Heterogeneity of circulating epithelial cells in breast cancer at single-cell resolution: identifying tumor and hybrid cells

Abstract

Circulating tumor cells (CTCs) and tumor hybrid cells, being the leading players in metastasis, have prognostic relevance and are potential antimetastatic targets. CTCs are identified as epithelial-positive and CD45 (leukocyte)-negative cells, whereas tumor hybrid cells usually have epithelial and leukocyte components. However, epithelial and hybrid cells are also observed in healthy subjects that complicate the detection of CTCs and tumor hybrid cells in cancer patients. This study evaluated the diversity of CD45-negative and CD45-positive circulating epithelial cells (CECs) in breast cancer patients (n=20) using single-cell RNA sequencing. We also tried to detect CTCs and tumor hybrid cells among CD45− CECs and CD45+ CECs by analyzing DNA ploidy since aneuploidy is a hallmark of cancer cells. Aneuploid cells were predominantly detected in CD45− CECs, whereas CD45+ CECs were mainly diploid. Most transcriptional cancer features, including many cancer-associated signaling pathways, were specific to aneuploid cells of one CD45− CEC cluster. These cells were also enriched by platelet genes and signaling pathways that may indicate their increased potential to adhere with thrombocytes. In CD45+ CECs, only one cluster had many aneuploid cells that were surprisingly negative for transcriptional cancer features. Thus, CD45− and CD45+ CECs are highly heterogeneous in breast cancer patients and consist of transcriptionally-distinct cell populations with varying degrees of DNA ploidy where aneuploid cells are likely CTCs and tumor hybrid cells.