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Quantitating multiple signaling pathway proteins in preclinical and clinical studies
October 2010
by Clifford C. Hoyt & Darren Lee  |  Email the author

The common need in cancer research and pharmaceutical drug development is to reveal the configurations of active signaling pathways in diseased tissues, to support target validation, trial design, patient selection, response assessment and if trials are successful, the diagnostic component of theranostics. Importantly, the predictive power of measurements of signaling protein expression depends on the precision and accuracy of tissue analysis tools.  
For example, many techniques deployed today, such as those based on microarray detection, or analysis of sample lysates, provide data that are in fact averages from volumes of tissue, including many cells not of interest. These methods blur out key proteomic information that reside at the cellular level, and relate to the signaling states of individual cells.  
Role of signal transduction pathways in cancer    
During the course of tumor progression, cancer cells acquire a number of characteristic alterations. These include the capacity to proliferate independently of exogenous growth-promoting or growth-inhibitory signals, the tendency to invade surrounding tissues and metastasize to distant sites, the penchant for eliciting an angiogenic response and the ability to evade mechanisms that limit cell proliferation, such as inflammatory response, apoptosis and replicative senescence. These properties reflect alterations in key cellular signaling pathways that in normal cells control cell proliferation, motility, and survival.  
Many of the proteins currently under investigation as possible targets for cancer therapy are signaling proteins that are components of these pathways. The nature of these signaling pathways and their roles in tumorigenesis are the subject of intense study by pharmaceutical companies, motivated by the hope that progress in understanding these signaling pathways will accelerate drug development. It is a complex research task to identify relevant pathways, understanding them and demonstrating correlation with outcome. An additional level of complexity arises from the fact that it is often the interrelationship between pathway proteins and their localization that help characterize the pathway, rather than the mere presence of a protein.  
Example: Detecting phospho-epitopes of AKT, ERK and S6  
These three pathway markers are widely studied and play a vital role in cancer pathogenesis. In this particular example, the goal is to detect the activation of PI3K/AKT, RAS, and MEK signaling pathways.   
AKT has recently been found to play a paradoxical role: on one hand, it increases cancer cells' survival capability, while on the other hand, it blocks their motility and invasion abilities, thereby preventing cancer from spreading [1]. It had been presumed that one could promote cancer cell death by inhibiting AKT that controls the synthesis of proteins involved in proliferation. Yet now, with this added complexity, the role of AKT must be understood further, so as not to promote metastases by inhibiting AKT expression.  
Activation of the MEK pathway up-regulates ERK protein levels, promoting cell division. This pathway is often up-regulated in human tumors and is thought to fulfill multiple roles in the acquisition of a complex malignant phenotype. Accordingly, a specific blockade of the MEK pathway is expected to result in not only an anti-proliferative effect, but also in anti-metastatic and anti-angiogenic effects in tumor cells. Recently, potent small-molecule inhibitors targeting components of the MEK pathway have been developed. Among them, BAY 43-9006 (Raf inhibitor), and PD184352, PD0325901 and ARRY-142886 (MEK1/2 inhibitors) have reached the clinical trial stage. The combination of MEK pathway inhibitors and conventional anticancer drugs might provide an excellent basis for the development of new chemotherapeutic strategies against cancer.  
Finally, s6 is a ribosomal protein involved in translation of mRNAs. It is thought to play an important role in controlling cell growth and proliferation.

