Circulating tumor cells (CTC), released by primary tumors into the bloodstream, represent a valuable source to inform on cancer heterogeneity, cancer progression, metastatic disease and therapy efficacy without the need of invasive tumor biopsies. However, the extreme rarity and heterogeneity of CTCs, occurring at genotypic, phenotypic and functional levels, poses a major challenge for the study of this population and explains the lack of standardized strategies of CTC isolation.

Lung cancer, the leading causes of cancer-related death worldwide, is a paradigmatic example of how CTC heterogeneity can undermine the clinical utility of this biomarker, since contrasting data have been reported using different isolation technologies.

Some evidences suggest that only a fraction of CTC, characterized by stem-like feature and partial epithelial–mesenchymal transition (EMT) phenotype, can sustain metastasis initiation.

Cancer stem cells (CSCs) have the potential to maintain primary tumors, initiate metastasis and escape both chemotherapy and immunotherapy treatments. Moreover, a close connection has been reported in several tumor types among hybrid phenotype, characterized by retention of epithelial and mesenchymal traits, acquisition of CSC feature and increased metastatic potential.

This review focuses on the phenotypic and functional heterogeneity of CTCs and the resulting implications for their isolation and clinical validation, especially in the setting of non-small cell lung cancer (NSCLC). In particular, we discuss the most relevant studies providing evidence for the presence and prognostic/predictive value of CTC subsets characterized by stem-like and hybrid EMT phenotype.

Despite technical and conceptual issues, tracking circulating CSCs has the potential to improve the prognostic/predictive value of CTCs in NSCLC setting and could provide novel insights into the comprehension of the metastatic process and identification of novel therapeutic targets.