Abstract
Uncovering CTCs phenotypes offer the promise to dissect their heterogeneity related to metastatic competence. CTC survival rates are highly variable and this can lead to many questions as yet unexplored properties of CTCs responsible for invasion and metastasis vs dormancy. We isolated CTC subsets from peripheral blood of patients diagnosed with or without breast cancer brain metastasis. CTC subsets were selected for EpCAM negativity but positivity for CD44+/CD24− stem cell signature; along with combinatorial expression of uPAR and int β1, two markers directly implicated in breast cancer dormancy mechanisms. CTC subsets were cultured in vitro generating 3D CTC tumorspheres which were interrogated for biomarker profiling and biological characteristics. We identified proliferative and invasive properties of 3D CTC tumorspheres distinctive upon uPAR/int β1 combinatorial expression. The molecular characterization of uPAR/int β1 CTC subsets may enhance abilities to prospectively identify patients who may be at high risk of developing BCBM.
Highlights
Tumor relapse is a significant clinical problem which is relevant in breast cancer where patients are asymptomatic because disseminated cells appear to become dormant, are undetectable by clinical tools, and residual disease remains dormant for periods longer than 20 years[1,2]
Transcriptional profiles of circulating tumor cells (CTCs) directly isolated from breast cancer patients are distinct from ones of breast cancer cell lines that are widely used for drug discovery, a finding which raises issues regarding the appropriateness of using cell lines to model breast cancer therapy[7,8]
We have previously reported the discovery of CTCs that do not express the common carcinoma epithelial cell adhesion molecule (EpCAM-negative CTCs) and possess high competence to generate breast cancer brain metastasis (BCBM) in xenografts[13]
Summary
Tumor relapse is a significant clinical problem which is relevant in breast cancer where patients are asymptomatic because disseminated cells appear to become dormant, are undetectable by clinical tools, and residual disease remains dormant for periods longer than 20 years[1,2]. We report the isolation of subsets of EpCAM-negative breast cancer CTCs containing stem-cell properties (CD44+/CD24−) by multiparametric flow cytometry with a combinatorial uPAR and int β 1 expression and their abilities to grow long-term in vitro. We characterized CTC subsets possessing six cell surface expression markers (CD45−/EpCAM-negative/CD44+/CD24−/uPAR+/−/int β 1+/−) to determine the expression profiling of candidate genes related to breast cancer and embryonic stem-cell pathways and demonstrate their tumor origin as putative CTCs. Third, we investigated CTC subsets for cell adhesion, proliferation properties, and for subset abilities to generate in vitro 3D CTC tumorspheres (3D-spheroids) and invade into extracellular matrix. We provide first-time evidence for the isolation of intra/inter-patient EpCAM-negative, uPAR/int β 1 CTCs subsets with distinct capabilities for long-term in vitro growth; along with mechanistic link of these CTC subsets to cell adhesion, proliferative and invasive properties relevant to BCBM onset
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