Related Experiment Video
Updated: Mar 1, 2026
Microfluidics-based High-throughput Circulating Tumor Cell Sorting and Single-cell Sequencing Technology
Published on: November 14, 2025
Development of a Protocol for Single-Cell Analysis of Circulating Tumor Cells in Patients with Solid Tumors
Carolina Reduzzi1, Rosita Motta1, Giulia Bertolini2
1Biomarker Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, 20133, Milano, Italy.
Abstract:
Genomic characterization of circulating tumor cells (CTCs) enables the monitoring of tumor progression and of adaption occurring during treatment. CTC molecular characterization represents indeed a precious tool to implement in the clinical practice for better dealing with acquired resistance to systemic treatment and tumor evolution. Unfortunately CTCs are very rare and enrichments from blood samples and subsequent identification of these cells are technically very challenging. We describe here the main steps leading to the development of a technical protocol for visualization, enumeration and recovery of single CTCs exploiting the recently developed DEPArray™platform. Our description of the technical workflow starts with evaluation of pre-analytical aspects related to blood sample collection warning about the possible effects on immunoreactivity profiles which may bias the interpretation. Subsequently, other CTC-enrichment approaches are critically discussed and compared in relation to their performances with the DEPArray™. Identification of CTCs represents another critical point due to their heterogeneity and due to the still-to-be clarified role of different subpopulations, typically epithelial, mesenchymal or mixed. Finally, issues related to single cell analysis are illustrated. The chapter ends with an overview of results obtained on real clinical samples which support the reliability of the protocol and its transferability to the daily clinical routine.
Insights
Genomic characterization of circulating tumor cells (CTCs) is crucial for monitoring cancer progression and treatment adaptation. This study details a reliable protocol for CTC visualization, enumeration, and recovery using the DEPArray™ platform for improved clinical application.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Circulating tumor cells (CTCs) offer insights into tumor progression and treatment resistance.
- Analyzing CTCs is challenging due to their rarity and heterogeneity.
- Current methods require robust protocols for accurate CTC characterization.
Purpose of the Study:
- To develop and validate a technical protocol for the visualization, enumeration, and recovery of single CTCs.
- To evaluate the DEPArray™ platform for CTC analysis.
- To address pre-analytical, enrichment, identification, and single-cell analysis challenges in CTC research.
Main Methods:
- Development of a workflow for CTC analysis using the DEPArray™ platform.
- Evaluation of pre-analytical factors affecting CTC immunoreactivity.
- Critical comparison of CTC enrichment techniques with DEPArray™.
- Addressing CTC heterogeneity (epithelial, mesenchymal, mixed) during identification.
- Illustration of single-cell analysis methodologies.
Main Results:
- A reliable protocol for CTC visualization, enumeration, and recovery was established.
- The DEPArray™ platform demonstrated effectiveness in handling CTCs.
- Pre-analytical considerations and enrichment strategies were optimized.
- Challenges in CTC identification due to heterogeneity were addressed.
- Successful application of the protocol on clinical samples was demonstrated.
Conclusions:
- The developed protocol ensures reliable CTC analysis, supporting clinical implementation.
- The DEPArray™ platform facilitates accurate enumeration and recovery of single CTCs.
- This workflow aids in managing treatment resistance and understanding tumor evolution through CTC genomic characterization.
More Related Videos
05:17Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment
Published on: May 14, 2019
14:14Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications
Published on: February 28, 2014