each array and place into individual plastic containers with 20 ml of 1x Wash Buffer. Rinse the 4-well Multi-dish with deionized or distilled water and dry thoroughly. 
    10. Wash each array with 1x Wash Buffer for 10 min on a rocking platform shaker. Repeat two times for a total of three washes. 
    11. Dilute the Anti-Phospho-Tyrosine-HRP Detection Antibody in 1x Array Buffer 2 using the dilution factor on the vial label. Pipette 2.0 ml into each well of the 4-well Multi-dish. 
    12. Carefully remove each array from its wash container. Allow excess buffer to drain from the array. Return the array to the 4-well Multi-dish containing the Anti-Phospho-Tyrosine-HRP and cover with the lid. 
    13. Incubate for 2 h at room temperature on a rocking platform shaker. 
    14. Wash each array as described in Steps C9 and C10.
      Note: Complete the remaining steps without interruption. 
    15. Carefully remove each membrane from its wash container. Allow excess Wash Buffer to drain from the membrane by blotting the lower edge onto paper towels. Place each membrane on the bottom sheet of the plastic sheet protector with the identification number facing up. 
    16. Pipette 1 ml of the prepared Chemi Reagent Mix evenly onto each membrane.
      Note: Using less than 1 ml of Chemi Reagent Mix per membrane may result in incomplete membrane coverage. 
    17. Carefully cover with the top sheet of the plastic sheet protector. Gently smooth out any air bubbles and ensure Chemi Reagent Mix is spread evenly to all corners of each membrane. Incubate for 1 min. 
    18. Position paper towels on the top and sides of the plastic sheet protector containing the membranes and carefully squeeze out excess Chemi Reagent Mix. 
    19. Remove the top plastic sheet protector and carefully lay an absorbent lab wipe on top of the membranes to blot off any remaining Chemi Reagent Mix. 
    20. Leaving membranes on the bottom plastic sheet protector, cover the membranes with plastic wrap taking care to gently smooth out any air bubbles. Wrap the excess plastic wrap around the back of the sheet protector so that the membranes and sheet protector are completely wrapped. 
    21. Place the membranes with the identification numbers facing up in an autoradiography film cassette.
      Note: Use an autoradiography cassette that is not used with radioactive isotope detection.
    22. Expose membranes CL-XPosureTM Film. Multiple exposure times are recommended (Figure 1).


      Figure 1. Proteomic analysis of Receptor tyrosine kinases in mouse KSHV-induced KS-like tumors shows activation of PDGF receptor-alpha. Mouse Phospho-Receptor Tyrosine Kinase (RTK) Array Kit used to quantify levels of phosphorylation of 39 RTKs in mouse KSHV-induced KS-like tumors. Note the major activation spot corresponding to PDGF receptor alpha chain (Cavallin et al., 2018).

Data analysis

The positive signals seen on developed film can be quickly identified by placing the transparency overlay template on the array image and aligning it with the pairs of reference spots in three corners of each array. The stamped identification number on the array should be placed on the left-hand side. The location of controls and capture antibodies is listed in the Appendix.

Notes:

  1. Reference spots are included to align the transparency overlay template and to demonstrate that the array has been incubated with Anti-Phospho-Tyrosine-HRP during the assay procedure. 
  2. Pixel densities on developed X-ray film can be collected and analyzed using a transmission mode scanner and image analysis software.
  1. Using ImageJ Protein Array Analyzer tool create a template to analyze pixel density in each spot of the array. 
  2. Export signal values to a spreadsheet file for manipulation in a program such as Microsoft Excel or GraphPad Prism. 
  3. Determine the average signal (pixel density) of the pair of duplicate spots representing each RTK. 
  4. Subtract an averaged background signal from each RTK. Use a signal from a clear area of the array or the PBS negative control spots as a background value (Figure 2).


    Figure 2. Quantification of the 45 min exposure array from Figure 1. Bar graph and pie chart from densitometry for the higher-exposure blot are equally color coded for the most prominent signals (Cavallin et al., 2018).

Western Blotting
Twenty micrograms of proteins of the same tumor lysates used for the Mouse Phospho-RTK Array were mixed with Laemmli buffer, boiled for 5 min, resolved by SDS-PAGE in 4-20% Mini-PROTEAN® TGX Stain-FreeTM Protein Gels, 12-well. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane by blotting overnight at 300 mA. Membranes were blocked with 5% nonfat milk/TBS/Tween 20 for 1 h and incubated with primary antibodies (4 °C, 16 h). After 3 TBS/Tween 20 washes, membranes were incubated with HRP-labeled secondary antibodies (1:10,000 dilution) for 1 h at room temperature. Protein bands were developed using SuperSignalTM West Pico PLUS Chemiluminescent Substrate. To analyze multiple proteins on the same membrane, membranes were washed with Restore PLUS Western Blot Stripping Buffer according to the manufacturer’s protocol (Figure 3).


