Clinical Significance of Circulating Tumor Cell Induced Epithelial-Mesenchymal Transition in Patients with Metastatic Colorectal Cancer by Single-Cell RNA-Sequencing

SIMPLE SUMMARY: Circulating tumor cell (CTC) detection is a non-invasive and easy way to collect and separate single cells to use as tumor biomarkers. In this article, we show the feasibility and clinical value of RNA sequencing of CTCs at the single-cell level by exploiting CTC-FIND with SMART-Seq v4 technologies in patients with metastatic colorectal cancer (mCRC). CTCs were classified by epithelial, epithelial-mesenchymal transition (EMT), and stem cell-related gene expression. A patient who had EMT gene expressing CTCs showed significantly shorter PFS and OS regardless of epithelial CTCs presence. Therefore, the presence of CTCs expressing EMT-related genes at the single-cell level may be ofprognostic value and a potential biomarker for risk stratification in patients with refractory mCRC who are considered to have highly heterogeneous features. ABSTRACT: Background: Circulating tumor cells (CTCs) are a prognostic marker in patients with metastatic colorectal cancer (mCRC). However, little is known about the characterization of CTCs in mCRC at the single-cell level using RNA sequencing. The purpose of this study was to validate the capability to detect and isolate single CTCs for single-cell RNA sequencing (scRNA-seq) and to identify clinical significance at a single CTC level. Methods: Single CTCs from 27 mCRC patients were collected by CTC-FIND, which is comprised of filter separation and immunomagnetic depletion to collect ultra-pure CTC samples. To address tumor heterogeneity, CTCs were collected without relying on any traditional CTC markers, such as epithelial and mesenchymal cell antigens, and were undertaken by scRNA-seq using SMART-Seq v4. Results: We identified 59 single CTCs which were classified into four groups by epithelial, epithelial-mesenchymal transition (EMT) and stem cell-related gene expression. Patients receiving second or later-line treatment who had EMT gene expressing CTCs had a significantly shorter PFS and OS. Conclusions: Exploiting CTC-FIND with SMART-Seq v4 showed that scRNA-seq of CTCs may shed new insight into tumor heterogeneity of mCRC and that the presence of CTCs expressing EMT-related genes at the single-cell level could have prognostic value in mCRC patients.