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.