Detection of Receptor Activation in FFPE Specimens MET ligand receptor complexes in H661, MCF7, HGF stimulated MCF7, Ln18, Ln229, U138, U118, U87MG cell lines. C. Immunoprecipitation/Western blot analysis of c-MET (Tyr1003) phosphorylation and c-MET in Ln18, Ln229, U138, U118, and U87 MG glioma cells. The H596 cell line with exon 14 (L964-D1010) deletion was included as a negative control for c-MET (Tyr1003) phosphorylation. doi:10.1371/journal.pone.0015932.g007 applied our antibody proximity technology to the development of a quantitative assay that measures HGF expression levels in FFPE tumor specimens. A large panel of HGF antibodies was screened for specificity using a stable cell line (HEK293/HGF (clone 1) that overexpress HGF and the HGF negative parental cell line (HEK 293). Using these cell lines, we identified a goat polyclonal anti- human HGF antibody and a mouse monoclonal anti-human HGF antibody (clone SBF5) that exhibited high specificity. The proximity assay was then optimized in two antibody format using A549 cells in the presence of increasing amounts of HGF (Fig. 5A). In agreement with a previous report [24], unstimulated A549 cells exhibited low HGF expression (Fig. 5A). HGF measurements using isotype antibody matched controls were less than 5% of the HGF specific assay signals (Fig. 5A). Next, we quantified HGF in cell lines that exhibit autocrine HGF/c-MET signaling. We assessed endogenous level of HGF in FFPE pellets generated from a panel of glioma cell lines (Ln229, Ln18, U138, U118, and U87MG cells). U138, U118, and U87MG possess an HGF/c-MET autocrine loop and secrete HGF in the culture medium [23]. U118, Ln18, and U87MG exhibited ,12–40 fold higher levels of HGF than U138 cell line (Fig. 5Bi), while Ln229 cells produced no detectable HGF signal. These measurements correlated well with the HGF ELISA measurements we obtained using the corresponding glioma cell lysates (Fig. 5Bii) and are in agreement with previous reports on endogenous HGF expression in HGF in U138 and U118 cells [25]. The dynamic range of the HGF proximity assay was defined as ,3 log10 by comparing the assay signals of the HEK293/HGF cell line against the U138 cell line (Fig. 5Ci) and this is consistent with detection of HGF by IHC in corresponding cell lines (Fig. 5Cii). Detection and quantification of hepatocyte growth factor (HGF) in human tumors by antibody proximity assays We utilized the same NSCLC tumor panel that was used for the evaluation of the c-MET proximity assay to evaluate the HGF proximity assay (Fig. 6A). NL7 was the only sample that exhibited high HGF expression levels in this panel. Isotype antibody controls signals were consistently less than 20% of HGF antibody-specific signals (Fig. 6A). To compare HGF proximity assay measurements to independent biochemical methods, we performed Western blot and ELISA assays on the corresponding tissue lysates. HGF was immuno-precipitated from 2 mg of cellular protein and probed for HGF by Western blot analysis. Consistent with the HGF proximity assay, sample NL7 exhibited high levels of HGF compared to the other samples (Fig. 6B). ELISA analysis of the HGF levels in these samples was also consistent with both the proximity assay and Western blot measurements (Fig. 6C). Furthermore, the results of the HGF proximity assays, ELISA, and Western blot were all concordant with IHC staining (Fig. 6D). Detection and quantification of HGF/c-MET ligand- receptor interaction in FFPE cell line pellets by antibody proximity-based assay and correlation with c-MET (pY1003) phosphorylation We used anti c-MET and anti-HGF antibodies that worked well in the c-MET and HGF assays to develop a quantitative assay for detection of HGF/c-MET complexes in FFPE specimens. As illustrated in Fig. 7Ai, the assay is performed by (a) incubation of FFPE samples with a rabbit anti-MET antibody (clone SP44) together with a human anti-HGF antibody that is conjugated to a VeraTag reporter, (b) addition of biotin-conjugated goat anti- rabbit antibody which binds to the rabbit anti-MET (clone SP44) antibody, (c) addition of streptavidin-methylene blue, which serves as photosensitizer, and (d) illumination of the sample, which activates the photosensitizer and releases VeraTag reporters in close proximity (see Fig. 1A). This secondary antibody format enhances signal strength which in turn improves assay sensitivity and reproducibility (Wallweber J, Unpublished data). A similar format has been applied to the quantitative measurements of the truncated form (p95) of the HER2 receptor in breast cancer [11]. To demonstrate the ability of our new assay to detect the HGF/c- MET complex, we performed the assay on unstimulated and HGF stimulated A549 cells. As illustrated in Fig. 7Aii, we observed increases in the HGF/c-MET complex in the HGF stimulated A549 cells which were proportional to the dosage of HGF. To assess the extent of assay background resulting from non-specific antibody binding, replicate samples (adjacent sections) were incubated with the VeraTag labeled anti-HGF antibody and an isotype (IgG DA1E) in place of the anti-MET antibody. In this case, isotype control signals were less than 20% of the HGF/c- MET complex specific signals (Fig. 7Aii (inset)). Additionally, we could demonstrate a decrease in the HGF/c-MET complex signal in HGF treated A549 cells when HGF was pre-incubated with an HGF neutralizing antibody (clone 24612) (Fig. 7Aii). To rule out that this decrease in HGF/c-MET signal was not due to assay interference by neutralizing antibody binding, we verified that the addition of a 10-fold excess of HGF neutralizing antibody did not interfere with the assay (data not shown). Furthermore, we also observed a corresponding decrease in c-MET phosphorylation when HGF was pre-incubated with the HGF neutralizing antibody (data not shown). Next, we evaluated whether the FFPE assay could be used to detect endogenous levels of HGF/c-MET complexes in glioma cell lines that activate c-MET signaling through the autocrine production of HGF [25]. As illustrated in Fig. 7B, the HGF/c- MET complex was detected in the Ln18, U138, U118, and U87MG cell lines but not in the Ln229 cells (Fig. 7B). This result is consistent with the HGF and c-MET data we present in Fig. 2A and Fig. 5Bi, respectively, i.e. Ln229 cells express intermediate levels of c-MET but not endogenous HGF. Once again, we verified that the signals from isotype control assays were less than 20% of the HGF/c-MET specific assay signals (Fig. 7B (inset)). Furthermore, there was no significant difference in the HGF/c- MET and isotype control signals for MCF7 and H661 cell lines which express very low or no c-MET and do not express endogenous HGF (Fig. 7B). Next, we sought to correlate the presence of autocrine driven HGF/c-MET complexes in glioma cells with a direct indicator of c-MET activation. Upon ligand activation, c-MET is phosphor- ylated, most notably at amino acid positions Y1003, Y1234/1235, and Y1349. In addition, c-MET phosphorylation (pY1003) is a marker of HER1 inhibitor (gefitinib) resistance in NSCLC patients [26]. We measured c-MET pY1003 levels by immuno-precipita- tion in lysates prepared from the Ln18, Ln229, U118, U138, and U87MG glioma cell lines to compare with the HGF/c-MET levels PLoS ONE | www.plosone.org 12 January 2011 | Volume 6 | Issue 1 | e15932