Fig. 4.

NT5C1A expression and function in PDAC stroma in vitro and in vivo. A) and B) Tissue microarray analysis for NT5C1A expression revealed very low expression in the tumor stroma of resected PDAC patients. A semi-quantitative scoring system indicated no stromal expression with score 0 and strong stromal expression with score 3. TMA-1 from Goettingen (A) with n = 77 patients (none scored with 3) and TMA-2 from Erlangen (B) with n = 330 patient samples. C) Representative immunohistochemistry of NT5C1A expression showing no NT5C1A expression (score 0), low expression (score 1), and robust stromal expression (score 2) of TMA-1. Scale bars 20 μm. D) Murine PSCs were treated with increasing concentrations of gemcitabine for six days and crystal violet assays were performed. The staining was more pronounced in NT5C1A expressing cell lines. Representative images of two independent experiments, with each two technical replicates, are shown. E) Crystal violet stain intensity was quantified and results were normalized to untreated control cells. Graph indicates mean ± SEM, two-way ANOVA with Sidak's multiple comparisons test was performed (7 nM: p < .035, 12 nM: p < .009, 20 nM: p < .0001, 35 nM: p = .007). F) and G) MTT cell viability assay for KPC cell lines treated with conditioned medium (CM) of NT5C1A expressing PSCs and control CM. F) Schematic experimental overview. CM from PSCs (+NT5C1A) and control PSCs (+vector) was obtained by preincubation with 25 nM gemcitabine-hydrochloride for 24 h. Subsequently, tumor cells were treated for 72 h with CM of PSCs and viability was assessed using MTT cell viability assay. G) Tumor cell viability of two different murine KPC cell lines was significantly decreased following treatment with CM of +NT5C1A-expressing PSCs (KPCa: 73%; p = .003 and KPCb: 75%; p = .047). Graphs indicate mean ± SEM of four biological replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)