IL-17A can contribute to CICs maintenance through IL-17A receptor. (A) Autocrine expression of IL-17A was assessed using a 120 human cytokine antibody array from RayBio on 4 CICs treated with vehicle (DMSO) or with chemotherapy for 3 d. The mean concentration of IL-17A was statistically significant in the four CICs treated with chemotherapy compared to vehicle. (n = 1); P < 0.01. Student’s t test was used to assess the significance. The experiment was performed once. (B) The autocrine IL-17A production was examined by studying the effects of IL-17A blockade on the tumor-initiating capacity of CICs in the absence of CAFs. The effects were assessed in two different specimens by 10 d of co-culture. 10⁴ CICs were treated with vehicle (DMSO), chemotherapy alone, or along with IL-17A blocking antibody (15 ng/ml). Number of viable cells was measured by Cell Titer-Glo assay. No statistically significant difference in viability was noted as a result of IL-17A blocking antibody. Data are presented as mean ± SD (n = 3); unlabeled, P > 0.05. Student’s t test was used to assess the significance. The experiment was performed twice, and representative data are shown. (C) IL-17RA was expressed in CICs as demonstrated by immunofluorescence staining. Nuclei were counterstained with DAPI. Bar, 10 µm. (D) Confirmation of IL-17RA expression was assessed in CICs by FACS analysis of single cells from three freshly dissociated xenografts co-stained for FITC-CD44 and APC-IL17RA.