Figure 3. Validation of the mouse Phospho-receptor Tyrosine Kinase (RTK) array kit. PDGFRA and phospho-PDGFRA (A) or c-KIT and phospho-cKIT (B) determined in 3 different samples of Mouse Normal Skin and mouse KSHV-induced KS-like tumors from 3 different mice by immunoblotting (Cavallin et al., 2018). (C) GAPDH used as loading control.

Recipes

  1. 1x Tris/Glycine running buffer (1 L)
    100 ml 10x Tris/Glycine
    900 ml distilled water
  2. Transfer buffer (1 L)
    100 ml of 10x Tris/Glycine/SDS
    200 ml Methanol
    700 ml distilled water
  3. 1x TBS (1 L)
    50 ml 20x TBS
    950 ml distilled water
  4. TBS/Tween (500 ml)
    25 ml TBS 20x
    1 ml Tween 20
    Distilled water to 500 ml
  5. 5% Blotting-Grade Blocker, nonfat dry milk/TBS/Tween (10 ml)
    500 mg Blotting-Grade Blocker, nonfat dry milk
    10 ml TBS/Tween

Note: Bring all reagents to room temperature before use.

  1. Lysis Buffer 17
    Add 10 μg/ml Aprotinin, 10 μg/ml Leupeptin, and 10 μg/ml Pepstatin to the volume of Lysis Buffer 17 required for cell lysate preparation
    Prepare fresh for each use
  2. 1x Array Buffer 2
    Add 2 ml of Array Buffer 2 Concentrate to 8 ml of deionized or distilled water
    Prepare fresh for each use
  3. 1x Wash Buffer
    If crystals have formed in the concentrate, warm the bottles to room temperature and mix gently until the crystals have completely dissolved
    Dilute 40 ml of 25x Wash Buffer Concentrate into 960 ml of deionized or distilled water
  4. Chemi Reagent Mix
    Chemi Reagents 1 and 2 should be mixed in equal volumes within 15 min of use (protect from light)
    1 ml of the resultant mixture is required per membrane

Acknowledgments

This work was supported by NIH grants CA75918, and CA136387; and by NCI/OHAM supplements from the Miami CFAR grant 5P30AI07396, and by the Florida Biomedical Foundation, Bankhead Coley Foundation Grant 3BB05.

Competing interests

The authors do not have any conflicts of interests or competing interests.

Ethics

The animal experiments have been performed under UM IACUC approval number 13±093. The University of Miami has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health. Additionally, UM is registered with USDA APHIS. The Council on Accreditation of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International) has continued the University of Miami's full accreditation.

