Detection of Receptor Activation in FFPE Specimens Figure 6. VeraTag FFPE quantification of HGF and correlation with Western blot, ELISA, and IHC in human tumor specimens. A. VeraTag FFPE quantification of HGF in NSCLC tumors. Isotype IgG control signals (inset) were subtracted from HGF signals. B. Immunoprecipitation/ Western blot analysis of HGF in corresponding NSCLC tumor lysates. C. ELISA determinations of HGF in corresponding NSCLC specimens. D. IHC detection in NSCLC specimens. doi:10.1371/journal.pone.0015932.g006 100 nm from the source of 1O2 [10]. In a final step, cleaved fluorescein reporters are collected from tissue sections, resolved by capillary electrophoresis, and quantified using customized VeraTag software. Proximity assays can be configured to detect either receptor and ligand expression or ligand-receptor protein complexes by the appropriate selection of antibodies that target receptor and/or ligand. In proximity assays, receptor or ligand is detected and quantified by fluorescein-reporter and biotin-conjugated antibodies targeting different epitopes on the same protein (Fig. 1B). Converse- ly, to detect and quantify ligand-receptor complexes, the fluorescein- reporter conjugated antibody targets the receptor while the biotin- conjugated antibody targets the ligand (Fig. 1B) or vice versa. Detection and quantification of c-MET receptor in FFPE cell lines by antibody proximity assays The expression of c-MET has been studied in many epithelial and mesenchymal cancers. In general, high expression of c-MET is associated with poorer prognosis. High c-MET expression in glioblastoma, breast cancer, gastric cancer, and ovarian cancer is associated with poor survival [16–18]. Despite the availability of semi-quantitative IHC assays for c-MET detection in FFPE tissues, a method that can quantify a continuous measurement of c-MET expression in tissues has not been developed. We have developed a proximity assay for quantification of total c-MET expression in FFPE specimens. We procured multiple c- MET antibodies and assessed the specificity of each antibody in FFPE samples. For the initial assessment of antibody specificity, we used the lung cancer cell lines H1680 and H522 that express high and undetectable levels of c-MET, respectively. From these analyses, the c-MET (CVD13) rabbit polyclonal antibody and c- MET (clone 3D4) mouse monoclonal antibody were selected for further assay optimization. Proximity assays were performed on a panel of FFPE specimens prepared Non-Small Cell Lung, Breast, and Glioma Cancer cell lines, using c-MET antibody pairs CVD13 and clone 3D4 that were conjugated with either fluorescent reporters or biotin. Cell lines were selected based on their variable levels of c-MET protein expression: H441 express high levels of c-MET, H226 and H2170 express intermediate levels [19,20], MCF7 cells express low, but detectable levels of c-MET [21] and H661 do not express c-MET [20]. We also used Ln18, U138, U118, and Ln229 glioma cell lines. Cell line pellets were prepared as FFPE specimens and analyzed for c-MET levels (Fig. 2A). The H441 cell line exhibited the highest signals while H226, Ln18, U138, U118, Ln229, and H2170 exhibited interme- diate signals and the lowest c-MET were measured in MCF7 and H661 cells. Isotype control assays using antibody matched IgG generated signals that were less than 10% of the c-MET specific signals (Fig. 2A). Together, these data indicate that the dynamic range of c-MET proximity assays extends over a 2 log10 range. In order to correlate FFPE proximity assay data with other independent biochemical methods, we prepared soluble cell lysates from the same panel of cell lines and conducted Western blot and ELISA analysis. FFPE proximity assay measurements (Fig. 2A) were generally concordant with Western blot (Fig. 2B) and ELISA (Fig. 2C) results. We also performed IHC on FFPE cell line pellets and as expected the c-MET staining pattern was highest in H441 cells and intermediate to low in the H226 and MCF7 cells (Fig. 2D). Taken together, the c-MET FFPE assay exhibited a broad range of specificity and data was consistent with independent assays including Western blot, ELISA and IHC. To further characterize the c-MET antibodies used in our proximity assay, we mapped their binding epitopes in an ELISA assay using a panel of overlapping peptides spanning the cytoplasmic domain of c-MET. This analysis indicated that the c-MET (clone 3D4) monoclonal antibody binds the epitope (1227RDMYDKEYYSVHNKT1241) in the C-terminus of c-MET, while the c-MET (CVD13) rabbit polyclonal binds the epitope (1378DEVDTRPASFWETS1391) at the extreme C-terminus (Fig. 3A, 3B). We also characterized a c-MET (clone SP44) rabbit monoclonal antibody in the c-MET FFPE assay. These data indicate that this rabbit monoclonal antibody (clone SP44) has c- MET specificity similar to the c-MET rabbit polyclonal (CVD13) antibody (data not shown). Characterization of this c-MET (clone SP44) antibody by epitope mapping analysis indicated that it binds to the epitope (1378DEVDTRPASFWETS1391) at the extreme C- terminus (Fig. 3A, 3B) similar to the c-MET (CVD13) rabbit polyclonal antibody and this enabled us to use this monoclonal antibody (clone SP44) as an alternative to the polyclonal c-MET (CVD13) antibody. Detection and quantification of c-MET receptor in human tumors by antibody proximity assay In order to characterize c-MET levels in human tumors, we obtained 15 human Non-Small Cell Lung (NSCLC) cancer specimens. Portions of each tumor were prepared as formalin- fixed paraffin embedded slides and soluble cell lysates (excluding sample NL2 and NL8 due to small tumor volumes). These NSCLC tumor samples displayed a ,20-fold range of c-MET levels while isotype matched controls generated less than 10% of the c-MET specific signal (Fig. 4A). To compare our FFPE measurements with independent biochemical methods, we ana- lyzed soluble lysates from the same panel of tumors using Western blot (Fig. 4B) and ELISA (Fig. 4Ci). Generally, c-MET FFPE measurements were concordant with Western blot and ELISA data. A two-tailed comparison of c-MET protein levels measured in FFPE specimens by proximity assay and by ELISA in soluble lysates was statistically significant (Spearmen r: 0.91; p,0.0001) (Fig. 4Cii). We also evaluated c-MET levels by IHC staining (Fig. 4Di) and the comparison of FFPE c-MET levels by IHC H- score and the proximity assay correlated with statistical signif- icance (Spearmen r: 0.89; p,0.0001) (Fig. 4Dii). Taken together, our results demonstrate that the c-MET FFPE assay can detect and quantify c-MET receptor levels in FFPE tumor specimens, and these measurements correlate well with measurements obtained by conventional methods. Detection and quantification of hepatocyte growth factor (HGF) in cell lines by antibody proximity assays Several studies have shown an association of serum or tumor associated HGF with the progression of various carcinomas [22,23]. High levels of HGF in resected NSCLC are correlated with poor survival [23]. Despite the clinical significance of HGF overexpression, a quantitative assay that measures HGF in FFPE specimens has not been reported. To address this unmet need, we PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e15932