SOX2-driven stemness as a key determinant for long-term maintenance of pre-treatment patient-derived organoids in esophageal squamous cell carcinoma

Background: Patient-derived organoids (PDOs) provide an advanced in vitro model that faithfully recapitulates tumor heterogeneity than conventional two-dimensional cultures. A major advantage of PDOs is their ability to directly link experimental findings with clinical treatment outcomes, making them an attractive platform for translational research. Previous studies have reported high establishment and passaging efficiencies of PDOs in adenocarcinomas, including colorectal and gastric cancers. In contrast, the establishment and long-term maintenance of PDOs derived from esophageal squamous cell carcinoma (ESCC) remain challenging, with lower success rates than those observed in other tumor types. The biological determinants underlying this variability in PDO-forming capacity among ESCC tumors remain poorly understood. Methods:PDOs were established from pretreatment biopsy specimens obtained from patients with advanced ESCC. Successful establishment was defined as organoid formation after initial seeding, and long-term maintenance was defined as continued growth through at least three passages. Cases were grouped according to long-term organoid maintenance capability, and clinical characteristics and immunohistochemical features were analyzed. SOX2 expression was evaluated by immunohistochemistry using an intensity-based scoring system. Results:Among 57 enrolled patients, Initial PDO establishment was achieved in 53 of 57 cases (93.0%), but only 15 cases (26.3%) met the criteria for long-term maintenance. On average, 48 days were required to reach three passages following PDO establishment. Cell viability in biopsy specimens showed no correlation with PDO establishment or long-term passaging rates. Similarly, TNM classification parameters, including tumor invasion depth and the number of lymph node metastases, were not associated with PDO establishment or long-term maintenance. In contrast, immunohistochemical analysis of biopsy specimens from PDO-cultured cases revealed that higher SOX2 expression in tumor cells was significantly associated with increased rates of long-term organoid maintenance (p < 0.05). These findings suggest that tumor cell-intrinsic stemness characteristics, exemplified by SOX2 expression, may play a critical role in PDO establishment and long-term passaging. Conclusion:Tumor cell-intrinsic stemness, such as SOX2 expression, appears to be a key determinant of successful establishment and long-term maintenance of ESCC patient-derived organoids.Our findings suggest that while initial formation is common, long-term modeling of ESCC is biologically restricted. Overcoming this barrier in SOX2-low tumors will be essential for the universal application of PDOs in ESCC translational research.