Exogenous IL-17A promotes chemoresistance through the NF-κB pathway. (A) Chemosensitivity of 3 × 10³ CICs from the three independent human samples stimulated with exogenous IL-17A and treated with chemotherapy was evaluated by CellTiter-Glo assay. Data are mean ± SD (n = 3); *, P < 0.05; **, P < 0.01. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (B) The effect of the media conditioned by CAFs on CICs from two different specimens was assessed by ten-day co-culture. 10⁴ CICs were cultured with 2 × 10⁴ CAFs treated with vehicle (DMSO), chemotherapy alone or along with IL-17A blocking antibody (15 ng/ml), or an isotype IgG control. Cell growth was measured by CellTiter-Glo assay. Data are mean ± SD (n = 3); **, P < 0.01; ***, P < 0.001. Student’s t-test was used to assess the significance. The experiment was performed three times and representative data are shown. (C) CICs gene profile from three independent human samples stimulated with vehicle or exogenous IL-17A was performed on an Illumina expression array. Signal data were filtered to include only genes that were seen to have changes in at least two of the three matched pairs and were subjected to hierarchical clustering. Signal ratio > 1.5 and P < 0.05. (D) Network analysis of the most significant genes up-regulated in CICs stimulated with IL-17A versus vehicle was generated using IPA (Ingenuity Systems). (E) The alteration of the NF-κB pathway and its downstream target ERK1/2 was assessed at the protein level through the analysis of the levels of the phosphorylation status of NF-κB p65 (RelA) and p42-44-ERK1/2 in one sample treated with vehicle or with IL-17A with and without the IL-17A blocking antibody for 12 h.