The Identification and Characterization of Breast Cancer CTCs Competent for Brain Metastasis

Abstract

Brain metastatic breast cancer (BMBC) is uniformly fatal and increasing in frequency. Despite its devastating outcome, mechanisms causing BMBC remain largely unknown. The mechanisms that implicate circulating tumor cells (CTCs) in metastatic disease, notably in BMBC, remain elusive. Here we characterize CTCs isolated from peripheral blood mononuclear cells (PBMCs) of patients with breast cancer, and also develop CTC lines from three of these patients. In epithelial cell adhesion molecule (EpCAM)–negative CTCs, we identified a potential signature of brain metastasis comprising “brain metastasis selected markers (BMSM)” HER2+/EGFR+/HPSE+/Notch1+. These CTCs—which are not captured by the CellSearch platform because of their EpCAM negativity—were analyzed for cell invasiveness and metastatic competency in vivo. CTC lines expressing the BMSM signature were highly invasive and capable of generating brain and lung metastases when xenografted in nude mice. Notably, increased brain metastatic capabilities, frequency, and quantitation were detected in EpCAM− CTCs overexpressing the BMSM signature. The presence of proteins of the BMSM CTC signature was also detected in the metastatic lesions of animals. Collectively, we provide evidence of isolation, characterization, and long-term culture of human breast cancer CTCs, leading to the description of a BMSM protein signature that is suggestive of CTC metastatic competency to the brain.

Circulating Tumor Cells from Different Vascular Sites Exhibit Spatial Heterogeneity in Epithelial and Mesenchymal Composition and Distinct Clinical Significance in Hepatocellular Carcinoma

“…Recently, CTCs have garnered significant interest as the potential "seeds" that initiate cancer metastasis (2,3,30). Prior to the current study, the spatial heterogeneity of CTCs was only assumed on the basis of animal models.…”

“…The results of prior animal studies have suggested that trafficking of tumor cells via bloodstream is involved in the induction of metastatic disease (2)(3)(4)(5)(6). However, the evidence derived from animal models differs significantly from human cancer.…”

SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

“…Affibodies are a nonimmunoglobulin scaffold, consisting of a three-helix bundle, selected for high-affinity binding through phage display, and easily expressed in E. coli (21). The initial affibody we modified bound strongly and specifically to epidermal growth factor receptor (EGFR) (21), a tyrosine kinase expressed on many cancer cells and a target antigen in CTC isolation (22,23). We inserted KTag at the N terminus of the affibody and SpyTag at the C terminus (i.e., KTag-AffiEGFRSpyTag) so that covalent ligation between KTag and SpyTag would generate chains (Fig.…”

Role of Circulating Tumor Cells and Cell-Free Tumor Deoxyribonucleic Acid Fragments as Liquid Biopsy Materials in Breast Oncology

“…[12] For example, EpCAM-negative breast CTCs, which metastasize to brain have recently been identified. [50] Identification of these CTCs suggests that, EpCAM-negative CTC sub-populations may be present in LB samples of BC patients that need to be identified. In order to detect all heterogeneous types of breast CTCs novel antigenindependent CTC enrichment techniques need to be developed in the future.…”