Male C57BL/6 mice were fed with a Western diet to induce obesity. Daily treatment with the aromatase inhibitor letrozole was started 4 weeks before subcutaneous injection of B16-F10 tumor cells. Letrozole treatment was continued until the end of the experiment. After the development of palpable tumors, mice received either anti–PD-1 or isotype control. (A) Expression of the 9 most significant genes from the GSEA Hallmark “Estrogen_Response_Late,” measured by qPCR in isotype-treated mice (n = 9–10/group). Unpaired 2-tailed Student’s t test was used. *P < 0.05, **P < 0.01. Data are depicted as a heatmap and as box plots for 3 representative genes. Box plots show the interquartile range (IQR), median (line), and the most extreme values within 1.5 × IQR (whiskers). Points beyond the whiskers represent outliers. (B) Tumor growth of vehicle-treated (dotted line) or letrozole-treated (solid line) mice receiving anti–PD-1 (red) or isotype control (blue). Black arrows indicate anti–PD-1 or isotype treatment (n = 9–10/group). Two-way ANOVA with Tukey’s post hoc test was used. *P < 0.05, **P < 0.01, ****P < 0.0001. (C) Infiltration of CD3⁺ cells and gp100-specific CD8⁺ T cells in tumors, quantified by flow cytometry (n = 9–10/group). Unpaired 2-tailed Student’s t test was used. *P < 0.05. (B and C) Data are depicted as mean ± SEM. (D) Expression of selected immune genes, measured by qPCR (n = 9–10/group). Data are depicted as Tukey box plots and Mann-Whitney test was used. *P < 0.05.