Dissecting the Heterogeneity of Circulating Tumor Cells in Metastatic Breast Cancer: Going Far Beyond the Needle in the Haystack

Abstract

Although the enumeration of circulating tumor cells (CTC) defined as expressing both epithelial cell adhesion molecule and cytokeratins (EpCAM⁺/CK⁺) can predict prognosis and response to therapy in metastatic breast, colon and prostate cancer, its clinical utility (i.e., the ability to improve patient outcome by guiding therapy) has not yet been proven in clinical trials. Therefore, scientists are now focusing on the molecular characterization of CTC as a way to explore its possible use as a "surrogate" of tumor tissues to non-invasively assess the genomic landscape of the cancer and its evolution during treatment. Additionally, evidences confirm the existence of CTC in epithelial-to-mesenchymal transition (EMT) characterized by a variable loss of epithelial markers. Since the EMT process can originate cells with enhanced invasiveness, stemness and drug-resistance, the enumeration and characterization of this population, perhaps the one truly responsible of tumor recurrence and progression, could be more clinically useful. For these reasons, several devices able to capture CTC independently from the expression of epithelial markers have been developed. In this review, we will describe the types of heterogeneity so far identified and the key role played by the epithelial-to-mesenchymal transition in driving CTC heterogeneity. The clinical relevance of detecting CTC-heterogeneity will be discussed as well.

Keywords: circulating tumor cells; epithelial-to-mesenchymal transition; metabolism; metastatic breast cancer; spatial and temporal heterogeneity; stemness.

Figure 1

CTC in EMT can predict prognosis. CD45-depleted blood samples of MBC patients were assessed for the presence of nucleated cells (recognized by the blue staining of DAPI) expressing epithelial (green fluorescence), mesenchymal (red fluorescence) and leukocyte (cyan fluorescence) markers. As depicted on the left panels, besides CD45-positive leukocytes (L), 4 subsets of CD45-negative cells were detected: cells expressing only epithelial markers (E-CTC), cells co-expressing epithelial and mesenchymal markers (EM-CTC), cells expressing only mesenchymal markers (MES) and cells negative for all the assessed markers (NEG). Increased (↑) or decreased (↓) number or proportion of these subsets was significantly associated with specific clinical-pathological features and patient outcome. OS, overall survival; PFS, progression free survival; TN, triple negative; CNS, central nervous system [46].

Figure 2

EMT can enhance tumor heterogeneity driving, in interested cells, phenotypic as well as functional changes increasing biological aggressiveness.