Figure 3.

(See previous page). Temozolomide treatment of RETAAD mice induces intratumoral upregulation of CXCL9 and CXCL10. (A-E) RETAAD mice were treated with Temazolomide (TMZ) or dimethyl sulfoxide (DMSO) vehicle only after they were determined to have developed genitourinary (GU) and cutaneous tumors by clinical examination. (A) In vitro activated CD45.1 T cells were transferred into RETAAD mice at day 10 following DMSO or TMZ treatment. Recipient mice were sacrificed 5 days after T-cell transfer. Dissociated tumor cells were immunostained and flow cytometry was performed to detect transferred CD45.1 T cells in GU tumors. (B) CXCR3 expression on the surface of infiltrating T cells in GU tumors from DMSO or TMZ-treated mice was examined by immunostaining and flow cytometry analysis. (C) qRT-PCR analysis of gene expression of CXCL9 and CXCL10 in GU tumors at various time points after TMZ treatment. (D) Immunofluorescence imaging was performed to examine CXCL9 and CXCL10 protein expression in GU tumors at day 10 after TMZ treatment. (E) Mean fluorescence index (MFI) quantification of CXCL9 and CXCL10 staining in GU tumors at day 10 after TMZ treatment. Data from panel: (A) are pooled from 2 separate experiments with 3 mice per group in each experiment (n = 6/group); (B) is representative of 4 separate experiments (n = 4/group); (C and E) are pooled from 4-6/group. Bars represent mean ± SD. Statistical analyses were performed using the unpaired two-tailed Student's t-test in (A and E) and the one-way ANOVA test with Bonferroni's post-test analysis in (C); *p<0.05, **p<0.01. Images from (D) are representative of 4-6 independent experiments (n = 4-6/group). Scale bars in (D) represent 200 μm.