Figure 3

Expression of MME in NSCLC samples, NSCLC cell lines, and fibroblasts. (A) MME and HIF-1α were stained in consecutive sections of fresh NSCLC. Representative areas from a sample with high HIF-1α staining in tumor (arrow) and stroma cells (arrowhead) are shown (images A-F). While MME positive stroma cells were found predominantly in HIF-1α positive areas, no association of HIF-1α and MME was found in tumor cells. Note the intensely MME positive stroma cells (asterix) surrounding an islet of tumor cells, which were MME negative. Images G and H show a sample from a different patient. In this patient MME staining in stroma cells was apparently unrelated to HIF-1α. Scale bar: 200 μm. (B) Immunohistochemistry for MME in normoxic and hypoxic fragments from a single patient. (C) NSCLC cells were cultured in hypoxia (1% oxygen) or ambient oxygen for three days and mRNA levels of hexokinase 2 and of the four overlapping hypoxia genes were analyzed. Expression levels in hypoxia relative to normoxia are shown. Results are mean +/- SEM from three independent experiments. (D) Human lung fibroblasts from three different donors and carcinoma-associated fibroblasts (CAFs) isolated from NSCLC from three different patients were cultured in hypoxia (1% oxygen) or ambient oxygen for three days and MME mRNA levels were analyzed. Results are mean +/- SEM from n = 5 to 7 independent experiments. (C and D) Groups were compared with one-sample Student’s t-test. HK2, hexokinase 2; MME, membrane metallo-endopeptidase; KCTD11, potassium channel tetramerisation domain containing 11; PPP1R3C, protein phosphatase 1 regulatory subunit 3C; FAM115C, family with sequence similarity 115 member C; nox, normoxia; hox, hypoxia. *P < 0.05, **P < 0.01, ***P < 0.001.