American Association for Cancer Research
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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

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journal contribution
posted on 2023-10-02, 07:41 authored by Dario P. Anobile, Iris C. Salaroglio, Fabrizio Tabbò, Sofia La Vecchia, Muhlis Akman, Francesca Napoli, Maristella Bungaro, Federica Benso, Elisabetta Aldieri, Paolo Bironzo, Joanna Kopecka, Francesco Passiglia, Luisella Righi, Silvia Novello, Giorgio V. Scagliotti, Chiara Riganti

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).


Fondazione AIRC per la ricerca sul cancro ETS (AIRC)

Cassa di Risparmio di Torino

Compagnia di San Paolo (CSP)

European Cooperation in Science and Technology (COST)



The response to immune checkpoint inhibitors (ICI) often differs between genders in non–small cell lung cancer (NSCLC), but metanalyses results are controversial, and no clear mechanisms are defined. We aim at clarifying the molecular circuitries explaining the differential gender-related response to anti–PD-1/anti–PD-L1 agents in NSCLC. We prospectively analyzed a cohort of patients with NSCLC treated with ICI as a first-line approach, and we identified the molecular mechanisms determining the differential efficacy of ICI in 29 NSCLC cell lines of both genders, recapitulating patients’ phenotype. We validated new immunotherapy strategies in mice bearing NSCLC patient-derived xenografts and human reconstituted immune system (“immune-PDXs”). In patients, we found that estrogen receptor α (ERα) was a predictive factor of response to pembrolizumab, stronger than gender and PD-L1 levels, and was directly correlated with PD-L1 expression, particularly in female patients. ERα transcriptionally upregulated CD274/PD-L1 gene, more in females than in males. This axis was activated by 17-β-estradiol, autocrinely produced by intratumor aromatase, and by the EGFR-downstream effectors Akt and ERK1/2 that activated ERα. The efficacy of pembrolizumab in immune-PDXs was significantly improved by the aromatase inhibitor letrozole, which reduced PD-L1 and increased the percentage of antitumor CD8+T-lymphocytes, NK cells, and Vγ9Vδ2 T-lymphocytes, producing durable control and even tumor regression after continuous administration, with maximal benefit in 17-β-estradiol/ERα highfemale immune-xenografts. Our work unveils that 17-β-estradiol/ERα status predicts the response to pembrolizumab in patients with NSCLC. Second, we propose aromatase inhibitors as new gender-tailored immune-adjuvants in NSCLC.See related commentary by Valencia et al., p. 3832

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