Generation of 3D-Organoid Model of Ameloblastoma

Objectives: Due to the complex microenvironment within solid tumors, it is accepted that the traditional two-dimensional (2D) monolayer culture model has inherent limitations in mimicking the in vivo tumor cell behaviors. To date, numerous studies have contributed to a paradigm shift in modeling diseases, including tumor, by moving from the traditional 2D culture system to three-dimensional (3D) cultures. Even though much progress has been made in developing 3D organoids of malignant tumors, little has been done in benign tumors. In this study, we utilize human ameloblastoma (AM)-derived epithelial cells (AM-EpiCs) and mesenchymal stromal cells (MSCs) to generate 3D organoid models to recapitulate the histological and molecular phenotypes of human AM for further mechanistic and intervening studies.
Methods: Two distinct cell populations, epithelial and MSCs, were isolated from human solid AM tissues and expanded in vitro. For 3D-organoid culture, AM-EpiCs alone or mixtures of AM-EpiCs and MSCs were mixed with Matrigel and cultured in defined 3D culture medium. The expression of LGR5 was compared between the organoids and human AM tissues by immunofluorescence study.
Results: The 3D-organoids generated from AM-EpiCs alone or mixtures of AM-EpiCs and AM-MSCs recapitulated different histopathological subtype structures and molecular properties of AM as determined by H & E staining and immunofluorescence studies. AM-EpiC and AM-EpiC/MSC organoids expressed 53% to 71.1% LGR5 in epithelial islands while around 66.3% LGR5 expression in AM tissues.
Conclusions: The 3D-organoids formed by AM-EpiCs and AM-MSCs recapitulate the histological and molecular properties of human AM and are capable of remodeling the subpopulation of LGR5 stem-like AM-EpiCs to maintain homeostasis of AM. Generation of human tumor 3D-organoids with AM-EpiCs/AM-MSCs provide a useful platform to model different types of AM and allow drug screening for conservative management and tissue sparing of this aggressive benign tumor.