Supplementary Material for: Establishment of a novel organoid line from esophageal squamous cell carcinoma with cytoplasmic vacuoles: Association of autolysosome swelling with vacuole formation

Introduction: We experienced a case of esophageal squamous cell carcinoma (ESCC) exhibiting mucus-negative cytoplasmic vacuoles. The carcinoma cells with vacuoles were positive for p63, a marker of squamous cell carcinoma (SCC). Since the first description by Cramer and Heggeness in 1989, 24 cases of SCC with mucus-negative vacuoles have been reported to date. However, little is known about the clinical course of SCC with vacuoles (SCCVs) and the nature of the cytoplasmic vacuoles themselves, due to its rarity and the lack of suitable models.
Methods: We established a novel organoid line – designated ECO_Vac – from residual cancer tissue of a patient with esophageal SCCV (ESCCV) after neoadjuvant chemotherapy (NAC), evaluated its applicability as a model of ESCCV, and characterized its cytoplasmic vacuoles by electron microscopic and organelle-specific fluorescent probe uptake assays.
Results: ECO_Vac was successfully established and was found to be applicable to in vitro drug assays and experiments involving in vivo tumor formation. We also found that the vacuoles in ESCCV were enlarged autolysosomes.
Conclusion: We established a novel organoid line, ECO_Vac, as a useful model for investigating the molecular pathogenesis of ESCCV. By using ECO_Vac, we demonstrated that the cytoplasmic vacuoles in ESCCV were unphysiologically enlarged autolysosomes.