97 MET ECD shedding and poor DFS SCC-4, SCC-25, CAL-27, and UM-SCC-14C showed weak immunoreactivity for p70β, p90β, and p95β indicating that they are subjective to ectodomain shedding. In contrast, SCC-9 shows no immunoreactivity indicating ectodomain shedding (Fig. 3c). An independent nonreduced blot, generated using A2H2-3, confirms that all examined oral squamous cell carcinoma cell lines express p145β and p170 (Supplementary Fig. 2). An independent ELISA—using the same antibody—on the culture media used to grow the oral squamous cell carcinoma cell lines, shows that all examined cell lines shed the N-terminus of MET with varying levels (Fig. 3d). Subsequently, it was examined whether ectodomain shedding and regulated intramembrane proteolysis can be detected in patient derived fresh frozen tissues (n = 44) under reducing conditions using D1C2. Six of the 44 examined tissues were excluded from further analysis because of the absence of immunoreactivity of both C-terminal MET and the endogenous control (b-actin). The remaining 38 tissues represent 30 surgically removed primary cancers (Fig. 4). The immunoreactivity of (b-actin) was low (scores 0 and 1) in 10 of the 37 analyzed tissues (27%). The purple heatmap (Fig. 5a), depicting D1C2 immunoreactivity under reducing conditions, shows that C-terminal MET protein fragments can be detected that are consistent with ectodomain shedding (p95β, p90β, p70β, p60β, and p55β) and regulated intramembrane proteolysis (p50β). The observed immunoreactivity patterns can be classified into five categories: (1) negative for p145β and C-terminal fragments (n = 4), (2) positive for p145β and negative for C-terminal fragments (n = 18), (3) positive for p145β and C-terminal fragments consistent with ectodomain shedding (n = 2), (4) positive for p145β and C-terminal fragments consistent with regulated intramembrane proteolysis (n = 2), and (5) positive for p145β and C-terminal fragments consistent with ectodomain shedding and regulated intramembrane proteolysis (n = 11, Fig. 5a, b). The results for multiple samples derived from 1, 2, and 25 are consistent. They show no signs of ectodomain shedding and/or regulated intramembrane proteolysis. In contrast, the results for the biological duplicates of cancers 26, 27, and 30, show inconsistent results for the presence of ectodomain shedding and/or regulated intramembrane proteolysis. 4
RkJQdWJsaXNoZXIy MTk4NDMw