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.

1. Identification of Morphological and Molecular Tumor Heterogeneity
<ol>
 <li>Tumors removed from -80 &deg;C freezer and kept on dry ice.</li>
 <li>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 &deg;C until ready for use.</li>
 <li>Coat samples in OCT and cut in a cryostat at -22 &deg;C.</li>
 <li>Samples stained using Hematoxylin and Eosin counterstaining method.</li>
 <li>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&amp;b (H&amp;E staining of high low and mixed grade).</li>
 <li>Select up to 4 samples from each morphologically differing region within each tumor for protein extraction.</li>
</ol>
2. Protein Extraction from Tumor Samples
<ol>
 <li>Cut tumor sample from OCT.</li>
 <li>Place 50-75 mg of tissue into 2 ml tubes with 990 &mu;l 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).</li>
 <li>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.</li>
 <li>Transfer homogenized sample to new 2 ml tube using pipette leaving the steel ball behind.</li>
 <li>Add 10 &mu;l of Triton X-100 to each sample before centrifuging at 13,000 x g for 30 min at 4 &deg;C.</li>
 <li>Transfer supernatants to fresh microcentrifuge tubes.</li>
 <li>Determine protein concentration using BCA assay (Figure 2).</li>
 <li>Normalize protein concentrations at 1 mg/ml.</li>
</ol>
3. Antibody Validation
<ol>
 <li>Prepare protein samples (extracted from suitable cell lines or tissue) for western blot and run on a 10% SDS-PAGE gel.</li>
 <li>Transfer samples onto nitrocellulose membrane overnight at 4 &deg;C.</li>
 <li>Block the membrane in Li-Cor Odyssey Blocking Buffer (diluted 50:50 in PBS) for 1 hr at room temperature.</li>
 <li>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.</li>
 <li>Incubate membrane in primary antibodies overnight at 4 &deg;C.</li>
 <li>Make up 0.1% PBS-Tween20 (PBS-T; 1 ml Tween20 /1L PBS).</li>
 <li>Wash membrane in PBS-T at room temperature for 5 min (x3).</li>
 <li>Dilute fluorescently-labelled secondary antibodies in Odyssey Blocking Buffer (diluted 50:50 in PBS) 0.01% SDS at 1:10,000 dilution (1.5 &mu;l/15 ml).</li>
 <li>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.</li>
 <li>Wash membrane in PBS-T at room temperature for 5 min (x3), keeping the membrane in the dark.</li>
 <li>Wash membrane in PBS at room temperature for 5 min (x3) to remove residual Tween20, again keeping the membrane in the dark.</li>
 <li>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.</li>
 <li>Scan the membrane on the LiCor Odyssey scanner. Keep the membrane in the dark until such time as it has been scanned.</li>
 <li>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.</li>
</ol>
4. RPPA Printing
<ol>
 <li>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.</li>
 <li>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).</li>
</ol>
5. RPPA Protein Detection
<ol>
 <li>Wet slides in excess LiCor Blocking Buffer (diluted 50:50 in PBS). Incubate at RT for 1 hr on a rocking platform.</li>
 <li>Prepare 800 &mu;l of primary antibodies in LiCor Blocking Buffer (diluted 50:50 in PBS) at the desired concentrations and keep on ice.</li>
 <li>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.</li>
 <li>Remove residual buffer from wells and add 600 &mu;l primary antibody to respective wells.</li>
 <li>Place the slide and chamber into a sealed wet box and incubate on rocking platform overnight at 4 &deg;C.</li>
 <li>Make up 0.1% PBS-Tween20 (PBS-T; 100 &mu;l Tween 20 /100 ml PBS).</li>
 <li>Remove slides from cold room and carefully remove the primary antibodies from each well.</li>
 <li>Add 600 &mu;l of PBS-T and wash slides on rocking platform at RT for 5 min (X3).</li>
 <li>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 &mu;l/2 ml) in the first instance.</li>
 <li>Remove buffer from wells and add 600 &mu;l 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.</li>
 <li>Remove secondary antibodies from well and briefly wash (x3) in 600 &mu;l 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.</li>
 <li>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.</li>
 <li>Dry the Fastslide in 50 &deg;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.</li>
 <li>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.</li>
 <li>Image files are saved as .tiff files. Figure 4 (image of scanned files).</li>
</ol>
6. Data Analysis
<ol>
 <li>Launch the MicroVigene Software (VigeneTech, Carlisle, MA, USA).</li>
 <li>Open .tiff image file containing the scan of the RPPA slide.</li>
 <li>Select a predefined template file which will have a grid to overlay on the image of the RPPA slide.</li>
 <li>Click the Define Regions of Interest (ROI) button, to bring up the Grid.</li>
 <li>Position the Grid over the RPPA spots. Figure 6a (image of grid over image).</li>
 <li>Click the Select ALL button to highlight all the ROI.</li>
 <li>Click Find All. MicroVigene will automatically find the ROI, find the spots, subtract the background, remove any dust and quantify spots.</li>
 <li>Click the View Dilution Curve button to bring up the results for all the samples on the RPPA slide.</li>
 <li>Click Save Dilution Data.</li>
 <li>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 fitting10,11 
 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.</li>
 <li>Export the data in Microsoft Excel and plot y0 as in Figure 7.</li>
 <li>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.</li>
</ol>

