Fig. 5. KIRA6 affects the immunogenicity of chemotherapy.

A Scheme of the transwell migration experimental setup. B, C Transwell migration assay of human neutrophils exposed for 2 h to conditioned medium of A375 cells treated for 24 h with MTX in the presence or absence of KIRA6 (1 μM). D, E Representative pictures and relative quantification of the transwell migration assay of human macrophage-like THP1 cell line exposed for 4 h to conditioned medium of A375 cells treated for 24 h with MTX in the presence or absence of KIRA6 (1 μM). F Neutrophil migration was assessed after neutralization of CXCL8 secreted upon treatment with MTX with a αCXCL8-neutralizing antibody (0.5 μg/ml) or after addition of recombinant CXCL8 (10 ng/ml) in MTX + KIRA6 conditioned medium. G Dendritic cell maturation was assessed by measuring the surface expression of CD86 and HLA-DR by FACS 24 h after co-incubation with the ‘cell free’ medium of A375 cells treated for 24 h with CDDP, MTX, or Hyp-PDT in the presence or absence of KIRA6 (1 μM). H, I BALB/c mice immunized with PBS or with CT26 treated for 24 h with MTX in the presence or absence of KIRA6 (1 μM) were rechallenged with live CT26 tumor cells, and tumor growth (H) and survival (I) were monitored. N = 7-8mice/condition. Mice that did not develop tumor (n = 4 out 7 in MTX group and n = 1 out of 8 in MTX + KIRA6 group) are shown as a flat line on the x-axis in (H). In all graphs values are presented as mean ± SD of at least n = 3 independent biological replicates. Data are analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test in all the graphs except for two-tailed Student’s t-test in (G) to compare the effect of the inhibitor on the treatment, and Log-rank Mantel-Cox in (I). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.