Decoding CTCs in osteosarcoma: the molecular journey from initial tumor to metastasis

Abstract

Abstract Metastatic relapse remains the main cause of treatment failure in pediatric osteosarcoma (OS), as conventional imaging often misses minimal residual or low-burden disease. Circulating tumor cells (CTCs) represent a promising non-invasive biomarker for monitoring tumor progression and heterogeneity. We developed an integrated workflow to isolate, enrich, and molecularly characterize CTCs from six pediatric OS patients. Peripheral blood mononuclear cells were enriched using the Parsortix system. CTCs were classified by immunofluorescence using epithelial (EpCAM, CK8/18/19) and mesenchymal (Vimentin, TWIST1) markers. Single cells were isolated via DEPArray, followed by whole-genome amplification and low-pass sequencing to assess copy number alterations (CNAs). In one patient, whole-exome sequencing (WES) was performed on bulk CTCs, primary tumor, and metastatic tissue to confirm tumor origin and identify shared somatic mutations. Across the cohort, 908 CTCs were identified, predominantly mesenchymal, with a smaller epithelial subset showing higher genomic complexity and CNA burden. WES confirmed overlapping mutations and CNAs among CTCs, primary, and metastatic tumors. Epithelial CTCs exhibited greater genomic instability than mesenchymal cells. Phenotype correlations with treatment response suggested potential links to histological necrosis. This study provides the first comprehensive phenotypic and genomic characterization of CTCs in pediatric OS, confirming their neoplastic nature and molecular heterogeneity. These findings support CTC-based monitoring as a minimally invasive tool for guiding personalized therapy and improving disease surveillance in high-risk OS patients.