Introduction: Understanding tumor–vascular interactions requires experimental platforms that allow rapid, physiologically relevant assessment of tumor microenvironmental remodeling. The chick chorioallantoic membrane (CAM) model offers a highly vascularized in vivo system, yet its application for studying tumor secretome-driven vascular reprogramming remains underexplored. Here, we developed a combined secretome pretreatment and dual-reporter imaging strategy to investigate how tumor-cell-secreted soluble factors influence the vascularization during sarcoma progression.

Methods: Dual-reporter sarcoma (WEHI164, U2OS) cell lines stably expressing Katushka2S fluorescent protein and NanoLuc luciferase were generated for real-time monitoring of tumor growth and dissemination in the CAM model. CAM tissues were pretreated with a tumor cell-conditioned medium (TCM) prior to tumor cell implantation to simulate secretome-driven microenvironment priming. Tumor progression and vascular remodeling were evaluated using in ovo fluorescence imaging, immunofluorescence staining of tumor nodule sections, and multiplex secretome profiling. VEGFA inhibition with bevacizumab was applied to assess the functional impact of VEGFA in TCM.

Results: TCM pretreatment significantly enhanced tumor engraftment, vascular remodeling, and metastatic dissemination in the CAM model. Multiplex analysis identified enrichment of pro-angiogenic factors, particularly VEGFA, in TCM. VEGFA blockade partially reversed secretome-induced neovascularization and reduced tumor progression.

Conclusions: This study establishes a rapid experimental platform that utilizes secretome pretreatment and tumor cell implantation for dual-reporter imaging in CAM to investigate tumor-vascular interactions in vivo. The platform enables real-time assessment of tumor microenvironment changes and treatment effects, aiding cancer research and development of anti-angiogenic therapy.