Orthotopic Esophageal Cancer Xenograft Model in Immunosuppressed Microminipigs for Near-Infrared Fluorescence...

Esophageal squamous cell carcinoma (ESCC) often presents as flat lesions difficult to detect with conventional endoscopy, leading to poor prognosis. To overcome this, we developed a near-infrared (NIR) molecular imaging strategy using an epidermal growth factor receptor (EGFR)-targeted antibody and established a novel orthotopic microminipig (MMP) model for translational evaluation. First, cetuximab was labeled with Alexa Fluor 680 (AF680) and validated in vitro for specific binding. Among five ESCC cell lines profiled for proliferation and antigen density, KYSE410 was selected for in vivo studies due to its optimal balance of these factors. In mouse xenografts, AF680-cetuximab tumor fluorescence peaked at 96 h, achieving high signal-to-noise ratios. Subsequently, to establish a clinically relevant large-animal platform, we optimized an immunosuppression protocol for MMPs. Through iterative testing, we determined that a combination of neonatal thymectomy and pharmacologic immunosuppression (tacrolimus, mycophenolate mofetil, and prednisolone), supplemented with antibiotics and proton-pump inhibitors, was essential to prevent rejection, sepsis, and gastric ulcers. This optimized regimen enabled stable orthotopic engraftment of human ESCC via endoscopic submucosal injection in the MMP esophagus. Using a custom-developed NIR fluorescence endoscope mimicking a clinical setting, we successfully visualized orthotopic tumors with clear margins at 96 h post-injection, subsequently confirmed by immunohistochemistry. In conclusion, AF680-cetuximab enables specific, high-contrast detection of ESCC. Furthermore, we report the first successful orthotopic engraftment of human cancer cells in immunosuppressed MMPs. This model provides a powerful platform for developing fluorescence-guided endoscopic diagnostics and theranostics for early-stage esophageal cancer, bridging the gap to future clinical application.