Circulating tumor cell based ex vivo platform for characterizing circulating hybrid cell dynamics in multiple cancer types

Circulating hybrid cells (CHCs) arising from tumor-leukocyte fusion may contribute to metastasis and therapy resistance through enhanced DNA repair, immune evasion, and acquired migratory capacity. However, the prevalence, expansion dynamics, and clinical significance of CHCs across cancer types remain poorly characterized.Circulating tumor cells (CTC)-enriched samples from patients with breast cancer, NSCLC, and colorectal cancer were analyzed through extended ex vivo culture. CHCs were identified by dual pan-cytokeratin and CD45 immunofluorescence combined with morphological criteria. An AI-assisted image analysis platform is being developed for automated CHC quantification and characterization. A subset underwent parallel culture with cisplatin to assess chemoresistance and clinical correlations with treatment history and disease status were evaluated. Initial validation demonstrates reliable discrimination between immune cells and non-immune cells (potential CTCs/CHCs), enabling systematic quantification. Dual immunofluorescence staining revealed distinct CHC populations characterized by co-expression of epithelial (pan-CK + ) and hematopoietic (CD45 + ) markers with large cell size. CHCs were rarely observed in early culture across all cancer types but became prominent by extended culture, indicating progressive enrichment over time. Complete quantitative analysis with statistical validation is ongoing. These findings establish extended ex vivo culture as a platform for investigating CHC biology, assessing drug sensitivity, and determining clinical relevance.