485P - A novel approach to identify subpopulation of CTCs with metastatic potential using sc-RNA-seq

Background

Integrins are known to play a crucial role in the adhesion, invasion and migration of tumor cells. Recent studies revealed the association of certain integrin subunits in organotropic metastasis of breast cancer. In this study, we investigated the biological properties associated with different integrin subunits gene expression in CTCs.

Methods

The study included 81 patients with non-metastatic invasive breast carcinoma of no special type (T1-4N0-3M0, all molecular subtypes). Single-cell cDNA libraries were prepared using the Single Cell 3′ Reagent Kit v3.1 and a 10x Genomics Chromium Controller. The ready cDNA libraries were sequenced on NextSeq 500 and NextSeq 2000 (Illumina, USA).

Results

CTCs were identified based on the expression of one or more epithelial markers: KRT5, 7, 8, 14, 18, EPCAM, MUC1 and CDH1 (E-cadherin). In total, expression of 14 integrin subunits was detected in CTCs: nine α-subunits and five β-subunits. In each cell, the combination of different integrin subunits was evaluated, and if complementary α and β-subunits gene expression detected in one cell, we suggested that CTCs potentially could synthesize functionally complete integrin heterodimers. Depending on the combinations of different subunits in CTCs, 455 cells were divided into nine groups. In each group, we assessed the proportion of cells with expression of genes relevant to the most important functions that determine the malignant potential of tumor cells: stemness, EMT, invasion. The most differences were observed in the group of CTCs potentially capable to express the α6β4 heterodimer. Namely, integrins α6- and β4-positive CTCs more frequent expressed stemness (ALDH1A1, CD133, KLF4, MYC) and invasion markers (RAC1, RHOC, ROCK, MMP1,2). A combination of epithelial (KRT 8, 10, 18, 19 and MUC1) with mesenchymal features (absence of EPCAM expression, expression of CDH2, VIM, ZEB1) indicates a hybrid state of EMT.

Conclusions

A novel approach to scRNA sequence analysis has identified subpopulations of CTCs with pronounced biological properties which can play role of "seeds". The simultaneously presence of integrin α6 and β4-subunits in CTCs was associated with stemness, EMT and invasiveness of CTCs in breast cancer patients.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

The study was supported by the Russian Science Foundation (grant #21-15-00140).

Disclosure

All authors have declared no conflicts of interest.