References

  1. Cavallin, L. E., Goldschmidt-Clermont, P. and Mesri, E. A. (2014). Molecular and cellular mechanisms of KSHV oncogenesis of Kaposi's sarcoma associated with HIV/AIDS. PLoS Pathog 10(7): e1004154.
  2. Cavallin, L. E., Ma, Q., Naipauer, J., Gupta, S., Kurian, M., Locatelli, P., Romanelli, P., Nadji, M., Goldschmidt-Clermont, P. J. and Mesri, E. A. (2018). KSHV-induced ligand mediated activation of PDGF receptor-alpha drives Kaposi's sarcomagenesis. PLoS Pathog 14(7): e1007175.
  3. Chang, Y., Cesarman, E., Pessin, M. S., Lee, F., Culpepper, J., Knowles, D. M. and Moore, P. S. (1994). Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 266(5192): 1865-1869.
  4. Dittmer, D. P. and Damania, B. (2016). Kaposi sarcoma-associated herpesvirus: immunobiology, oncogenesis, and therapy. J Clin Invest 126(9): 3165-3175.
  5. Dittmer, D. P. and Krown, S. E. (2007). Targeted therapy for Kaposi's sarcoma and Kaposi's sarcoma-associated herpesvirus. Curr Opin Oncol 19(5): 452-457.
  6. Emuss, V., Lagos, D., Pizzey, A., Gratrix, F., Henderson, S. R. and Boshoff, C. (2009). KSHV manipulates Notch signaling by DLL4 and JAG1 to alter cell cycle genes in lymphatic endothelia. PLoS Pathog 5(10): e1000616.
  7. Ganem, D. (2010). KSHV and the pathogenesis of Kaposi sarcoma: listening to human biology and medicine. J Clin Invest 120(4): 939-949.
  8. Krown, S. E. (2003). Therapy of AIDS-associated Kaposi's sarcoma: targeting pathogenetic mechanisms. Hematol Oncol Clin North Am 17(3): 763-783.
  9. Labo, N., Miley, W., Benson, C. A., Campbell, T. B. and Whitby, D. (2015). Epidemiology of Kaposi's sarcoma-associated herpesvirus in HIV-1-infected US persons in the era of combination antiretroviral therapy. AIDS 29(10): 1217-1225.
  10. Liu, R., Li, X., Tulpule, A., Zhou, Y., Scehnet, J. S., Zhang, S., Lee, J. S., Chaudhary, P. M., Jung, J. and Gill, P. S. (2010). KSHV-induced notch components render endothelial and mural cell characteristics and cell survival. Blood 115(4): 887-895.
  11. Maurer, T., Ponte, M. and Leslie, K. (2007). HIV-associated Kaposi's sarcoma with a high CD4 count and a low viral load. N Engl J Med 357(13): 1352-1353.
  12. Mesri, E. A., Cesarman, E. and Boshoff, C. (2010). Kaposi's sarcoma and its associated herpesvirus. Nat Rev Cancer 10(10): 707-719.
  13. Mesri, E. A., Feitelson, M. A. and Munger, K. (2014). Human viral oncogenesis: a cancer hallmarks analysis. Cell Host Microbe 15(3): 266-282.
  14. Mutlu, A. D., Cavallin, L. E., Vincent, L., Chiozzini, C., Eroles, P., Duran, E. M., Asgari, Z., Hooper, A. T., La Perle, K. M., Hilsher, C., Gao, S. J., Dittmer, D. P., Rafii, S. and Mesri, E. A. (2007). in vivo-restricted and reversible malignancy induced by human herpesvirus-8 KSHV: a cell and animal model of virally induced Kaposi's sarcoma. Cancer Cell 11(3): 245-258.
  15. Nguyen, H. Q., Magaret, A. S., Kitahata, M. M., Van Rompaey, S. E., Wald, A. and Casper, C. (2008). Persistent Kaposi sarcoma in the era of highly active antiretroviral therapy: characterizing the predictors of clinical response. AIDS 22(8): 937-945.
  16. Sodhi, A., Chaisuparat, R., Hu, J., Ramsdell, A. K., Manning, B. D., Sausville, E. A., Sawai, E. T., Molinolo, A., Gutkind, J. S. and Montaner, S. (2006). The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor. Cancer Cell 10(2): 133-143.
  17. Sullivan, R. J., Pantanowitz, L. and Dezube, B. J. (2009). Targeted therapy for Kaposi sarcoma. BioDrugs 23(2): 69-75.

简介

卡波西氏肉瘤(KS)疱疹病毒(KSHV)是一种通过自分泌和旁分泌机制激活宿主致癌信号级联反应,引起KS(一种血管生成的艾滋病相关梭形细胞肿瘤)的病毒。 KSHV包括PI3K / AKT / mTORC,NFkB和Notch的许多宿主信号级联对于细胞特异性转化机制至关重要,并且它们的鉴定为治疗靶标发现铺平了道路。对人KS肿瘤和我们的小鼠KS样肿瘤共有的分子KS特征的分析显示KS标记物VEGF和PDGF受体的一致表达与体内其他血管生成配体及其受体的上调。这指向各种受体酪氨酸激酶(RTK)信号轴的自分泌和旁分泌激活。因此,我们描述了使用Mouse Phospho-RTK阵列试剂盒筛选KSHV诱导的KS样小鼠肿瘤的活化受体酪氨酸激酶并通过RTK蛋白质印迹验证的方案。我们发现该方法可以成功地用于以无偏见的方式对肿瘤中最活化的酪氨酸激酶受体进行排序。这使我们能够将PDGFRA鉴定为AIDS-KS中的致癌驱动因子和治疗靶标。
【背景】卡波西氏肉瘤疱疹病毒(KSHV)是卡波西肉瘤(KS)的病原体(Chang et al。,1994; Ganem,2010; Mesri et al。,2010; Dittmer和Damania,2016)。 KS是与艾滋病相关的主要癌症(AIDS-KS),是影响非洲男性和儿童的最普遍的癌症类型(Ganem,2010; Mesri et al。,2010; Cavallin et al。,2014)。虽然自从HAART(高效抗逆转录病毒疗法)的广泛实施以来,西方世界的AIDS-KS发病率明显下降,但很大比例的AIDS-KS患者从未达到完全缓解(Krown,2003; Nguyen 等人,,2008; Cavallin et al。,2014)。此外,即使在HAART治疗的受控病毒血症的HIV患者中,KSHV患病率和KS发病率似乎也在增加(Maurer et al。,2007; Labo et al。,2015) 。了解KS肿瘤发生中病毒和宿主因子的相互作用对于新疗法的合理开发至关重要(Dittmer和Krown,2007; Sullivan et al。,2009)。 KSHV包括PI3K / AKT / mTORC,NFkB和Notch的许多宿主信号级联对于细胞特异性转化机制至关重要,它们的鉴定为治疗靶标发现铺平了道路(Sodhi et al。,2006; Emuss et al。,2009; Liu et al。,2010; Mesri et al。,2014; Dittmer和Damania, 2016)。对人KS肿瘤和我们的小鼠KS样肿瘤共有的分子KS特征的分析显示KS标志物VEGFR 1,2,3,Podoplanin的一致表达与体内血管生成配体和受体的上调,指出各种受体酪氨酸激酶信号轴的上调(Mutlu 等人,2007; Mesri 等人,2010)。因此,我们开始使用KSHV依赖性肿瘤发生的小鼠模型对KSHV激活的宿主酪氨酸激酶信号级联进行排序,这有助于我们将PDGFRA鉴定为AIDS-KS中的致癌驱动因子和治疗靶标(Cavallin et al。 ,2018)。

