Detection of Receptor Activation in FFPE Specimens Figure 9. c-MET, HGF, and HGF/c-MET complexes in human carcinomas. A. NSCLC. B. Gastric carcinoma. C. Head and Neck carcinoma. c-MET, HGF, and HGF/c-MET assays were performed for all FFPE samples. Samples were rank ordered from low to high levels of HGF. The levels of c-MET receptor and HGF/c-MET were also tabulated. Red color-High $90th percentile; Pink color-Low #10th percentile. NSCLC and Gastric tumor exceeded 70% tumor content by pathologic analysis. In Head & Neck carcinoma samples, non-tumor tissue was removed by macrodissection. Di. c-MET was immunoprecipitated from NSCLC lysate samples and immunoblotted using c-MET (Tyr1003) or c-MET antibodies. Dii. c-MET was immunoprecipitated from gastric tumor lysates and immunoblotted using c-MET (Tyr1003) or c-MET antibodies. Samples that were c-MET phosphorylation (pY1003) positive (+) or c-MET (pY1003) negative (2) are summarized in Fig. A, B. doi:10.1371/journal.pone.0015932.g009 prognostic and predictive value of c-MET and/or HGF measurements [16,17,18,22,23]. Based on the AQUATM platform commercially available c-MET antibodies demonstrate lot to lot variation that result in poor reproducibility of c-MET measure- ments [29]. We screened a large panel of commercially available c- MET and HGF antibodies and optimized a pair of antibodies for use in proximity assays. The use of two antibodies that bind in close proximity undoubtedly contribute to the high specificity of detection of the assays described here. To develop a HGF/c-MET ligand-receptor assay, we utilized the c-MET and HGF antibodies that worked well in our FFPE assays. We also incorporated a secondary antibody detection format that enhances assay signal strength and reproducibility [11]. In cell lines and tumors that were positive for HGF/c-MET, signals were more than 2 fold higher than the isotype IgG control signals. In addition, detection of endogenous levels of HGF/c-MET in glioma cells was consistent between proximity FFPE and SPPICE biochemical assays. c-MET phosphorylation was used to demonstrate that HGF/c-MET detection can serve as a surrogate measure of c-MET activation. In several glioma cells that exhibit activation of HGF/c- MET signaling, we detected high levels of c-MET (pY1003) phosphorylation that are likely modulated through a c-MET and HGF autocrine loop. In contrast, our measurements of HGF, c- MET, HGF/c-MET, and c-MET phosphorylation suggest that c- MET activation in Ln229 cells is not the result of autocrine signaling. At present, we do not understand the mechanism of constitutive c-MET phosphorylation in this cell line, but it is conceivable that signaling is mediated through an alternative co- receptor or as yet unidentified c-MET activating mutations. c-MET activation has recently been shown to play important roles in HN carcinoma [2] via a paracrine mechanism in which HGF is secreted by surrounding stromal cells [30]. Consistent with this observation, we detected c-MET phosphorylation in NSCLC and gastric tumors that also contained measurable levels of HGF/ c-MET complex. In NSCLC, Zucali et al [1] reported a significant correlation between c-MET (pY1003) phosphorylation and resistance to gefitinib treatment and rapid disease progression. A similar association between gefitinib resistance and HGF induced c-MET activation has also been reported for lung adenocarcino- mas containing activating EGFR mutations [26]. Although, we detected c-MET (pY1003) phosphorylation in human tumors, our attempts to measure c-MET (pY1234/1235) and c-MET (pY1349) References 1. Baker AF, Dragovich T, Ihle NT, Williams R, Fenoglio-Preiser C, et al. (2005) Stability of phosphoprotein as a biological marker of tumor signaling. Clin Cancer Res 12: 4338–4340. 2. Jones RJ, Boyce T, Fennell M, Jacobs V, Pinto F, et al. (2008) The impact of delay in cryo-fixation on biomarkers of Src tyrosine kinase activity in human breast and bladder cancers. Cancer Chemother Pharmacol 61: 23–32. 3. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141: 1117–1134. 4. Comoglio PM, Giordano S, Trusolino L (2008) Drug development of Met inhibitors: targeting oncogene addiction and expedience. Nature Drug Discov 7: 504–516. 5. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF (2003) Met, metastasis, motility and more. Nature Mol cell Biol 4: 915–925. 6. Toschi L, Janne PA (2008) Single-agent and combination therapeutic strategies to inhibit hepatocyte growth factor/Met signaling in cancer. Clin Cancer Res 14: 5941–5946. phosphorylation were unsuccessful. It is possible that pY1003 c- MET phosphorylation is inherently more stable than c-MET (py1234/1235) phosphorylation. Alternatively, the kinetics of site- specific phosphorylation may be different. A study of tyrosine phosphorylation of c-MET in a renal carcinoma cell line suggested that c-MET (pY1349) phosphorylation occurs 5 minutes after HGF treatment and persists for 120 minutes while c-MET (pY1234/1235) phosphorylation occurs 30 minutes after HGF treatment and is subject to rapid turnover [31]. Strategies to identify relevant biomarkers in parallel with development of targeted therapeutics is essential for the successful selection of appropriate clinical trial subjects and eventual treatment of potentially responsive patients. A number of approaches for HGF/c-MET biomarker identification in a clinical setting have been proposed including the measurement of plasma HGF and shed c-MET [32]. Patients treated with a therapeutic antibody against HGF (AMG102), exhibited increased plasma HGF levels but no significant changes in shed c-MET [32]. In a phase I of c-MET MetMab (Anti-MET therapeutic antibody), a single patient that experienced a complete response exhibited a diagnostic profile consistent with autocrine biology [33]. In another study, c-MET phosphoryaltion was reduced in the post- operative tumor biopsies of patients treated with a c-MET kinase inhibitor (ARQ197) [34]. The VeraTag proximity assays de- scribed here present a unique opportunity to provide quantitative measurements of c-MET receptor activation that may contribute to the development of antagonistic c-MET therapeutics. Acknowledgments We wish to thank: Dr. Jerry Wallweber for sharing protocols for antibody screening. Drs. John Winslow and Jerry Wallweber for procurement of Head and Neck carcinoma tissues. Gundo Diedrich for assistance with peptide ELISA. David Stathas and Lise Wong for tumor sectioning and slide preparation. Drs Thomas Sherwood and Weidong Huang for histological assessments. Dr Hasan Tahir for synthesizing VeraTag reporter molecules. Drs. Jeff Sperinde, Michael Bates, and Christos J Petropoulos for critical review of the manuscript. Author Contributions Conceived and designed the experiments: RD. Performed the experiments: RD JZ. Analyzed the data: RD JZ GP EP. Contributed reagents/ materials/analysis tools: EP. Wrote the paper: RD. 7. Zucali PA, Ruiz MG, Giovannetti E, Destro A, Varella-Garcia M, et al. (2008) Role of c-Met expression in non-small cell lung cancer patients treated with EGFR tyrosine kinase inhibitors. Ann Onclogy 19: 1605–1612. 8. Seiwert TY, Jagadeeswaran R, Faoro L, Janamanchi V, Nallasura V, et al. (2009) The MET receptor tyrosine kinase is a potential novel therapeutic target for head and neck squamous carcinoma. Cancer Res 69: 3021–3031. 9. Nakamura Y, Niki T, Goto A, Morikawa T, Miyazawa K, et al. (2007) c-Met activation in lung adenocarcinoma tissues: An immunohistochemical analysis. Cancer Sci 98: 1006–1013. 10. Shi Y, Huang W, Tan Y, Jin X, Dua R, et al. (2009) A novel proximity assay for detection of proteins and protein complexes: quantitation of Her1 and Her2 total protein expression and homodimerization in formalin-fixed paraffin embedded cell lines and breast cancer tissue. Diag Mol Pathol 18: 11–21. 11. Sperinde J, Jin X, Banerjee J, Penuel E, Saha A, et al. (2010) Quantitation of p95Her2 in paraffin sections by using a p95-specific antibody and correlation PLoS ONE | www.plosone.org 16 January 2011 | Volume 6 | Issue 1 | e15932