Figure 4. Validation of NPPB expression in EOC models and primary tumours.

NPPB is the most significantly upregulated gene (ranked by fold change). Expression of (a) NPPB in stromal cells co-cultured for 7 days with 2 normal OSEC lines and 7 EOC cell lines. NPPB expression in stromal cells is significantly upregulated in response to co-culture with at least 50% of EOC cell lines; with expression levels of NPPB increasing up to 350-fold when INOFs and MSCs were co-cultured with EOC cells compared to controls. Expression of NPPB was significantly lower in stromal cells co-cultured with OSECs than the same cells co-cultured with the majority of EOC cell lines. Expression of NPPB is absent/negligible in 1BR3 skin fibroblasts, regardless of culture conditions. Note that for NPPB expression MSC values are shown on a log scale, whereas INOF and 1BR3 data are on a linear scale. In the microarrays and by qPCR, MSCs tended to show larger changes in gene expression than INOFs. (*P<0.05, two-tailed paired Student's T-test, α=0.05, compared to at least 2/3 controls: non-co-cultured stromal cells and OSEC-co-cultured stromal cells). Error bars = s.d. (b) NPPB protein is expressed in ovarian tumours. Right panel, example of a tumour staining positive for the active, secreted NPPB peptide. Expression is seen in the tumour stromal (TS) cells as well as, in some cases, diffuse staining in the tumour epithelium (TE), which may be due to diffusion of the secreted protein. Middle panel shows an example of a tumour that does not express NPPB. Left panel, NPPB expression was not detected in normal ovarian stromal cells. (c) Analysis of NPPB protein expression in tumour stromal cells of 145 primary EOCs stained by immunohistochemistry. (d) Measurement of NT-proBNP levels in patients with ovarian cancer. NT-proBNP is elevated in 4/8 EOC patients independently of cardiac failure. Dashed line = cutoff for elevated NT-proBNP (100pg/ml).