Detection of Receptor Activation in FFPE Specimens followed by Western blotting with anti-c-MET antibodies (Bii). C. c-MET and endogenous HGF in glioma cells were cross-linked as described above and SPPICE was used to measure HGF/c-MET complexes. doi:10.1371/journal.pone.0015932.g008 we detected using the VeraTag FFPE assay format. The H596 cell line containing a deletion in c-MET exon 14 that removes amino acids L964 through D1010 [27] was used as a negative control for our c-MET (pY1003) immuno-precipitation studies. As illustrated in Fig. 7C, higher basal c-MET (pY1003) phosphorylation was detected in Ln18 and U87MG cells relative to U138 and U118 cells. This observation is consistent with the detection of higher levels of HGF/c-MET complex in Ln18 and U87MG cells relative to U138 and U118 cells using the VeraTag FFPE assay format (Fig. 7B). Interestingly, the Ln229 cell line that lack measurable levels of the HGF/c-MET complex in the FFPE assay (Fig. 7B) exhibited elevated levels of c-MET (pY1003) phosphoryaltion in the lysate immunoprecipitation assay (Fig. 7C). Detection of HGF/c-MET ligand-receptor complexes by Surface Protein-Protein Interaction by Cross-linking ELISA (SPPICE) assay We developed a novel biochemical method for detection of the HGF/c-MET complex to cross-validate the VeraTag HGF/c- MET assay. Chemical cross-linking of proteins is commonly used to demonstrate protein-protein interactions, therefore, we com- bined chemical crosslinking with ELISA based detection to develop Surface Protein-Protein Interaction by Cross-linking ELISA (SPPICE). A schematic of the HGF/c-MET SPPICE assay is depicted in Fig. 8A, and details of the assay are described in Materials and Methods. Briefly, cell cultures expressing surface c-MET receptors in the presence of either exogenous or endogenous HGF are treated with a membrane impermeable sulfo-EGS cross-linker. Soluble cross-linker proteins are subse- quently extracted and the lysates are applied to 96 well microtiter plates pre-coated with c-MET antibody. A biotinylated HGF antibody is added and HGF/c-MET complexes are detected by further addition of streptavidin-HRP and colorimetric substrate. SPPICE was used to assess HGF/c-MET levels in unstimulated or HGF stimulated A549 cells. We detected HGF dose-dependent increases in the HGF/c-MET complex only when the cells were treated with the sulfo-EGS cross-linker (Fig. 8Bi). In the absence of the cross-linker treatment, the HGF/c-MET complex was not detected in either unstimulated and HGF stimulated A549 cells. The detection of HGF/c-MET ligand-receptor complexes by conventional SDS-PAGE and Western blot analysis further corroborated the SPPICE results (Fig. 8Bii). To assess endogenous levels of HGF/c-MET ligand-receptor complexes in glioma cells, we used SPPICE to characterize Ln18, Ln229, and U118 cell lysates. Consistent with the proximity FFPE assay (Fig. 7C), SPPICE detected HGF/c-MET complexes in Ln18 and U118 cells but not in Ln229 cells (Fig. 8C). Profiling c-MET, HGF, and HGF/c-MET ligand-receptor complexes in human NSCLC, Gastric, and Head and Neck tumors by antibody proximity assays We measured HGF/c-MET ligand-receptor complexes in the same NSCLC carcinoma tumor panel that was used for individual c-MET and HGF measurements. c-MET, HGF, and HGF/c- MET levels were also measured in six gastric carcinomas and 33 Head and Neck (HN) carcinomas. The HN tumors included 17 squamous cell and 16 non-squamous cell carcinomas. Tumors were rank ordered based on increasing HGF levels and corresponding c-MET and HGF/c-MET complex measures are represented as a heat map in Figs. 9A, B, C. The HGF/c-MET complex was detected in 7 of 13 (54%) NSCLC specimens (NL1, NL4, NL5, NL6, NL11, NL15), 3 of 6 (50%) gastric tumors (G2, G3, G5), and 11of 33 (33%) HN tumors (HN1, HN5, HN7, HN8, HN12, HN15, HN16, HN21, HN25, HN26, HN33). Within HN group, HGF/c-MET was detected in 7 of 17 (40%) squamous cell carcinomas (HN1, HN5, HN7, HN8, HN21, HN25, HN26), and 4 of 16 (25%) non-squamous cell carcinomas (HN12, HN15, HN16, HN33). Tumors identified positive for HGF/c-MET ligand-receptor complexes exhibited signals that were at least 2- fold greater than the isotype assay control signals (data not shown). A combined analysis of NSCLC, gastric, and HN tumor measurements did not reveal significant correlations between HGF/c-MET levels and c-MET expression (pearson r = 0.1782; p = 0.2063) or HGF expression (r = -0.021; p = 0.8794). It is important to note that HGF/c-MET measurements were not simply the products of the individual HGF and c-MET measurements (pearson r = 0.1164; p = 0.4112). We also compared our HGF/c-MET measurements in the proximity FFPE assay with the levels of c-MET (pY1003), c-MET (pY1234/1235), and c- MET (pY1349) phosphorylation detected in NSCLC and gastric tumors. Due to the low abundance of phosphorylated c-MET protein in tumor samples, c-MET was immunoprecipitated prior to immuno-detection by Western blot (Fig. 9Di, ii). c-MET (pY1003) phosphorylation was detected in 9 NSCLC tumor lysates (NL1, NL2, NL3, NL4, NL5, NL6, NL8, NL14, and NL15) and the HGF/c-MET was also detected in 5 of 6 corresponding FFPE sections by proximity FFPE assay (NL1, NL4, NL5, NL6, and NL15). Two tumors (NL2, NL8) were too small (,20mm2) to run in the FFPE format. c-MET (pY1003) phosphorylation was detected in only two gastric tumor lysates (G2, G3) and these same tumors were also positive for HGF/c-MET complexes by proximity FFPE assay. In one gastric tumor lysate (G6), a slightly higher molecular weight/slower migrating band that also reacted with the c-MET (pY1003), however, at the present time, the identity of this protein is uncertain (Fig. 9Dii). Our attempts to detect c-MET (pY1234/1235) and c-MET (pY1349) phosphory- lation in NSCLC and gastric tumors were unsuccessful (data not shown) suggesting that phosphorylation at these sites might be labile than c-MET (pY1003). Discussion The c-MET, HGF, and c-MET/HGF assays described in this report are based on the release of antibody conjugated fluorescein reporters by antibody conjugated molecular scissors that are bound in close proximity. This technology has been used to measure total HER2 receptor and HER2 homodimer levels in breast tumor FFPE specimens [10,28] and several studies have demonstrated the clinical utility of these measurements [11,12,28]. In this report, we used the HGF/c-MET ligand-receptor interaction as a model for the development of a ligand-receptor proximity assay as a method to measure receptor activation in FFPE specimens. We have generated assays to quantify the expression of c-MET, HGF, and HGF/c-MET ligand-receptor complexes in FFPE cell lines and human carcinomas and have cross-validated these measurements using independent biochemical methods. The expression of c-MET and HGF is associated with tumor progression and a number of clinical studies have implicated the PLoS ONE | www.plosone.org 14 January 2011 | Volume 6 | Issue 1 | e15932