在这里,我们描述了在小鼠KSHV诱导的KS样肿瘤中使用小鼠磷酸化RTK阵列试剂盒的测定方案,其可以用作以无偏方式对肿瘤中最活化的酪氨酸激酶受体进行排序的可靠方法。 Proteome Profiler小鼠Phospho-RTK阵列试剂盒(R&D Systems)是一种基于膜的夹心免疫分析。在硝酸纤维素膜上一式两份发现的捕获的抗体与样品中存在的特定靶蛋白结合。用HRP-缀合的泛磷酸酪氨酸抗体检测捕获的蛋白质的酪氨酸磷酸化,然后使用化学发光检测试剂进行可视化。产生的信号与结合分析物中磷酸化的量成比例。

令人惊讶的是,对肿瘤RTK活化模式的分析(图1和2)显示单个突出激活的RTK斑点,其占肿瘤的总酪氨酸激酶活化的显着部分。该信号对应于PDGF受体α链(PDGFRA)(Cavallin 等,,2018)。通过蛋白质印迹分析验证阵列(图3A)显示PDGFRA在肿瘤中稳健表达并且比在小鼠正常皮肤中磷酸化更多。另一方面,c-kit(图3B),相关的RTK,在皮肤和肿瘤中类似地表达,并且在肿瘤中显示低水平的活化;与皮肤对照组相似,与我们的磷酸酪氨酸激酶蛋白质组阵列中c-kit的低活化相关(Cavallin et al。,2018)。

关键字:血小板源性生长因子受体α多肽, 酪氨酸激酶, 蛋白质芯片, KSHV, 卡波西肉瘤

材料和试剂

常见实验室耗材

  1. 移液器提示 
  2. 手套 
  3. 容量为50毫升的塑料容器(用于清洗阵列)
  4. 保鲜膜
  5. 吸水实验室擦拭巾(KimWipes ®或同等产品) 
  6. 纸巾
  7. 塑料透明板材保护膜(修剪至10厘米x 12厘米,三面开口) 
  8. TissueRuptor一次性探针(QIAGEN,目录号:990890)

试剂制备(小鼠磷酸化RTK阵列试剂盒)
  1. Proteome Profiler Mouse Phospho-RTK Array Kit(R& D Systems,目录号:ARY014)
    套件内容:
    1. 4阵列膜
    2. 4井多盘
    3. 数组缓冲区:
      1)
      裂解缓冲液17(见食谱)
      2)
      数组缓冲区1
      3)
      1x阵列缓冲区2(参见食谱)
      4)
      1x洗涤缓冲液(见食谱)
      5)
      Chemi Reagent Mix(见食谱)
    4. 裂解缓冲液
    5. 洗涤缓冲液
    6. 抗磷酸酪氨酸-HRP检测抗体
    7. 化学发光检测试剂
    8. 透明度叠加模板
    9. 详细议定书
    注意:将未开封的试剂盒存放在2°C至8°C。请勿使用过去的工具包到期日期。
  2. 抑肽酶(西格玛,目录号:A6279)
  3. Leupeptin(Tocris,目录号:1167)
  4. Pepstatin(Tocris,目录号:1190)

