Heterogeneity of Circulating Tumor Cell Neoplastic Subpopulations Outlined by Single-Cell Transcriptomics

Abstract

Fatal metastasis occurs when circulating tumor cells (CTCs) disperse through the blood to initiate a new tumor at specific sites distant from the 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. The enumeration of CTCs allows an estimation of the overall metastatic burden in breast cancer patients, but challenges regarding CTC heterogeneity and metastatic propensities persist, and their decryption could improve therapies. CTCs from metastatic breast cancer (mBC) patients were captured using the RareCyteTM Cytefinder II platform. The Lin− and Lin+ (CD45+) cell populations isolated from the blood of three of these mBC patients were analyzed by single-cell transcriptomic methods, which identified a variety of immune cell populations and a cluster of cells with a distinct gene expression signature, which includes both cells expressing EpCAM/CK (“classic” CTCs) and cells possessing an array of genes not previously associated with CTCs. This study put forward notions that the identification of these genes and their interactions will promote novel areas of analysis by dissecting properties underlying CTC survival, proliferation, and interaction with circulatory immune cells. It improves upon capabilities to measure and interfere with CTCs for impactful therapeutic interventions.

TLR4 and pSTAT3 Expression on Circulating Tumor Cells (CTCs) and Immune Cells in the Peripheral Blood of Breast Cancer Patients: Prognostic Implications

“…This approach allows the analysis of the entire peripheral immune cell compartment with reduced cost and technical requirements. Nevertheless, future studies utilizing transcriptomic, and flow cytometric approaches would help to identify specific peripheral immune alterations associated with TLR4 and/or pSTAT3 expression on PBMCs, in order to delineate their role in the peripheral immune response in BC [ 61 , 62 ].…”

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

“…Probably, the scRNA-seq data is not the most appropriate resource to assess CNAs. Some previous scRNA-seq studies identified CTCs and differentiated them from normal peripheral blood mononuclear cells but only in metastatic BC patients 21,22 . It is well known that CTCs are significantly higher in advanced than in early cancers 37 .…”

“…Single-cell RNA sequencing (scRNA-seq) is an effective instrument for deciphering cell heterogeneity, identifying new cell populations, and analyzing their phenotypes. Previous scRNA-seq studies showed that breast CTCs coexpress epithelial and mesenchymal markers 7 and several specific transcripts 21 and can be in the state of estrogen responsiveness/increased proliferation or reduced proliferation/EMT 22 . Tumor hybrid cells exhibit gene expression patterns distinct from parental cells but retain the expression of critical genes of each parental cell 17 .…”

Single-Cell RNA Sequencing Uncovers Heterogeneous Circulating Tumor Cell Subsets in Breast Cancer

“…Pauken M.C. and co-authors [ 17 ], in an effort to describe the whole spectrum of CTC subsets that contribute to CTC heterogeneity and metastatic potency, utilized the transcriptomic analysis of white blood cells from 21 metastatic BC patients. Their experimental strategy included the isolation of cells based on lineage expression through flow cytometry, followed by analysis using the cutting-edge technologies of RNA-seq and scRNA-seq.…”

“…To summarize, Pauken M.C. et al [ 17 ], performed a comprehensive characterization of CTCs in BC patients by employing an unbiased transcriptomic analysis of Lin– and Lin+ cells via scRNA-seq. Their approach allowed the identification of a “CTC-candidate” cluster with a distinct gene expression signature enriched with genes promoting cell adhesion, proliferation, cell-to-cell communication, as well as with genes for TFs, oncogenes, and genes that drive EMT and CSCs, which collectively may foster cell migration and metastatic dissemination.…”