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

Summary

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.

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 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.

Protocol

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 method.
5. Microscopy analysis of frozen sections to ensure they are of ccRCC nature, viable tumor and for grading (low grade, high grade or mix....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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