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 mixed low/high grade, challenging to differentiate Fuhrman grades 1 to 4 on frozen section). Figure 1 a&b (H&E staining of high low and mixed grade).
6. Select up to 4 samples from each morphologically differing region within each tumor for protein extraction.

2. Protein Extraction from Tumor Samples

1. Cut tumor sample from OCT.
2. Place 50-75 mg of tissue into 2 ml tubes with 990 μl of lysis buffer [50 mM Tris-HCl (pH 7.5), 5 mM EGTA (pH 8.5), 150 mM NaCl] supplemented with aprotinin (Sigma A6279) (10 mg/ml), phosphatase inhibitor cocktail 2 (Sigma, P5716), phosphatase inhibitor cocktail 3 (Sigma, P0044) and a protease inhibitor cocktail (Roche, 11836153001).
3. Add a single 5 mm steel ball to each tube and homogenize at 50Hz for 5 min twice using a TissueLyser, checking the level of homogenization after each 5 min period.
4. Transfer homogenized sample to new 2 ml tube using pipette leaving the steel ball behind.
5. Add 10 μl of Triton X-100 to each sample before centrifuging at 13,000 x g for 30 min at 4 °C.
6. Transfer supernatants to fresh microcentrifuge tubes.
7. Determine protein concentration using BCA assay (Figure 2).
8. Normalize protein concentrations at 1 mg/ml.

3. Antibody Validation

1. Prepare protein samples (extracted from suitable cell lines or tissue) for western blot and run on a 10% SDS-PAGE gel.
2. Transfer samples onto nitrocellulose membrane overnight at 4 °C.
3. Block the membrane in Li-Cor Odyssey Blocking Buffer (diluted 50:50 in PBS) for 1 hr at room temperature.
4. Dilute the primary antibodies in Li-Cor Odyssey Blocking Buffer (diluted 50:50 in PBS) at the manufacturers recommended dilution typically 1 in 1,000.
5. Incubate membrane in primary antibodies overnight at 4 °C.
6. Make up 0.1% PBS-Tween20 (PBS-T; 1 ml Tween20 /1L PBS).
7. Wash membrane in PBS-T at room temperature for 5 min (x3).
8. Dilute fluorescently-labelled secondary antibodies in Odyssey Blocking Buffer (diluted 50:50 in PBS) 0.01% SDS at 1:10,000 dilution (1.5 μl/15 ml).
9. Incubate membrane in secondary antibodies at room temperature for 45 min with gentle shaking - it is important to protect the membrane from the light until such time as it has been finally scanned.
10. Wash membrane in PBS-T at room temperature for 5 min (x3), keeping the membrane in the dark.
11. Wash membrane in PBS at room temperature for 5 min (x3) to remove residual Tween20, again keeping the membrane in the dark.
12. Lie membrane flat on a piece of filter paper in the dark and allow to air dry - allowing the membrane to dry may enhance the signal and reduce background but render it useless for stripping and re-probing.
13. Scan the membrane on the LiCor Odyssey scanner. Keep the membrane in the dark until such time as it has been scanned.
14. Only select antibodies that generate a single predominant band at the correct molecular weight. Figure 3 shows examples of acceptable and unacceptable antibodies for use with RPPA.

4. RPPA Printing

1. Protein lysates were spotted onto nitrocellulose- coated glass slides (Fastslides-Whatman) using a MicroGrid II robotic spotter. The slides used contained 2 pads onto which the samples were spotted. Each pad was spotted with identical samples, in this case 100 samples. Other available formats include 1, 8 and 16 pad slides. The higher the number of pads the smaller the number of samples which can be spotted onto each.
2. A series of five 2-fold dilutions were made from each sample and each was then spotted in triplicate (resulting in a total of 15 spots per sample).

