89 MET ECD shedding and poor DFS the A2H2-3 antibody essentially as described in (28). Intensities of the relevant bands were evaluated as follows: absent, weak, moderate, and strong. ELISA The occurrence of MET ectodomain shedding was also examined by performing an ELISA on the culture media used to grow HeLa, PC3, LNCaP, HT-29, SCC-4, SCC-9, SCC-25, CAL-27, and UM-SCC-14C cell lines. The ELISA plate was coated with 2 μg/mL anti-MET antibody at 4 °C overnight. Following wash and block steps, either 100 μL of (a) MET ectodomain standard in diluent buffer or (b) tumor cell culture supernatants (dilutes 1:50 with diluent buffer) were added per well. After incubation, the plate was washed and 100 μL of 0.5 μg/mL biotinylated anti-MET antibody—that recognizes a different epitope than the anti-MET antibody used for coating—was added per well. Detection was carried out as described (28). Signal was read at 450 nm with 570 nm correction on a Spectramax plate reader (Molecular Devices, LLC, San Jose, CA, USA) and evaluated as follows: absent, weak, moderate, and strong. Immunohistochemistry Staining of cell lines and whole tissue sections using D1C2 and A2H2-3 was essentially performed as described before (18, 28). Two observers (SW and MDH) independently scored D1C2 and A2H2-3 immunoreactivity across the cell lines, whole tissue sections and tissue microarrays. Well-differentiated cancer cells that show no nuclei were omitted during scoring. Since the staining intensities for both antibodies were comparable for internal positive controls—e.g., endothelial cells lining veins (Supplementary Fig. 1)—present in the whole tissue sections and tissue microarrays, it was assumed that D1C2 and A2H2-3 membranous immunoreactivities can be compared when examining formalin-fixed paraffin-embedded tissues processed in a routine diagnostic setting. Scoring of D1C2 and A2H2-3 immunoreactivity across cell lines and whole tissue sections as well as MET ectodomain shedding across whole tissue sections For the cell lines, staining intensity was evaluated for the nucleus, cytoplasm, and membrane separately on a scale from 0 to 3 (Table 1). Each location was scored separately. For the patient derived whole tissue sections, intensities and scores were only evaluated for the membrane. Representative images of membranous A2H2-3 immunoreactivities observed on whole tissue sections are depicted in Fig. 1. Representative images of D1C2 intensities have been reported (18). 4
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