Abstract 23P
Background
Metastasis is initiated by tumor cells detached from the primary tumor and entering the bloodstream. Circulating tumor cells (CTCs) are presented by clusters and individual cells which consist of phenotypically and genetically distinct subpopulations. However, only a few CTCs can survive in the bloodstream and form metastases. Deciphering CTC heterogeneity represents a promising instrument for the identification of the metastasis-initiating cells and developing therapeutic strategies against metastatic disease. In this study, we used single-cell RNA sequencing to assess CTC composition in patients with breast cancer.
Methods
CTCs were enriched by RosetteSep Human CD45 Depletion Cocktail (STEMCELL Technologies Inc.) and sequenced using Chromium Single Cell 3' Reagents v3.1 (10x Genomics) on NextSeq 500 instrument (Illumina). The data were analyzed using Cell Ranger and Loupe Browser (10x Genomics).
Results
In total, 100 CD45-negative CTCs expressing different epithelial genes (EpCAM, KRT5, 8, 17-19, and E-cadherin) have been identified. These CTCs were represented by two subpopulations one of which was enriched by NF-kB, Notch, and HIF-1 signaling pathways, whereas another one showed hematopoietic cell lineage features. Surprisingly, despite the CD45-negative selection of CTCs used in the study, we detected 67 circulating hybrid cells (CHCs) positive for CD45 and epithelial genes. CHCs were represented by two subpopulations expressing either CD4 or CD14. Both CTCs and CHCs demonstrated significant heterogeneity in the expression of mesenchymal (VIM and CDH2) and stem (CD44, CD133, and ALDH1) genes. However, only CTCs and CHCs with mesenchymal and stem features were enriched in signaling pathways involved in transcriptional misregulation in cancer and inflammation.
Conclusions
CTCs and CHCs are represented by transcriptionally distinct subpopulations of which mesenchymal stem-like cells are characterized by multiple cancer and inflammation signaling pathways.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Russian Science Foundation, grant number 19-75-30016.
Disclosure
All authors have declared no conflicts of interest.