Complex Analysis of Single-Cell RNA Sequencing Data.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool for studying the physiology of normal and pathologically altered tissues. This approach provides information about molecular features (gene expression, mutations, chromatin accessibility, etc.) of cells, opens up the possibility to analyze the trajectories/phylogeny of cell differentiation and cell-cell interactions, and helps in discovery of new cell types and previously unexplored processes. From a clinical point of view, scRNA-seq facilitates deeper and more detailed analysis of molecular mechanisms of diseases and serves as a basis for the development of new preventive, diagnostic, and therapeutic strategies. The review describes different approaches to the analysis of scRNA-seq data, discusses the advantages and disadvantages of bioinformatics tools, provides recommendations and examples of their successful use, and suggests potential directions for improvement. We also emphasize the need for creating new protocols, including multiomics ones, for the preparation of DNA/RNA libraries of single cells with the purpose of more complete understanding of individual cells.

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

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.