Breast cancer brain metastases (BM) affect younger women disproportionally, including those lacking estrogen receptor (ER), progesterone receptor, and HER2 (known as triple-negative breast cancer; TNBC). Previous studies in preclinical models showed that pre-menopausal levels of estradiol (E2) promote TNBC-BM through incompletely understood mechanisms involving reactive astrocytes. Herein, a novel mechanism involving E2-dependent upregulation of brain-derived neurotrophic factor (BDNF) in astrocytes, and subsequent activation of tumor cell tropomyosin kinase receptor B (TrkB), is identified. E2 increased experimental BM of TNBC 4T1BR5 and E0771 cells by 21 and 3.6 fold, respectively, compared to E2-depleted mice. ERα + reactive astrocytes were found at early and late stages of BM, and E2 upregulated BDNF in ER + reactive astrocytes in vitro and in vivo. TrkB was expressed in TNBC brain-trophic cell lines, BM-patient-derived xenografts, and breast cancer BM. Conditioned media from E2-treated astrocytes (CM-E2) activated TrkB and downstream AKT, ERK, and PLC-γ signaling in TNBC cells, increasing their invasiveness and tumor-initiating capability in vitro. The promotion of BM by E2-activated astrocytes was found to be more complex, involving feedback loops and other receptor tyrosine kinases. In 4T1BR5 cells, there was a positive feedback loop whereby astrocytic BDNF induced cancer cell BDNF translation. Upregulation of cancer cell BDNF was required to promote full invasiveness of 4T1BR5 in response to CM-E2, and was observed in brain metastatic cells in E2-treated mice in vivo. Moreover, the non-competitive BDNF/TrkB inhibitor ANA-12 reduced E2-induced 4T1BR5 BM to levels similar to OVX mice. BDNF also activated EGFR in TrkB + EGFR + TNBC cells, suggesting that E2 action through astrocytes activates redundant pathways promoting BM. These findings have important therapeutic implications, as they provide a rationale to use E2-depletion therapies or TrkB inhibitors to prevent or delay development of BM in younger women.

Purpose: Patients with human EGFR2-positive (HER2 þ ) breast cancer have a high incidence of brain metastases, and trastuzumab emtansine (T-DM1) is often employed. Stereotactic radiosurgery (SRS) is frequently utilized, and case series report increased toxicity with combination SRS and T-DM1. We provide an update of our experience of T-DM1 and SRS evaluating risk of clinically significant radionecrosis (CSRN) and propose a mechanism for this toxicity.Experimental Design: Patients with breast cancer who were 45 years regardless of HER2 status or had HER2 þ disease regardless of age and underwent SRS for brain metastases were included. Rates of CSRN, SRS data, and details of T-DM1 administration were recorded. Proliferation and astrocytic swelling studies were performed to elucidate mechanisms of toxicity.Results: A total of 45 patients were identified; 66.7% were HER2 þ , and 60.0% were 45 years old. Of the entire cohort, 10 patients (22.2%) developed CSRN, 9 of whom received T-DM1. CSRN was observed in 39.1% of patients who received T-DM1 versus 4.5% of patients who did not. Receipt of T-DM1 was associated with a 13.5-fold (P ¼ 0.02) increase in CSRN. Mechanistically, T-DM1 targeted reactive astrocytes and increased radiation-induced cytotoxicity and astrocytic swelling via upregulation of Aquaporin-4 (Aqp4).Conclusions: The strong correlation between development of CSRN after SRS and T-DM1 warrants prospective studies controlling for variations in timing of T-DM1 and radiation dosing to further stratify risk of CSRN and mitigate toxicity. Until such studies are completed, we advise caution in the combination of SRS and T-DM1.

Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8–12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings.

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Purpose: The survival of women with brain metastases (BM) from breast cancer remains very poor with over 80% dying within a year of their diagnosis. Here we define the function of IL13Rα2 in outgrowth of breast cancer brain metastases (BCBM) in vitro and in vivo, and postulate IL13Rα2 as a suitable therapeutic target for BM. Experimental design:We performed IHC staining of IL13Rα2 in BCBM to define its prognostic value.Using inducible-shRNAs in TNBC and HER2+ breast-brain metastatic models we assessed IL13Rα2 function in vitro and in vivo. We performed RNAseq and functional studies to define the molecular mechanisms underlying IL13Rα2 function in BCBM.Results: High IL13Rα2 expression in BCBM predicted worse survival after BM diagnoses. IL13Rα2 was essential for cancer-cell survival, promoting proliferation while repressing invasion. IL13Rα2 KD resulted in FAK downregulation, repression of cell cycle and proliferation mediators and upregulation of Ephrin B1 signaling. Ephrin-B1 (i) promoted invasion of BC cells in vitro, (ii) marked micrometastasis and invasive fronts in BCBM, (iii) predicted shorter disease-free survival (DFS) and BM-free survival (BMFS) in breast primary tumors known to metastasize to the brain. In experimental metastases models, which bypass early tumor invasion, downregulation of IL13Rα2 prior or after tumor seeding and brain intravasation decreased BMs, suggesting that IL13Rα2 and the promotion of a proliferative phenotype is critical to BM progression.Conclusions: Non-genomic phenotypic adaptations at metastatic sites are critical to BM progression and patients' prognosis. This study opens the road to use IL13Rα2-targeting as a therapeutic strategy for BM.Research.