Figure 6.

miR-27a in CRC cells modulates monocyte-derived human DCs maturation and secretion of selected cytokines upon drug-mediated ICD. (a) Scheme of the experimental setting: hu-iDCs were generated from filtered buffy coat-derived monocytes donated by healthy donors and grown for 5/6 days in the presence of specific stimulatory factors. Hu-iDCs were identified by FACS analysis of CD1a⁺ cells, counted and co-cultured (1 : 2 ratio) with HCT116 or RKO cells previously transfected for 72 h with a miR-27a synthetic mimic (S), AS or scrambled control (C) RNA, respectively, and treated for the last 12 h with OXP (100 μM). (b) Flow cytometry analysis of specific DC maturation markers (CD86⁺/HLA-DR⁺, CD80⁺/CD83⁺) upon 20 h of co-cultures as described in (a); lipopolysaccharide stimulation (0.1 mg/ml) of hu-iDCs was used as positive control of DC maturation. Only the results with OXP treatment are reported, similar results were obtained with MTX. The histogram illustrates the results of five experiments. (c) A cytokine ELISA array was performed on the CM from S-, AS- or C-transfected HCT116, alone or from co-cultures of the same cells with hu-iDCs; the color-code reports the range of values of the cytokines detected. (d) The histogram shows quantification of IL-8, IL-4 and IL-6 detected with the cytokine ELISA array. Samples were analyzed in triplicate and data are mean±S.D. and representative of three experiments. *P⩽0.05; **P⩽0.01 (two-tailed Student's t-test)