Supplementary Figure S15 from Autocrine 17-β-Estradiol/Estrogen Receptor-α Loop Determines the Response to Immune Checkpoint Inhibitors in Non–Small Cell Lung Cancer

Letrozole sensitizes xenografts to a second cycle of pembrolizumab by modulating the tumor-associated immune-environment. 60 Hu-CD34+NSG mice bearing female-derived ERα lowNCI-H11385 and ERα highNCI-H1975 tumors, male-derived ERα lowA549 and ERα highNCI-H1650 tumors (15/tumor type) were treated for 5 weeks with pembrolizumab+letrozole with 10 mg/kg i.p. of 100 µL saline solution of pembrolizumab (day 1) followed by 5 mg/kg i.p. (days 7, 14, 21, 28, 35), and 1 mg/kg per os of 100 µL saline solution of letrozole daily (days 1-35), and then divided into three cohorts of 20 animals (n=5 mice/tumor type within each cohort). The first cohort of animals (left panel) was left untreated until week 15 (-Pemb-Letr); the second cohort (middle panel) was treated with 1 mg/kg per os of 100 µL saline solution of letrozole daily until week 15 (+Letr); the third cohort (right panel) was treated with 1 mg/kg per os of 100 µL saline solution of letrozole daily from week 6 to week 10, and then with pembrolizumab+letrozole from week 11 to week 15 (+Pemb+Letr). a. Tumor growth was monitored daily using calipers. Data are presented as mean volumes+ SD. **p<0.01, ***p<0.001: volume of female ERα lowNCI-H1385 and ERα highNCI-H1975 tumors (left panel), or ERα highNCI-H1975 tumors (middle-right panels) versus mean volume of male ERα lowA549 and ERα highNCI-H1650 tumors. b. PD-L1 expression was measured using flow cytometry on dissociated tumor cells after explants at week 15 (n=5/group). *p<0.05: +Pemb+Letr versus -Pemb-Letr (NCI-H1650 tumors), +Letr versus -Pemb-Letr (NCI-H1385 tumors); **p<0.01: +Pemb+Letr versus -Pemb-Letr (NCI-H1385 tumors); ***p<0.001: +Letr/+Pemb+Letr versus -Pemb-Letr (NCI-H1975 tumors) (ANOVA). c-e. On TILs isolated from tumor extracts (week 15), the percentage of CD8+Ki67+IFNγ+cells (panel c), CD56+Ki67+IFNγ+cells (panel d), and Vγ9+Ki67+IFNγ+cells (panel e) was measured by flow cytometry (n=5/group). CD8+Ki67+IFNγ+cells: *p<0.05: +Pemb+Letr versus -Pemb-Letr (NCI-H1975 and NCI-H1650 tumors), ***p<0.001: +Pemb+Letr versus -Pemb-Letr (NCI-H1385 tumors). CD56+Ki67+IFNγ+cells: *p<0.05: +Pemb+Letr versus -Pemb-Letr (NCI-H1385, NCI-H1975 and NCI-H1650 tumors). Vγ9+Ki67+IFNγ+cells: *p<0.05: +Pemb+Letr versus -Pemb-Letr (NCI-H1385, NCI-H1975 and NCI-H1650 tumors) (ANOVA).