Evaluating the RIST Molecular-Targeted Regimen in a Three-Dimensional Neuroblastoma Spheroid Cell Culture Model

Abstract

Background: The outcome for patients with high-risk neuroblastoma remains poor and novel treatment strategies are urgently needed. The RIST protocol represents a novel metronomic and multimodal treatment strategy for high-risk neuroblastoma combining molecular-targeted drugs as ‘pre-treatment’ with a conventional chemotherapy backbone, currently evaluated in a phase II clinical trial. For preclinical drug testing, cancer cell growth as spheroid compared to mo-nolayer cultures is of advantage since it reproduces a wide range of tumor characteristics, including the three-dimensional architecture and cancer stem cell (CSC) properties. The objective of this study was to establish a neuroblastoma spheroid model for the rigorous assessment of the RIST treatment protocol. Methods: Evaluation of CSC marker expression was performed by mRNA and protein analysis and spheroid viability by luminescence-based assays. Aberrant expression of RNA-binding protein La in neuroblastoma was assessed by tissue microarray analysis and patients’ data mining. Results: Spheroid cultures showed increased expression of a subgroup of CSC-like markers (CXCR4, NANOG and BMI) and higher Thr389 phosphorylation of the neuroblastoma-associated RNA-binding protein La when compared to monolayer cultures. Molecular-targeted ‘pre-treatment’ of spheroids decreased neoplastic signaling and CSC marker expression. Conclusions: The RIST treatment protocol efficiently reduced the viability of neuroblastoma spheroids characterized by advanced CSC properties.

Preclinical Models of Neuroblastoma—Current Status and Perspectives

“…The gap between 2D cell cultures and animal models can be bridged by complex three-dimensional systems that closely recapitulate the course of human cancer [ 2 , 116 , 144 , 156 , 171 ]. Recent developments in three-dimensional, microfluidic, and multicellular systems used for neuroblastoma research [ 150 ] are promising, especially for testing new therapeutic approaches [ 118 , 160 , 253 , 254 ], including natural product research [ 177 ], immunotherapy [ 172 , 255 , 256 ], and new multimodal strategies for high-risk neuroblastoma [ 149 ]. A heterogenous disease such as neuroblastoma may largely benefit from comprehensive approaches involving a combination of various biological models supplemented by mathematical simulations [ 146 , 235 , 257 , 258 ].…”

“…Spheroids are maintained in a scaffold-free and gel-free setting: they are grown in the hanging drop culture or on low-attachment plastic surfaces. The cells aggregate spontaneously, and the aggregates are composed of different subpopulations of cells, thus expressing the structural heterogeneity important for studies aimed for discovering anticancer therapy or the identification of pathways involved in tumor biology and cancer biomarkers [ 18 , 147 , 148 , 149 ]. The 3D multicellular cultures in vitro have many advantages over traditional cells maintained in the 2D setting, as they recapitulate the cellular complexity and heterogeneity of a tumor and the well-defined structural organization of the colony [ 150 ].…”