Mechanisms of fatal brain metastatic breast cancer (BMBC) are largely unknown. Similarly, properties and biomarker identification of circulating tumor cells (CTCs), the “seeds” of metastasis, remain elusive. Here we report novel strategies investigating CTCs isolated from peripheral blood mononuclear cells (PBMCs) of patients with BMBC, including the development and characterization of CTC lines. We identified a unique BMBC CTC signature (HER2+/EGFR+/HPSE+/Notch1+/EpCAM-) by investigating CTCs that could not be captured by the US Federal Drug Administration-approved Veridex CellSearch platform (EpCAM - negative CTCs). Second, we analyzed the invasive and metastatic competencies of isolated CTCs. Established CTC lines expressing the BMBC signature were highly invasive and capable to form brain metastasis in xenografts. Third, tumor cell morphologies of CTC - induced metastases closely resembled ones of pathologically assessed tumors of patients whose blood was source of CTCs. Fourth, the presence of BMBC signature protein – expressing cells was detected in CTC - induced BMBC. Collectively, we provide first-time evidence of human CTCs isolation and long-term growth, and the establishment of CTC lines. Further, we prove the metastatic competency of these CTC lines and necessity of the BMBC CTC signature to promote brain metastasis. These strategies and results can be of significance to develop new therapies against breast cancer metastasis in general, BMBC in particular.