5. RPPA Protein Detection

1. Wet slides in excess LiCor Blocking Buffer (diluted 50:50 in PBS). Incubate at RT for 1 hr on a rocking platform.
2. Prepare 800 μl of primary antibodies in LiCor Blocking Buffer (diluted 50:50 in PBS) at the desired concentrations and keep on ice.
3. Mount the slides in either (i) the single frame Chip Clip or (ii) the 'FastFrame' four bay slide holder so that a tight seal is formed between the slide and the incubation chamber.
4. Remove residual buffer from wells and add 600 μl primary antibody to respective wells.
5. Place the slide and chamber into a sealed wet box and incubate on rocking platform overnight at 4 °C.
6. Make up 0.1% PBS-Tween20 (PBS-T; 100 μl Tween 20 /100 ml PBS).
7. Remove slides from cold room and carefully remove the primary antibodies from each well.
8. Add 600 μl of PBS-T and wash slides on rocking platform at RT for 5 min (X3).
9. Prepare fluorescently-labelled secondary antibodies by diluting in Odyssey Blocking Buffer (diluted 50:50 in PBS) 0.01% SDS at 1:2,000 dilution (1 μl/2 ml) in the first instance.
10. Remove buffer from wells and add 600 μl fluorescently-labelled secondary antibodies to respective wells. Incubate secondary antibodies at room temperature for 45 min with gentle shaking - it is important to protect the membrane from the light until such time as it has been finally scanned.
11. Remove secondary antibodies from well and briefly wash (x3) in 600 μl PBS-T at room temperature. Remove slide from carrier, transfer to a suitable container and wash in excess PBS-T for 15 min, keeping the membrane in the dark.
12. Remove PBS-T and further wash membrane with PBS at room temperature for 15 min to remove residual Tween-20, again keeping the membrane in the dark.
13. Dry the Fastslide in 50 °C oven for 10 min and then scan on the Li-Cor Odyssey scanner. Keep the slide in the dark until it has been scanned.
14. Scan the slide at 680 nm and/or 800 nm depending on the secondary antibody/antibodies used. For two-color detection always use highly cross-adsorbed secondary antibodies to minimize cross reactivity. Careful selection of primary and secondary antibodies is necessary for two-color detection. Of primary importance is the selection of different host species (e.g. rabbit and mouse) for the two primary antibodies. This allows discrimination by anti-rabbit and anti-mouse secondary antibodies which are labelled with dye with easily distinguishable emission spectra.
15. Image files are saved as .tiff files. Figure 4 (image of scanned files).

6. Data Analysis

1. Launch the MicroVigene Software (VigeneTech, Carlisle, MA, USA).
2. Open .tiff image file containing the scan of the RPPA slide.
3. Select a predefined template file which will have a grid to overlay on the image of the RPPA slide.
4. Click the Define Regions of Interest (ROI) button, to bring up the Grid.
5. Position the Grid over the RPPA spots. Figure 6a (image of grid over image).
6. Click the Select ALL button to highlight all the ROI.
7. Click Find All. MicroVigene will automatically find the ROI, find the spots, subtract the background, remove any dust and quantify spots.
8. Click the View Dilution Curve button to bring up the results for all the samples on the RPPA slide.
9. Click Save Dilution Data.
10. As each sample is printed across 5 dilution points each in triplicate there are 15 points to analyze, which reduces the risk of errors and improves the quality of curve fitting. MicroVigene produces a 4-parameter logistic-log model "Supercurve" algorithm (Figure 6b), that incorporates all spots to produce a sigmoid curve of antigen-antibody binding kinetics. The assumption is that the same antibody-antigen binding kinetics is taking place at each sample spot, even in the different samples, thus by taking all spots on an array to fit a common response curve can increase the confidence of the curve fitting^10,11
    Y=a+ ((b-a)/(1+e(c*d-ln(x)))
    where x is the dilution factor and Y is the signal intensity.
    Samples can be comparatively analyzed by using the y0 value, which in our analysis corresponded to the y value at the midpoint of the x values after mapping those onto the supercurve.
11. Export the data in Microsoft Excel and plot y0 as in Figure 7.
12. Intratumoral protein variance was calculated separately for untreated and treated treated patients in an ANOVA framework. Variance distributions combining data from all the analyzed proteins were compared by a Mann-Whitney test (MWT). Intratumoral variances for individual proteins were compared by an F-test where assumptions of normality and homoscedasticity held, respectively assessed using the Lillefours and Fligner tests; false discovery rate (FDR) correction was applied¹². Differential protein expression between untreated and treated patient samples was tested for each protein using student's t-test where normality and homoscedasticity assumptions were met, otherwise MWT was performed; FDR was applied over combined t-test and MWT values ¹². Significance of protein expression and variance Pearson correlation for the proteins were estimated using a standard approach [R reference] and FDR applied ¹².

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

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