Introduction: Triple negative breast cancers (TNBC, which lack estrogen receptor (ER), progesterone receptor and HER2), occur three times more frequently in premenopausal women and metastasize expeditiously to brain and lungs. Clinical and experimental evidence shows that estrogen, the main premenopausal ovarian hormone, promotes metastases of TNBC, through their action on the tumor microenvironment. We have shown that 17-β-Estradiol (E2) promotes experimental brain metastases (BM) of TNBC cells by inducing estrogen-receptor positive (ER+) astrocytes in the brain niche to secrete growth factors that promote cancer cell migration, invasion and growth. Given that 53% of pre-menopausal women with TNBC develops BM and 80% die within a year, it is now critical to define whether blocking estrogen signaling could have therapeutic value to prevent or delay progression of BM. Results: To assess whether E2-depletion could prevent brain metastatic colonization, brain trophic sublines of human TNBC (231BR), murine TNBC (4T1BR5), a non-selected TN cell line syngeneic to C57BL mice (E0771-GFP-luc) were intracardially (ic) injected two-days post-endocrine initiation, in ovariectomized (OVX) female mice treated with a) E2 (levels equivalent to those found in pre-menopausal women), b) placebo (OVX), or c) the aromatase-inhibitor (AI) letrozole (OVX + Letrozole) to block brain and peripheral E2-synthesis. BM were quantified histologically, via MRI or ex-vivo imaging of brains at euthanasia. E2-treated mice injected with E0771-GFP-luc cells showed an average of 6.6± 3.5 metastatic clusters per mice, compared to 3.6 ± 3.2 and 1.8 ± 1.8 in OVX mice alone or OVX + letrozole treated mice (a 3.6 fold increase, P=0.0013, E2 vs OVX + Let). Imaging of excised brains showed comparable results, with a mean total photon flux of 5.1 × 106 ± 5.3 × 106 in E2 vs 1.1 × 106 ± 1.5 × 106 in OVX + letrozole (P=0.0056). Similar preventive effects of E2-depletion were found in 4T1BR5 and 231BR-cell injected mice, using histological quantification and MRI as endpoints. To assess whether E2-depletion therapies could decrease brain metastatic progression in a therapeutic model mimicking the clinical setting, E0771-GFP-luc cells were injected in OVX-mice that had been supplemented with E2 and metastasis allowed to develop for 6 days. Mice were then randomized to a) E2, b) E2-withdrawal (E2WD) or c) E2WD+ letrozole, and then all mice were irradiated (whole brain irradiation, single-dose, 15 Gy) to mimic current standard of care for BMs. All mice were euthanized 10 days later, when E2-treated mice showed signs of CNS impairment. Mice that received E2WD+letrozole showed the lowest BM burden compared to mice that remained in E2-treatments, as assessed by histological quantification (0.7 ± 1.2 vs 6.5 ± 5.3 metastatic clusters, respectively, P=0.0132) and ex-vivo brain imaging (mean total-photon flux 3.4 × 105 ± 4.1 × 105 in E2WD + let vs 3.1 × 106 ± 1.4 × 106 in E2, P=0.037). Brain metastasis in TNBC often occur in women who also present with systemic metastatic disease, and E0771-GFP-luc cells can colonize multiple organs following ic injection. Thus, non-brain metastatic burden (systemic metastasis) was measured by in vivo imaging before euthanasia in this cohort. Unlike brain metastasis, E2WD or E2WD+letrozole showed no statistically significant effect on systemic metastatic burden (mean total flux 1.41 × 106 ± 0.9 × 106 in E2, vs 0.54 × 106 ± 2.6 × 106 in E2WD vs 0.79 × 106 ± 1.0 × 106 in E2WD+let, P=0.416), supporting the notion that brain-specific rather than systemic mechanisms drive progression of brain metastasis in response to E2. Conclusions: These studies have important clinical implications as they provide pre-clinical evidence that ovarian suppression in combination with AIs can prevent and delay progression of established brain metastasis in younger women with TNBC.

Citation Format: Diana M. Cittelly, Maria J. Contreras-Zarate, Ricaurte A. Marquez-Ortiz, Nicole Day, Bebhinn Nagle, Ryan Ormond, Virginia F. Borges, Peter Kabos. Estrogen-depletion therapies prevent and delay progression of brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-03.