87 MET ECD shedding and poor DFS ding and regulated intramembrane proteolysis occur in vitro. The cell lines were obtained from different departments within the Erasmus Medical Center and the University Medical Center Utrecht. Detailed information concerning the properties of these cell lines is provided in Supplementary Table 1, while information on culture conditions is provided in Supplementary Table 2. Ethics statement Human tissues and patient data were used according to “The Code of Conduct for Responsible Use” and “The Code of Conduct for the Used of Data in Health Research” as stated by the Federation of Dutch Medical Scientific Societies (29). Patient tissues To examine whether proteolytic processing of MET can be detected in tissues that are processed in a diagnostic setting, the following approach was taken. Tissues from 34 primary oral squamous cell carcinoma that were surgically removed between 2004 and 2011, were collected from the tissue bank of the department of Pathology of the Erasmus Medical Center. Using a microtome, 10 × 10-μm-thick slices were cut from the fresh frozen cancer tissues for protein isolation in view of western blotting and 3-μm-thick whole tissue sections were cut from the formalin-fixed paraffin-embedded cancer tissues in view of immunohistochemical analyses. To examine the association between MET ectodomain shedding and survival, a tissue microarray representing 212 formalin-fixed paraffin-embedded primary oral squamous cell carcinomas—surgically removed between 1996 and 2005 in the University Medical Center Utrecht—was included in the study. In total, six tissue cores (0.6-mm diameter) were sampled from each cancer; three from the center and three from the periphery of the tumor. Further details concerning tissue microarray construction and histopathological scoring of the sampled cancers are described in (20). Using a microtome, 4-μm-thick slices were cut from the formalin-fixed paraffin-embedded cancer tissues for immunohistochemical analyses. The primary form of treatment for all included cancers (Erasmus Medical Center and University Medical Center Utrecht) was surgery with or without post-operative radiotherapy. RNA isolation, cDNA generation, and quantitative real-time PCR Culturing of the cell lines, RNA isolation, cDNA generation, quantitative real-time PCR were performed as previously described (18). Included positive controls for MET 4
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