Bridging the gap: a genetically validated avian chorioallantoic membrane platform for investigation of spontaneous circulating tumor cells.

Circulating tumor cells (CTCs) are clinically relevant markers of metastasis and prognosis in gastrointestinal (GI) cancers. However, their extreme rarity and the limitations of current in vivo models severely restrict mechanistic studies of tumor cell dissemination. Here, we demonstrate for the first time that the avian chorioallantoic membrane (CAM) assay can serve as a biologically relevant and ethically advantageous 3R-compliant (Replacement, Reduction, and Refinement) in vivo model for investigating spontaneous CTC generation. Six green fluorescent protein (GFP)-labeled human colorectal, pancreatic, and cholangiocarcinoma cell lines were xenografted onto the CAM. Using a standardized workflow, we achieved reliable detection and isolation of GFP-positive single CTCs from small-volume CAM blood samples. Critically, we reproducibly confirmed the presence of spontaneously shed CAM-derived CTCs independent of GFP expression using three established, clinically relevant CTC detection platforms. Subsequent single-cell whole-genome amplification enabled downstream short-tandem repeat (STR) profiling to authenticate CTC identity, alongside detection of tumor-specific point mutations and copy number alterations (CNAs). Isolated CTCs exhibited high genetic concordance with their parental cell lines, validating the technical robustness of our workflow and establishing the CAM assay as a versatile, scalable platform for investigating hematogenous dissemination and CTC biology outside of mammalian models. Consequently, the CAM assay opens new avenues for early mechanistic and pharmacological CTC research, including functional studies of patient-derived material.