Western Blots的材料和试剂
  1. PDVF膜
  2. CL-XPosure TM 薄膜,5 x 7英寸(13 x 18厘米)(Thermo Fisher Scientific,目录号:34090)
  3. 4x Laemmli样品缓冲液(Bio-Rad Laboratories,目录号:1610747)
  4. 4%-20%Mini-PROTEAN ® TGX无染色 TM 蛋白凝胶,12孔,20μl(Bio-Rad Laboratories,目录号:4568095)
  5. 用于蛋白质印迹和天然凝胶的10x Tris /甘氨酸缓冲液(Bio-Rad Laboratories,目录号:1610771)
  6. 10x Tris / Glycine / SDS(Bio-Rad Laboratories,目录号:1610772)
  7. 甲醇ACS试剂,≥99.8%(Sigma,目录号:179337)
  8. Pierce TM BCA蛋白质分析试剂盒(Thermo Scientific,目录号:23227)
  9. Blotting级阻滞剂,脱脂奶粉(Bio-Rad Laboratories,目录号:1706404)
  10. 吐温20,100%非离子洗涤剂(Bio-Rad Laboratories,目录号:1706531)
  11. TBS Buffer,20x液体(VWR,目录号:97064-338)
  12. 恢复 TM PLUS Western Blot Stripping Buffer(Thermo Scientific,目录号:46430)
  13. SuperSignal TM West Pico PLUS化学发光底物(Thermo Scientific,目录号:34577)
  14. 原发性抗体:
    cKIT和p-cKIT(Cell Signaling Technology,目录号:9370)
    PDGFRA和p-PDGFRA(R& D Systems,目录号:AF307和AF2114)
  15. HRP标记的二抗(Promega,目录号:W4011)
  16. 去离子水或蒸馏水
  17. 1x Tris / Glycine运行缓冲液(见食谱)
  18. 转移缓冲区(见食谱)
  19. 1x TBS(1升)(见食谱)
  20. TBS / Tween(见食谱)
  21. 5%吸墨级阻滞剂,脱脂奶粉/ TBS / Tween(见食谱)

设备

  1. 移液器(Gilson,Pipetman Classic)
  2. 扁镊子
  3. 放射自显影盒
  4. 摇摆平台振动筛
  5. TissueRuptor II(QIAGEN,目录号:9002755)
  6. 离心机5810R(Eppendorf,目录号:5811000010)
  7. 电影开发商Alphatek AX-390-SE(Alphatek)
  8. 摇摆平台振动器

软件

  1. 用于ImageJ的蛋白质阵列分析仪(作者:Gilles Carpentier,Faculte des Sciences et Technologies,Universite Paris Est Creteil Val de Marne,France, https://imagej.nih.gov/ij/macros/toolsets/Protein%20Array%20Analyzer.txt
  2. GraphPad Prism 7.0a(GraphPad Software,Inc., www.graphpad.com

程序

  1. 试剂制备(小鼠磷酸化RTK阵列试剂盒)
    注意:使用前将所有试剂置于室温下。
    1. 小鼠Phospho-RTK阵列
      四个硝酸纤维素膜,每个含有39种不同的抗RTK抗体,一式两份印刷。只戴戴手套的手和平头镊子处理阵列。 
    2. 抗磷酸酪氨酸-HRP检测抗体:50μl与HRP结合的小鼠抗磷酸酪氨酸抗体
      在使用前,立即使用1x阵列缓冲液2将检测抗体稀释至样品瓶标签上指定的工作浓度。
      注意:根据需要准备尽可能多的检测抗体来进行每个实验。
    3. 裂解缓冲液17(参见配方5)
    4. 1x阵列缓冲器2(参见配方6)
    5. 1x洗涤缓冲液(参见配方7)
    6. Chemi Reagent Mix(见食谱8)

  2. 样品制备
    1. 通过解剖切除并使用TissueRuptor II在1ml用蛋白酶抑制剂制备的裂解缓冲液17中破坏1-5mg肿瘤组织(约0.2-0.3cm 3 )。 
    2. 在Eppendorf离心机5810R中,在4℃下以10,621 x g 离心样品10分钟。 
    3. 使用Pierce TM BCA蛋白质测定试剂盒收集肿瘤裂解物中的上清液和定量的蛋白质浓度。
    注意:
    1. 组织裂解液的建议起始范围为100-300μg,最大允许裂解液体积为250μl/阵列。
    2. 组织裂解液应立即使用或等分并在≤-70°C下储存。
    3. 解冻裂解液应在使用前保存在冰上。

