Cytokine-Induced TSLP Expression in CAFs

CAFs were seeded at 1.5-3 × 10⁴ cells/well in IMDM 10% FBS in 96-well-plates and starved overnight. The next day, medium was replaced and left for 72 h with the following stimuli: recombinant human IL-1α, IL-1β and TNF-α (R&D System), IL-18 (MBL) (all in IMDM 2% FBS at the indicated concentrations), and the supernatant of necrotic PDAC cells (100 μl). In inhibition experiments, 10 μg/ml anti-IL-1α (R&D System), anti-IL-1β, anti-TNF-α or isotype-matched antibodies (Abs) (BD), and 10 μg/ml Anakinra (Kineret, Amgen Europe) were added. mRNA expression of short and long TSLP isoforms by CAFs untreated or activated by recombinant cytokines was assessed by Real Time PCR (SYBR green, Applied Biosystems) using the primers described in [18 (link)]. In another set of experiments, CAFs were stimulated with the supernatant of THP1 cells treated with the supernatant of tumor cells after treatment with mock-transfected or ASC siRNA or negative control siRNA. TSLP secretion in CAF supernatants was measured by ELISA (DY1398, R&D System).