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

Fiach C O'Mahony, Jyoti Nanda, Alexander Laird, Peter Mullen, Helen Caldwell, Ian M Overton, Lel Eory, Marie O'Donnell, Dana Faratian, Thomas Powles, David J Harrison, Grant D Stewart

Research output: Contribution to journalArticlepeer-review

Abstract

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 carcinoma(5,6) 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 RCC(8) 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.
Original languageEnglish
JournalJournal of Visualized Experiments (JoVE)
Issue number71
DOIs
Publication statusPublished - 22 Jan 2013

Keywords

  • Blotting, Western
  • Carcinoma, Renal Cell
  • Humans
  • Kidney Neoplasms
  • Neoplasm Proteins
  • Protein Array Analysis

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