  3. 阵列程序
    注意:使用前将所有试剂置于室温。将样品保存在冰上。为避免污染,请在执行操作时戴上手套。
    1. 按照前面部分的说明准备所有试剂和样品。 
    2. 移取2.0ml阵列缓冲液1到将要使用的4孔Multi-dish的每个孔中。数组缓冲区1用作块缓冲区。 
    3. 使用平头镊子,从保护板之间移除要使用的每个阵列。 
    4. 将一个阵列放入4孔多盘的每个孔中。阵列号应朝上。
      注意:与阵列缓冲液1接触后,蓝色染料将从斑点消失。捕获抗体保留在其特定位置。 
    5. 在摇床平台振荡器上在室温下孵育1小时。调整托盘的方向,使每个阵列在其井中从一端到另一端摇摆。 
    6. 当阵列阻塞时,通过在1.25ml阵列缓冲液1中稀释所需量的裂解物来制备样品。根据需要用裂解缓冲液17调节至终体积1.5ml。最大允许裂解液体积为250μl/阵列。 
    7. 从4孔多盘中吸出阵列缓冲液1。加入准备好的样品,盖上4孔Multi-dish。 
    8. 在摇动平台振荡器上于2-8°C孵育过夜。
      注意:如果不需要最佳灵敏度,可以使用较短的孵育时间。 
    9. 小心地取出每个阵列,放入装有20毫升1x洗涤缓冲液的塑料容器中。用去离子水或蒸馏水冲洗4孔Multi-dish并彻底晾干。 
    10. 在摇动平台振荡器上用1x洗涤缓冲液洗涤每个阵列10分钟。重复两次,共洗三次。 
    11. 使用样品瓶标签上的稀释因子稀释1x阵列缓冲液2中的抗磷酸酪氨酸-HRP检测抗体。移取2.0毫升到4孔多盘的每个孔中。 
    12. 小心地从洗涤容器中取出每个阵列。允许多余的缓冲液从阵列中排出。将阵列放回含有抗磷酸酪氨酸-HRP的4孔Multi-dish中,盖上盖子。 
    13. 在室温下在摇动平台振荡器上孵育2小时。 
    14. 按照步骤C9和C10中的描述清洗每个阵列。
      注意:不间断地完成剩余的步骤。 
    15. 小心地从洗涤容器中取出每个膜。通过将下边缘吸到纸巾上,让多余的洗涤缓冲液从膜上排出。将每个薄膜放在塑料薄膜保护膜的底部薄片上,标识号朝上。 
    16. 将1ml制备的Chemi Reagent Mix均匀地移液到每个膜上。
      注意:每个膜使用少于1 ml的Chemi Reagent Mix可能导致膜覆盖不完整。
    17. 小心地盖上塑料薄膜保护膜的顶层。轻轻抚平任何气泡,确保Chemi Reagent Mix均匀涂抹在每个膜的各个角落。孵育1分钟。 
    18. 将纸巾放在含有膜的塑料薄膜保护膜的顶部和侧面,并小心地挤出多余的Chemi Reagent Mix。 
    19. 取下顶部的塑料薄膜保护膜,小心地在薄膜顶部铺上吸收性实验室擦拭物,以去除任何残留的Chemi Reagent Mix。 
    20. 将薄膜留在底部塑料薄膜保护膜上,用保鲜膜覆盖薄膜,小心地平滑任何气泡。将多余的塑料包裹物缠绕在纸张保护装置的背面,使膜和纸张保护装置完全包裹。 
    21. 将识别号朝上的膜置于放射自显影胶片盒中。
      注意:使用不与放射性同位素检测一起使用的放射自显影盒。
    22. 暴露膜CL-XPosure TM 膜。建议多次曝光时间(图1)。


      图1.小鼠KSHV诱导的KS样肿瘤中受体酪氨酸激酶的蛋白质组学分析显示PDGF受体-α的激活。小鼠磷酸受体酪氨酸激酶(RTK)阵列试剂盒用于量化磷酸化水平在小鼠KSHV诱导的KS样肿瘤中的39个RTK。注意对应于PDGF受体α链的主要活化斑点(Cavallin 等人,,2018)。

数据分析

通过将透明度覆盖模板放置在阵列图像上并将其与每个阵列的三个角中的参考点对对齐,可以快速识别在显影胶片上看到的正信号。阵列上标记的标识号应放在左侧。控制和捕获抗体的位置列于附录中。

注意:

  1. 包含参考斑点以对齐透明度覆盖模板,并证明在测定过程中阵列已与抗磷酸酪氨酸-HRP一起孵育。 
  2. 可以使用传输模式扫描仪和图像分析软件收集和分析开发的X射线胶片上的像素密度。
  1. 使用ImageJ Protein Array Analyzer工具创建一个模板来分析数组每个点的像素密度。 
  2. 将信号值导出到电子表格文件,以便在Microsoft Excel或GraphPad Prism等程序中进行操作。 
  3. 确定代表每个RTK的一对重复点的平均信号(像素密度)。 
  4. 从每个RTK中减去平均背景信号。使用来自阵列清晰区域或PBS阴性对照点的信号作为背景值(图2)。


    图2.图1中45分钟曝光阵列的定量。高密度测定法的条形图和饼图对于最显着的信号同样采用颜色编码(Cavallin et al 。,2018)。

Western Blotting
将20微克用于小鼠磷酸化RTK阵列的相同肿瘤裂解物的蛋白质与Laemmli缓冲液混合,煮沸5分钟,通过SDS-PAGE在4-20%Mini-PROTEAN ® TGX中分离无染色 TM 蛋白凝胶,12孔。通过在300mA下印迹过夜将蛋白质转移到聚偏二氟乙烯(PVDF)膜上。用5%脱脂乳/ TBS / Tween 20封闭膜1小时,并与一抗孵育(4℃,16小时)。在3次TBS / Tween 20洗涤后,将膜与HRP标记的第二抗体(1:10,000稀释)在室温下孵育1小时。使用SuperSignal TM West Pico PLUS化学发光底物开发蛋白质条带。为了分析同一膜上的多种蛋白质,根据制造商的方案用Restore PLUS Western Blot Stripping Buffer洗涤膜(图3)。


图3.验证小鼠磷酸受体酪氨酸激酶(RTK)阵列试剂盒。 PDGFRA和磷酸化PDGFRA(A)或c-KIT和磷酸化cKIT(B)在3个不同的样品中测定通过免疫印迹从小鼠正常皮肤和小鼠KSHV诱导的来自3只不同小鼠的KS样肿瘤(Cavallin 等,,2018)。 (C)GAPDH用作加载对照。

食谱

  1. 1x Tris /甘氨酸缓冲液(1 L)
    100毫升10倍Tris /甘氨酸
    900毫升蒸馏水
  2. 转移缓冲液(1升)
    100毫升10倍Tris /甘氨酸/ SDS
    200毫升甲醇
    700毫升蒸馏水
  3. 1x TBS(1升)
    50毫升20倍TBS
    950毫升蒸馏水
  4. TBS /吐温(500毫升)
    25毫升TBS 20x
    1毫升吐温20
    蒸馏水至500毫升
  5. 5%印迹级阻滞剂,脱脂奶粉/ TBS / Tween(10 ml)
    500毫克印迹级阻滞剂,脱脂奶粉
    10毫升TBS / Tween

注意:使用前将所有试剂置于室温下。

  1. Lysis Buffer 17
    将10μg/ ml抑肽酶,10μg/ ml Leupeptin和10μg/ ml Pepstatin加入到细胞裂解液制备所需的裂解缓冲液17中。
    每次使用都要准备新鲜的
  2. 1x阵列缓冲器2
    将2 ml Array Buffer 2浓缩液加入8 ml去离子水或蒸馏水中 为每次使用准备新鲜
  3. 1x洗涤缓冲液
    如果在浓缩物中形成晶体,将瓶子加热至室温并轻轻混合直至晶体完全溶解 将40ml 25x洗涤缓冲液浓缩物稀释到960ml去离子水或蒸馏水中
  4. Chemi Reagent Mix
    Chemi试剂1和2应在使用15分钟内以相同的体积混合(避光)
    每个膜需要1ml所得混合物

致谢

这项工作得到NIH拨款CA75918和CA136387的支持;并通过迈阿密CFAR资助5P30AI07396的NCI / OHAM补充,以及佛罗里达生物医学基金会,Bankhead Coley Foundation Grant 3BB05。

利益争夺

作者没有任何利益冲突或竞争利益。

伦理

动物实验已在UM IACUC批准号13±093下进行。迈阿密大学向美国国立卫生研究院实验室动物福利办公室(OLAW)提交动物福利保证。此外,UM已在USDA APHIS注册。实验动物管理评估和认证协会(AAALAC International)认证委员会继续对迈阿密大学进行全面认证。

