Abstract Fatal metastasis occurs when circulating tumor cells (CTCs) disperse through the blood to initiate a new tumor at specific sites distant from their primary tumor. CTCs have been classically defined as nucleated cells positive for epithelial-cell adhesion molecule and select cytokeratins (EpCAM/CK/DAPI), while negative for the common lymphocyte marker CD45. Although the enumeration of CTCs allowed the estimation of overall metastatic burden in breast cancer patients, the thorough interrogation of circulatory neoplastic cells at single-cell level has never been achieved, and is considered critical to improve therapies. We report that CTCs from metastatic breast cancer (mBC) patients were identified using the RareCyteTM Cytefinder II platform. Second, comprehensive transcriptomic analyses at single-cell level of Lin- and Lin+ blood cell populations isolated from patients identified a unique and heterogeneous cluster of cells which expressed EpCAM/CK and an array of genes not previously associated with classical CTCs. The differences in gene expression suggest that the neoplastic cell population in blood is distributed across heterogeneous cell profiles. This study proposes that the identification of these genes and neoplastic states will promote novel areas of analysis dissecting properties underlying CTC survival, proliferation, and interactions with immune cells. It improves abilities to measure and interfere with CTC states, their plasticity, and CTC functionalities for impactful therapeutic interventions. Fatal metastasis occurs when circulating tumor cells (CTCs) disperse through the blood to initiate a new tumor at specific sites distant from their primary tumor. CTCs have been classically defined as nucleated cells positive for epithelial-cell adhesion molecule and select cytokeratins (EpCAM/CK/DAPI), while negative for the common lymphocyte marker CD45. Although the enumeration of CTCs allowed the estimation of overall metastatic burden in breast cancer patients, the thorough interrogation of circulatory neoplastic cells at single-cell level has never been achieved, and is considered critical to improve therapies. Here we report that CTCs from metastatic breast cancer (mBC) patients were identified using the RareCyteTM Cytefinder II platform. Second, comprehensive transcriptomic analyses at single-cell level of Lin- and Lin+ blood cell populations isolated from patients identified a unique and heterogeneous cluster of cells which expressed EpCAM/CK and an array of genes not previously associated with classical CTCs. The differences in gene expression suggest that the neoplastic cell population in blood is distributed across heterogeneous cell profiles. This study proposes that the identification of these genes and neoplastic states will promote novel areas of analysis dissecting properties underlying CTC survival, proliferation, and interactions with immune cells. It improves abilities to measure and interfere with CTC states, their plasticity, and CTC functionalities for impactful therapeutic interventions. Citation Format: Dario Marchetti. Heterogeneity of circulating tumor cell neoplastic subpopulations interrogated by single-cell transcriptomics [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P155.