Fig. 8
Cancer Associated Fibroblast are the main source of IL-6 after 5-FU treatment and may signal to gp130pos;EpCAM.pos cells. CAFs were isolated as described in methods from the same specimens used for PDO generation and cocultured at different ratios with matched, early passage PDO#1 and PDO#4, to assess the levels of IL-6 released after ctrl, pentoxifylline, 5-FU or combined (pentoxifylline + 5-FU) treatment. a Representative micrographs of the CAF cultures derived from the same specimens of the PDO#1 (CAF#1) and PDO#4 (CAF#4). Left: bright field micrographs. Right: immunofluorescence staining with anti-SMA antibody. Size bar: 30 µm. b Overlay histogram plot of the CAF#1 and CAF#4 cultures stained with anti-SMA (Left) and with Anti-EpCAM (Right) antibodies and analyzed by flow cytometry. The background fluorescence from isotype-matched antibodies is also reported (c) Histograms showing the levels of IL-6 detected by an ELISA assay in the indicated samples. Results are expressed in nanograms per milliliter of IL-6, adjusted for 10^6 PDO-derived cells, after 48 h of medium conditioning and representative of three independent experiments. d Representative dot plots of the cocultures (1:1) stained with an anti-EpCAM antibody and with anti-gp130 antibody. Right panels. Quantitation of the results from the left panel for two independent experiments. Statistics: * p < 0.05; ** p < 0.01