Automated, multiplexed tissue cytometry  
Detecting pathway markers using conventional histology or immunofluorescence is a challenge, given the need to observe many markers simultaneously (i.e., to multiplex) in order to gain a full understanding of the pathways involved and the phenotypes. Conversely, conventional multiplexing techniques, such as microarrays or flow cytometry, fail to provide the contextual information needed to confirm intracellular localization; also a requirement in order to confirm pathway state. What is needed is simultaneous measurement of multiple proteins, on a per-cell basis, set in the context of the original anatomy.  
New platform technologies now offer us the opportunity to access this level of information, by utilizing an effective, practical and reliable platform for cytometric analysis of intact tissue sections. This can be conceptualized as "tissue cytometry." The platform supports preclinical and clinical studies through the integration of multiplexed immunohistochemical (IHC) or immunofluorescent (IF) labeling strategies, robotic slide handling, and automated multispectral image acquisition and analysis. Multispectral imaging systems and advanced image analysis software together provide the ideal platform for this application.  
The ideal imaging platform integrates: a) easy-to-implement multiplexed staining protocols; b) an automated slide analysis system that can isolate marker signals from one another and from autofluorescence; and c) pattern recognition-based image analysis software for automatically segmenting images and extracting quantitative data from cells of interest.  
Multispectral imaging and automated image analysis accelerates preclinical and clinical studies  
Quantitative, independent and specific multi-label protocols have been developed that in conjunction with easy-to-use multispectral imaging systems and advanced learn-by-example software, can greatly accelerate clinical and preclinical studies [2].    
For example, today, approximately one-third of small-molecule kinase inhibitors in development or trial target pathways are associated with EGFR activation. Analysis of EGFR activation in tumor xenographs is typically done by immunohistochemical staining of tissue sections for phosphor-epitopes of EGFR. Samples are analyzed by eye by pathologists, either under the microscope or on the computer screen as digital slides.  
Typically, pathologists can process slides at an average rate of 100 samples per day. A study of hundreds of samples takes days or weeks. On the other hand, if samples are stained with multiple color protocols that help guide image analysis and provide internal controls, such as a stain for total EGFR, slides can be analyzed automatically with image analysis software. Such software can then present segmentation results and associated marker intensity scores to pathologists for review, modification if necessary, and final approval.
In benchmark studies, results have shown that an analysis process that takes many days, at 100 slides per day, can be reduced to hours, at a rate of 200 to 300 slides per hour. The pathologists remain central to the process by training the image analysis algorithms to identify important tissue areas, and as a final quality control gate on image analysis results. 
In a recent study performed at one pharmaceutical company, a trained image analysis solution accurately segmented tissue into regions of interest for 98 percent of samples in a large, 3,000-sample study.  
There is another benefit to this approach, in addition to increased productivity and shorter study durations. Data is more consistent, since stain intensity scores are based on measured signal levels from a digital camera instead of human visual perception, which can vary over time based on changing ambient environments and is not well suited to capture the non-linear signal levels inherent in chromogenic absorption.  
Pharmaceutical companies are motivated by the hope that progress in understanding signaling pathway activity will accelerate drug development. The tasks of revealing activated pathways, understanding their interrelationships and determining correlation with outcome are challenging. The complexity inherent in signaling pathway activity can only be elucidated by revealing key marker localization and distribution within tumor cells, rather than the mere presence of a protein independent of morphological context.  
Combining multispectral imaging with advanced image analysis tools to perform tissue cytometry rapidly and on a large scale and using many markers at once has proven to enable a better understanding of the mechanism of disease and potentially better, more precise avenues of treatment.  
Clifford C. Hoyt, a founder of Cambridge Research & Instrumentation Inc. (CRi) in Woburn, Mass., joined the company in 1987 as a staff scientist. He has played a central role in the development of many of CRi's core technologies, including the liquid crystal tunable filter for multispectral and polarized light imaging and the integration of these core technologies into analytical instruments for applications such as in vitro fertilization, high-throughput drug screening, stem cell research, in vivo small-animal imaging, live-cell biology and tissue-based immunohistochemical analysis. He holds 12 patents, has numerous patents pending and is the author or co-author of 20 publications in technical journals.  
Darren Lee is vice president of marketing at CRi. He has more than 20 years of experience in marketing management, business development and engineering in the life sciences and clinical diagnostics industries. He has held senior-level management positions at Primera Biosystems, a molecular diagnostics company, and Decision Biomarkers, a life-science systems developer. He has also served in senior management positions at PerkinElmer and Packard Bioscience.  
 1. Yoeli-Lerner, Yiu, Rabinovitz, Erhardt, Jauliac and Toker: "Akt blocks breast cancer cell motility and invasion through the transcription factor NFAT." Molecular Cell, November 2005.
2. Levenson, Fornari and Loda: "Multispectral imaging and pathology: Seeing and doing more." Expert Opinion on Medical Diagnostics, 2008.



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