Detection of Hepatocyte Growth Factor (HGF) Ligand-c-MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels o

c-MET, HGF, and HGF/c-MET complexes in human carcinomas.

<p>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 ≥90^th percentile; Pink color-Low ≤10^th 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 (−) are summarized in Fig. A, B.</p

VeraTag FFPE quantification of HGF and correlation with Western blot, ELISA and IHC in cell lines.

<p>A. VeraTag FFPE quantification of HGF in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF signals. Bi. VeraTag quantification of HGF in Ln18, Ln229, U138, U118, and U87MG glioma cell lines. Isotype IgG control signals (inset) were subtracted from HGF assay signals. ND: Not detectable. Bii. ELISA determinations of HGF in A549, Ln18, Ln229, U138, U118, and U87MG cell lysates. ND: Not detectable. Ci. VeraTag FFPE quantification of HGF in HEK293/HGF (clone 1) and U138 glioma cell lines. Cii. IHC detection of HGF in HEK293/HGF stable clone (clone 1) and U138 glioma cells.</p

VeraTag FFPE quantification of HGF and correlation with Western blot, ELISA, and IHC in human tumor specimens.

<p>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.</p

Measurement of the HGF/c-MET complex by Surface Protein-Protein Interaction by Cross-linking ELISA (SPPICE) assay.

<p>A. SPPICE schemetic. c-MET was chemically cross-linked to expgenous HGF using the membrane impermeable sulfo-EGS cross-linker. Membrane proteins were solublized and HGF/c-MET complexes were measured using the SPPICE procedure (Bi) or by immunoprecipitation with HGF antibodies 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.</p

VeraTag FFPE c-MET quantification and correlation with Western blot, ELISA and IHC in cell lines.

<p>A. Quantification of c-MET expression in FFPE preparations of H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, U87, and H661 FFPE cell lines using the VeraTag assay. Background signals generated using an isotype control IgG (shown in inset) were subtracted from the c-MET assay signals. B. Western blot analysis of c-MET expression in H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, and H661 cell lysates. β-actin levels in the blot serves as a loading control. C. ELISA measurements of c-MET expression in H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, and H661 cell lysates. ND: Not detectable. D. IHC detection of c-MET expression in FFPE preparations of H441, H226, MCF7, Ln18, U118, and H661 cell lines.</p

Epitope mapping of c-MET antibodies by peptide scanning.

<p>A. Peptide ELISA. A series of 43 overlapping peptides, each 14–15 amino-acid residues in length, spanning the complete intracellular domain of c-MET (K955-S1370), were evaluated to map the binding epitope of c-MET antibodies. Peptides were spotted on microtiter plates and ELISA assays were performed using standard protocols. The binding properties of peptide 28 and 43 localized the binding sites of c-MET antibody 3D4 and c-MET antibodies CVD13 and SP44, respectively. The sequence of peptide #28 and #43 are shown (inset). B. A schematic representing the c-MET receptor and binding sites for c-MET antibodies.</p