Digital Quantitative Detection for Heterogeneous Protein and mRNA Expression Patterns in Circulating Tumor Cells

Area of Science:

• Oncology
• Molecular Biology
• Biotechnology

Background:

• Hepatocellular carcinoma (HCC) circulating tumor cells (CTCs) display heterogeneity due to epithelial-mesenchymal transition (EMT).
• Existing detection methods struggle with simultaneous multidimensional biomarker quantification, limiting insights into tumor biology and dynamics.
• A comprehensive understanding of HCC CTCs requires methods capable of analyzing both transcriptional and phenotypic characteristics.

Purpose of the Study:

• To introduce CTC Digital Simultaneous Cross-dimensional Output and Unified Tracking (d-SCOUT) technology for simultaneous quantification of HCC CTC biomarkers.
• To evaluate the diagnostic potential and clinical utility of d-SCOUT in assessing HCC.
• To demonstrate d-SCOUT's capability in monitoring treatment efficacy and metastatic risk.

Main Methods:

• Development of multi-real-time digital PCR (MRT-dPCR) and algorithms for unified quantification.
• Simultaneous measurement of Asialoglycoprotein Receptor (ASGPR), Glypican-3 (GPC-3), and Epithelial Cell Adhesion Molecule (EpCAM) proteins and their corresponding mRNAs.
• Integration of machine learning for clustering CTC characteristics and assessing metastatic risk.

Main Results:

• d-SCOUT achieved high sensitivity (LOD of 3.2 CTCs/mL) and reproducibility (mean %CV = 1.80-6.05%).
• Molecular signatures from HCC CTCs showed strong diagnostic potential (AUC=0.950, sensitivity=90.6%, specificity=87.5%) in 99 clinical samples.
• Machine learning analysis enabled clustering of CTC profiles and assessment of metastatic risk; dynamic tracking visualized treatment efficacy.

Conclusions:

• CTC d-SCOUT technology enables simultaneous quantification and interpretation of HCC CTC transcriptional and phenotypic biomarkers.
• The technology demonstrates significant diagnostic potential and utility in assessing HCC metastatic risk.
• d-SCOUT provides a powerful tool for dynamic monitoring of therapeutic effects, enhancing HCC management.