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Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P

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.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

Symptomatic brain metastases develop in 10 to 16% of patients with metastatic breast cancer and the prognosis of these patients is dismal with a mortality rate of near 80% within 1 year of diagnosis. Understanding the mechanisms by which breast cancer cells adapt to the brain microenvironment is key to design therapies that could be used to prevent or treat brain metastases. To date, the field has relied almost exclusively on intracardiac (ic) injection of brain-homing cells derived from highly aggressive triple negative (TN) MDA-MB-231 and HER2+ BT474 (BT474BR) cell lines to study these interactions. Although these are well characterized models, these are far from representing the heterogeneity observed in breast tumors and their metastases. Purpose: The goal of this study was to develop and characterize novel human brain metastases patient-derived xenografts (BM-PDX) to study the biology of brain metastases and to serve as tools for testing novel therapeutic approaches. We have also developed a labeling method to tag these PDXs with traceable markers for in vivo studies. Experimental procedures: Metastatic tissue was removed from brain parenchyma during surgery (n=5) or from cerebral spinal fluid (CSF) via catheter (n=1) from consenting donors harboring TN (n=3) or HER2+ (n=3) metastatic breast cancer. Tissue was immediately implanted in the mammary fat pad of NOD/SCID/GAMMA female mice and tumor expanded as solid tumors (BM-PDX). Upon reaching a volume of >200 mm3, partitioned pieces were transplanted into new recipient mice and growth kinetics measured. In order to track metastatic spread using PDXs, we developed a protocol to label PDX-dissociated cells with GFP-luciferin followed by re-implantation in mice. Tumor sections before and after labeling were immunostained for hormone receptors (estrogen, progesterone and androgen receptors –ER, PR, AR), HER2, and the basal cell marker cytokeratin 5 (CK5). These were scored by a pathologist. Dissociated cells from an established BM-PDX were cultured under different conditions in vitro, to generate a BM-derived cell line. Gene expression profiling was performed in some PDX before and after GFP-luciferase labeling, and in derived cell lines. Dissociated cells from BM-PDXs (E22-1) were injected intracardially (n=5) to measure their ability to colonize the brain in an experimental model of brain metastasis. Summary: Three of 3 TN brain metastases were successfully established as transplantable PDXs, while 0 of 3 HER2+ tumors grew in mice. Immunohistochemical analyses demonstrated that BM-PDXs retain morphology and markers comparable to those of donor samples. Labeled tumors grew in the mammary fat pad without spontaneous metastatic spread as measured via in vivo imaging (IVIS). Intracardiac injection of dissociated cells from BM-PDX E22-1 resulted in micro or macrometastases in 5/5 (100%) injected mice, indicating that BM-PDXs remain capable of colonizing the organ of origin. Metastases developed in brain parenchyma, surrounding vessels, with associated microglia and astroglia activation, similar to what is observed in human brain metastases. BM-PDX dissociated cells were cultured under different conditions to derive BM-cell lines. Two cell lines have been derived from a TN BM-PDX (F2-6) after in vitro culture using brain-like media or primary cell media. RNAseq analyses to fully characterize the genetic makeup of BM-PDX and cell line derivatives are currently in progress. Conclusions: We have developed novel PDXs derived from breast cancer brain metastases, which retain their ability to colonize the brain when disseminated in circulation. These novel GFP-luciferase labeled xenografts and cell lines, represent unique tools to study brain metastasis biology and to test novel therapeutic treatments for brain metastasis in clinically relevant models. Citation Format: Colton Hanna, Britta M. Jacobsen, Carol A. Sartorius, Peter Kabos, Kevin Lillehei, David R. Ormond, Michael Graner, Virginia F. Borges, Diana M. Cittelly. Development of novel breast cancer brain-metastases-derived xenografts and cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A09.