JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Published: January 22nd, 2013

DOI:

10.3791/50221

1Edinburgh Urological Cancer Group, University of Edinburgh, 2School of Medicine, University of St Andrews, 3Division of Pathology, University of Edinburgh, 4MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, 5Department of Pathology, Western General Hospital, 6Breakthrough Breast Cancer Research Unit, University of Edinburgh, 7St Bartholomew's Cancer Institute, Experimental Cancer Medicine Centre, Queen Mary University of London

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.

In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.

Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

1. Identification of Morphological and Molecular Tumor Heterogeneity

  1. Tumors removed from -80 °C freezer and kept on dry ice.
  2. Divide tumors into sections of approximately 1 cm3. Map the original position of each tumor section relative to each other and label with a unique name. Store samples in individual cryovials at -80 °C until ready for use.
  3. Coat samples in OCT and cut in a cryostat at -22 °C.
  4. Samples stained using Hematoxylin and Eosin counterstaining m.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

An example of a scanned RPPA slide can be seen in Figure 4(i) with both 680 and 800 nm channels shown. Separating the images by wavelength, Figure 4(ii) enables each pad on the RPPA slide to be analyzed and individual protein expression determined Figure 4(iii). As can be seen in Figure 4(iii) the expression of individual proteins across the samples is unique with Gelsolin having a high level of expression across the slide compared to cMYC which ha.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The RPPA method presented here represents a high throughput alternative to the widely used but comparatively low throughput western blot technique of protein analysis. The method allows hundreds of samples to be semi-quantitatively analyzed and compared simultaneously allowing for direct comparison of key proteins across a wide selection of cell lines and tissue samples. Multiplexing with different antibody species further increases the power of the technique allowing multiple antibodies to be used simultaneously. The ex.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The work of authors FCO, DF, JN, DJH and GDS mentioned above is funded by the Chief Scientist Office, grant number: ETM37 and supported by the Cancer Research UK Experimental Cancer Medicine Centre. The work of AL is funded by the Royal College of Surgeons of Edinburgh Robertson Trust, the Melville Trust for the care and cure of cancer and the Medical Research Council. IO is supported by a Royal Society of Edinburgh Scottish Government Fellowship cofunded by Marie Curie Actions and the UK Medical Research Council. The authors would like to thank SCOTRRCC co-applicants and collaborators for their useful discussions on some of the topics discussed in this paper.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Name of the reagent Company Catalogue number
aprotinin Sigma A6279
phosphatase inhibitor cocktail 2 Sigma P5726
phosphatase inhibitor cocktail 3 Sigma P0044
protease inhibitor cocktail Roche 11836153001
Triton X-100 Triton-X T8787
Li-Cor Odyssey Blocking Buffer Li-Cor 927-40000
TissueLyser Qiagen 85600
MicroGrid II robotic spotter Biorobotics  
FastFrame' four bay slide holder Whatman 10486001
FAST Slide - 2-Pad Whatman 10485317
IRDye 680LT Goat anti-Mouse IgG Licor 926-68020
IRDye 800CW Goat anti-Rabbit IgG Licor 926-32211

  1. Stewart, G. D., O'Mahony, F. C., Powles, T., Riddick, A. C., Harrison, D. J., et al. What can molecular pathology contribute to the management of renal cell carcinoma. Nat. Rev. Urol. 8, 255-265 (2011).
  2. Rini, B. I., Michaelson, M. D., Rosenberg, J. E., Bukowski, R. M., Sosman, J. A., et al. Antitumor activity and biomarker analysis of sunitinib in patients with bevacizumab-refractory metastatic renal cell carcinoma. J. Clin. Oncol. 26, 3743-3748 (2008).
  3. Swanton, C., Larkin, J. M., Gerlinger, M., Eklund, A. C., Howell, M., et al. Predictive biomarker discovery through the parallel integration of clinical trial and functional genomics datasets. Genome Med. 2, 52 (2010).
  4. Cortes, J., Jabbour, E., Kantarjian, H., Yin, C. C., Shan, J., et al. Dynamics of BCR-ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors. Blood. 110, 4005-4011 (2007).
  5. Gerlinger, M., Rowan, A. J., Horswell, S., Larkin, J., Endesfelder, D., et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883-892 (2012).
  6. Fisher, R., Larkin, J., Swanton, C. Inter and intratumour heterogeneity: a barrier to individualized medical therapy in renal cell carcinoma. Front. Oncol. 2, 49 (2012).
  7. Yachida, S., Jones, S., Bozic, I., Antal, T., Leary, R., et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature. 467, 1114-1117 (2010).
  8. O'Mahony, F. C., Faratian, D., Varley, J., Nanda, J., Theodoulou, M., et al. The use of automated quantitative analysis to evaluate epithelial-to-mesenchymal transition associated proteins in clear cell renal cell carcinoma. PLoS One. 7, e31557 (2012).
  9. Spurrier, B., Ramalingam, S., Nishizuka, S. Reverse-phase protein lysate microarrays for cell signaling analysis. Nat. Protoc. 3, 1796-1808 (2008).
  10. Hu, J., He, X., Baggerly, K. A., Coombes, K. R., Hennessy, B. T., et al. Non-parametric quantification of protein lysate arrays. Bioinformatics. 23, 1986-1994 (2007).
  11. Wang, X., Dong, Y., Jiwani, A. J., Zou, Y., Pastor, J., et al. Improved protein arrays for quantitative systems analysis of the dynamics of signaling pathway interactions. Proteome Sci. 9, 53 (2011).
  12. Benjamini, Y., Hochberg, Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological). 57, 289-300 (1995).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved