Human monocyte derived DCs were pulsed for three days with graded concentrations of DAMPs obtained from necrotic material from lysed HCT-116 colorectal tumour cells, or with a fixed concentration of S100A4 (1μg/ml). Thus, pulsed DCs were co-cultured with autologous lymphocytes for 5 (±1) days.
Panel A shows the mean percentage (±SEM) of proliferated lymphocytes compared to total lymphocyte count with asterisk indicating p ≤ 0.05.
Panel B shows increased metabolic activity of above mentioned lymphocytes compared to lymphocytes which were co-cultured with non-pulsed autologous DCs. Metabolic activity was assessed by measuring the amount of WST-1 cleaved per hour and increased activity was shown in percent increase. Asterisks indicate p ≤ 0.05.
Panel C confirms the expression of S100A4 by HCT-116 cells which were used to obtain DAMPs. Shown are results from RT-PCR analysis of S100A4 expression in three proliferating and 70-80% confluent colorectal tumour cell lines (HCT-116, COLO-678, CACO-2) and a cervical carcinoma cell line (HeLa). HCT-116 shows the highest and CACO-2 the lowest S100A4 expression, while CACO-2 lysate containes about double as much total protein compared to HCT-116 lysate (Panel D).