参考

  1. Cavallin,L.E.,Goldschmidt-Clermont,P。和Mesri,E.A。(2014)。 与HIV / AIDS相关的Kaposi肉瘤KSHV肿瘤发生的分子和细胞机制。 PLoS Pathog 10(7):e1004154。
  2. Cavallin,LE,Ma,Q.,Naipauer,J.,Gupta,S.,Kurian,M.,Locatelli,P.,Romanelli,P.,Nadji,M.,Goldschmidt-Clermont,PJ and Mesri,EA(2018) )。 KSHV诱导的配体介导的PDGF受体-α激活驱动Kaposi的肉瘤发生。 PLoS Pathog 14(7):e1007175。
  3. Chang,Y.,Cesarman,E.,Pessin,M。S.,Lee,F.,Culpepper,J.,Knowles,D。M. and Moore,P。S.(1994)。 鉴定艾滋病相关卡波西肉瘤中的疱疹病毒样DNA序列。 科学 266(5192):1865-1869。
  4. Dittmer,D。P.和Damania,B。(2016)。 卡波西肉瘤相关疱疹病毒:免疫生物学,肿瘤发生和治疗。 J Clin Invest 126(9):3165-3175。
  5. Dittmer,D。P.和Krown,S.E。(2007)。 针对Kaposi肉瘤和Kaposi肉瘤相关疱疹病毒的靶向治疗。 Curr Opin Oncol 19(5):452-457。
  6. Emuss,V.,Lagos,D.,Pizzey,A.,Gratrix,F.,Henderson,S.R。和Boshoff,C。(2009)。 KSHV操纵DLL4和JAG1的Notch信号转导以改变淋巴内皮细胞的细胞周期基因。 PLoS Pathog 5(10):e1000616。
  7. Ganem,D。(2010)。 KSHV和卡波西肉瘤的发病机制:倾听人类生物学和医学。 J Clin Invest 120(4):939-949。
  8. Krown,S.E。(2003)。 治疗与艾滋病相关的卡波西肉瘤:针对致病机制。 Hematol Oncol Clin North Am 17(3):763-783。
  9. Labo,N.,Miley,W.,Benson,C.A.,Campbell,T.B。和Whitby,D。(2015)。 抗逆转录病毒联合治疗期间HIV-1感染美国人卡波西肉瘤相关疱疹病毒的流行病学治疗。 艾滋病 29(10):1217-1225。
  10. Liu,R.,Li,X.,Tulpule,A.,Zhou,Y.,Scehnet,J。S.,Zhang,S.,Lee,J。S.,Chaudhary,P.M。,Jung,J。和Gill,P。S.(2010)。 KSHV诱导的缺口成分可提供内皮细胞和壁细胞特征以及细胞存活。 血 115(4):887-895。
  11. Maurer,T.,Ponte,M。和Leslie,K。(2007)。 艾滋病毒相关的卡波西肉瘤,CD4计数高,病毒载量低。 N Engl J Med 357(13):1352-1353。
  12. Mesri,E。A.,Cesarman,E。和Boshoff,C。(2010)。 Kaposi肉瘤及其相关疱疹病毒。 Nat Rev Cancer 10(10):707-719。
  13. Mesri,E.A.,Feitelson,M。A.和Munger,K。(2014)。 人类病毒肿瘤发生:癌症标志物分析。 细胞宿主微生物 15(3):266-282。
  14. Mutlu,AD,Cavallin,LE,Vincent,L.,Chiozzini,C.,Eroles,P.,Duran,EM,Asgari,Z。,Hooper,AT,La Perle,KM,Hilsher,C.,Gao,SJ, Dittmer,DP,Rafii,S。和Mesri,EA(2007)。人类疱疹病毒-8 KSHV诱导的 体内 - 限制性和可逆性恶性肿瘤:病毒诱导的卡波西肉瘤的细胞和动物模型。 癌细胞 11(3):245-258。
  15. Nguyen,H。Q.,Magaret,A。S.,Kitahata,M。M.,Van Rompaey,S.E.,Wald,A。和Casper,C。(2008)。 高活性抗逆转录病毒治疗时代的持续性卡波西肉瘤:表征临床反应的预测因素。 艾滋病 22(8):937-945。
  16. Sodhi,A.,Chaisuparat,R.,Hu,J.,Ramsdell,A.K.,Manning,B.D.,Sausville,E.A.,Sawai,E.T.,Molinolo,A.,Gutkind,J。S. and Montaner,S。(2006)。 TSC2 / mTOR途径驱动由卡波西氏肉瘤相关疱疹病毒G蛋白偶联诱导的内皮细胞转化受体。 癌细胞 10(2):133-143。
  17. Sullivan,R。J.,Pantanowitz,L。和Dezube,B。J.(2009)。 针对卡波西肉瘤的靶向治疗。 BioDrugs 23(2 ):69-75。

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引用:Naipauer, J., Cavallin, L. E. and Mesri, E. A. (2019). Unbiased Screening of Activated Receptor Tyrosine Kinases (RTKs) in Tumor Extracts Using a Mouse Phospho-RTK Array Kit. Bio-protocol 9(8): e3216. DOI: 10.21769/BioProtoc.3216.
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