Heterogeneity of Circulating Epithelial Cells in Breast Cancer at Single‐Cell Resolution: Identifying Tumor and Hybrid Cells

Abstract Circulating tumor cells and hybrid cells formed by the fusion of tumor cells with normal cells are leading players in metastasis and have prognostic relevance. This study applies single‐cell RNA sequencing to profile CD45‐negative and CD45‐positive circulating epithelial cells (CECs) in nonmetastatic breast cancer patients. CECs are represented by transcriptionally‐distinct populations that include both aneuploid and diploid cells. CD45 − CECs are predominantly aneuploid, but one population contained more diploid than aneuploid cells. CD45 + CECs mostly diploid: only two populations have aneuploid cells. Diploid CD45 + CECs annotated as different immune cells, surprisingly harbored many copy number aberrations, and positively correlated to tumor grade. It is noteworthy that cancer‐associated signaling pathways areabundant only in one aneuploid CD45 − CEC population, which may represent an aggressive subset of circulating tumor cells. Thus, CD45 − and CD45 + CECs are highly heterogeneous in breast cancer patients and include aneuploid cells, which are most likely circulating tumor and hybrid cells, respectively, and diploid cells. DNA ploidy analysis can be an effective instrument for identifying tumor and hybrid cells among CECs. Further follow‐up study is needed to determine which subsets of circulating tumor and hybrid cells contribute to breast cancer metastasis.