<ol>
	<li>Stewart, G. D., O'Mahony, F. C., Powles, T., Riddick, A. C., Harrison, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+can+molecular+pathology+contribute+to+the+management+of+renal+cell+carcinoma.">What can molecular pathology contribute to the management of renal cell carcinoma.</a> Nat. Rev. Urol. 8, 255-265 (2011).</li><li>Rini, B. I., Michaelson, M. D., Rosenberg, J. E., Bukowski, R. M., Sosman, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+activity+and+biomarker+analysis+of+sunitinib+in+patients+with+bevacizumab-refractory+metastatic+renal+cell+carcinoma.">Antitumor activity and biomarker analysis of sunitinib in patients with bevacizumab-refractory metastatic renal cell carcinoma.</a> J. Clin. Oncol. 26, 3743-3748 (2008).</li><li>Swanton, C., Larkin, J. M., Gerlinger, M., Eklund, A. C., Howell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+biomarker+discovery+through+the+parallel+integration+of+clinical+trial+and+functional+genomics+datasets.">Predictive biomarker discovery through the parallel integration of clinical trial and functional genomics datasets.</a> Genome Med. 2, 52 (2010).</li><li>Cortes, J., Jabbour, E., Kantarjian, H., Yin, C. 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Med. 366, 883-892 (2012).</li><li>Fisher, R., Larkin, J., Swanton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter+and+intratumour+heterogeneity:+a+barrier+to+individualized+medical+therapy+in+renal+cell+carcinoma.">Inter and intratumour heterogeneity: a barrier to individualized medical therapy in renal cell carcinoma.</a> Front. Oncol. 2, 49 (2012).</li><li>Yachida, S., Jones, S., Bozic, I., Antal, T., Leary, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467, 1114-1117 (2010).</li><li>O'Mahony, F. C., Faratian, D., Varley, J., Nanda, J., Theodoulou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+automated+quantitative+analysis+to+evaluate+epithelial-to-mesenchymal+transition+associated+proteins+in+clear+cell+renal+cell+carcinoma.">The use of automated quantitative analysis to evaluate epithelial-to-mesenchymal transition associated proteins in clear cell renal cell carcinoma.</a> PLoS One. 7, e31557 (2012).</li><li>Spurrier, B., Ramalingam, S., Nishizuka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse-phase+protein+lysate+microarrays+for+cell+signaling+analysis.">Reverse-phase protein lysate microarrays for cell signaling analysis.</a> Nat. Protoc. 3, 1796-1808 (2008).</li><li>Hu, J., He, X., Baggerly, K. A., Coombes, K. R., Hennessy, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-parametric+quantification+of+protein+lysate+arrays.">Non-parametric quantification of protein lysate arrays.</a> Bioinformatics. 23, 1986-1994 (2007).</li><li>Wang, X., Dong, Y., Jiwani, A. 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No conflicts of interest declared.

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...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr>
			<td>phosphatase inhibitor cocktail 2</td>
			<td>Sigma</td>
			<td>P5726</td>
		</tr>
		<tr>
			<td>phosphatase inhibitor cocktail 3</td>
			<td>Sigma</td>
			<td>P0044</td>
		</tr>
		<tr>
			<td>protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836153001</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Triton-X</td>
			<td>T8787</td>
		</tr>
		<tr>
			<td>Li-Cor Odyssey Blocking Buffer</td>
			<td>Li-Cor</td>
			<td>927-40000</td>
		</tr>
		<tr>
			<td>TissueLyser</td>
			<td>Qiagen</td>
			<td>85600</td>
		</tr>
		<tr>
			<td>MicroGrid II robotic spotter</td>
			<td>Biorobotics</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FastFrame' four bay slide holder</td>
			<td>Whatman</td>
			<td>10486001</td>
		</tr>
		<tr>
			<td>FAST Slide - 2-Pad</td>
			<td>Whatman</td>
			<td>10485317</td>
		</tr>
		<tr>
			<td>IRDye 680LT Goat anti-Mouse IgG</td>
			<td>Licor</td>
			<td>926-68020</td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-Rabbit IgG</td>
			<td>Licor</td>
			<td>926-32211</td>
		</tr>
	</tbody>
</table>

the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers

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.

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...

Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

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

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.

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

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 ...

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33.5K Views.  University of Edinburgh. 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.

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

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...