P09 | EFFECTS OF NUTLIN-3A ACTIVITY ON 2D AND 3D RETINOBLASTOMA MODELS

F. Bompan¹, R. Foschi², A. Dipinto³, F. Casciano², G. Lodi⁴, A. Sanvido⁵, P. Severi⁴, L. Giari¹, L. C. Cosenza⁵, R. Voltan², A. Romani⁴ | ¹Department of Environmental and Prevention Sciences, University of Ferrara, Ferrara, Italy; ²Department of Environmental and Prevention Sciences, University of Ferrara, Ferrara, Italy; LTTA Centre, University of Ferrara, Ferrara, Italy; ³Department of Environmental and Prevention Sciences, University of Ferrara, Ferrara, Italy; ⁴Department of Translational Medicine, University of Ferrara, Ferrara, Italy; LTTA Centre, University of Ferrara, Ferrara, Italy; ⁵Department of Translational Medicine, University of Ferrara, Ferrara, Italy

Retinoblastoma is the predominant paediatric tumor impacting the retina.¹ To effectively mimic the in vivo primary tumor, novel therapeutic approaches must be examined in increasingly complex models. The primary aim of this study was to evaluate the anticancer efficacy of MDM2 inhibitors in the treatment of retinoblastoma using preclinical 2D and 3D bio-printed innovative models. As retinoblastoma exhibits a p53 wild-type phenotype, the use of Nutlin-3a, a molecule known to free p53 from its inhibitor MDM2, can activate the p53 apoptotic pathway limiting retinoblastoma growth and survival.² We first assessed Nutlin-3a in 2D models, using the retinoblastoma cell lines Y79 and Weri-Rb1, both expressing p53wt, demonstrating a concentration dependent response as early as 24 h. We observed significant cell viability reduction, cell cycle blockade in the G2/M phase, apoptosis increases and p53 pathway activation, by cytofluorometric and western blotting analysis. Next, we used the same cell lines to create and characterize innovative 3D bioprinted models, using 2% alginate and 5% gelatine bioink and printing 10x106/mL Y79 and 20x106/mL Weri Rb1 cells; crosslinking with 50 mM CaCl2 stabilized the model. Two days after bioprinting, the 3D structures were treated with Nutlin-3a and assessed for viability using MTT 72 h after. In parallel, the 3D structures were fixed and embedded to paraffin and subjected to histological and immunohistochemical investigation. Haematoxylin and eosin staining revealed a 3D cellular architecture similar to the primary retinoblastoma tissue, with cells having a typically rosette distribution. Interestingly, in the 3D tissues the number and dimension of rosettes were significantly reduced after Nutlin-3a treatment, associated with a decrease expression of Ki-67. Our successful creation of a 3D model that closely resembles tumor tissue might potentially be used as a platform for future co-culture of tumor and healthy cells to assess innovative drugs and/or to study the administration of Nutlin-3a via nanoparticles.