Cell-Laden Hydrogel as a Clinical-Relevant 3D Model for Analyzing Neuroblastoma Growth, Immunophenotype, and Susceptibility to Therapies

Abstract

High risk Neuroblastoma (NB) includes aggressive, metastatic solid tumors of childhood. The survival rate improved only modestly, despite the use of combination therapies including novel immunotherapies based on the antibody-mediated targeting of tumor-associated surface ligands. Treatment failures may be due to the lack of adequate in vitro models for studying, in a given patient, the efficacy of potential therapeutics, including those aimed to enhance anti-tumor immune responses. We here propose a 3D alginate-based hydrogel as extracellular microenvironment to evaluate the effects of the three-dimensionality on biological and immunological properties of NB cells. NB cell lines grown within the 3D alginate spheres presented spheroid morphology, optimal survival, and proliferation capabilities, and a reduced sensitivity to the cytotoxic effect of imatinib mesylate. 3D cultured NB cells were also evaluated for the constitutive and IFN-γ-induced expression of surface molecules capable of tuning the anti-tumor activity of NK cells including immune checkpoint ligands. In particular, IFN-γ induced de novo expression of high amounts of HLA-I molecules, which protected NB cells from the attack mediated by KIR/KIR-L matched NK cells. Moreover, in the 3D alginate spheres, the cytokine increased the expression of the immune checkpoint ligands PD-Ls and B7-H3 while virtually abrogating that of PVR, a ligand of DNAM-1 activating receptor, whose expression correlates with high susceptibility to NK-mediated killing. Our 3D model highlighted molecular features that more closely resemble the immunophenotypic variants occurring in vivo and not fully appreciated in classical 2D culture conditions. Thus, based on our results, 3D alginate-based hydrogels might represent a clinical-relevant cell culture platform where to test the efficacy of personalized therapeutic approaches aimed to optimize the current and innovative immune based therapies in a very systematic and reliable way.

Multiparametric flow cytometry highlights B7-H3 as a novel diagnostic/therapeutic target in GD2neg/low neuroblastoma variants

“…In these patients, the B7-H3 surface expression could be even increased since it is upregulated by IFN-γ. 34 In the perspective of B7-H3-based immunotherapy, it is relevant to point out that B7-H3 shows low or null protein surface expression in most normal tissues. 48 An exception is represented by the MSC compartment.…”

Emerging Neuroblastoma 3D In Vitro Models for Pre-Clinical Assessments

“…Neuroblastoma cells can be successfully grown in cellulose and alginate-based hydrogels, demonstrating the applicability of FRESH bioprinting for the generation of microsized 3D neuroblastoma tumors (74). The same type of hydrogel has been explored in the immunology field as well showing the changes in immunophenotype profiles in neuroblastoma cells surrounded by biomimetic ECM (75). Beta tricalcium phosphate (b-TCP) scaffolds used as bone mimetic, have been obtained by combining high resolution 3D printing with manually cast slurry (76).…”

“…Immunotherapies have recently attracted great interest as a novel approach for cancer treatment, but the lack of adequate in vitro models for testing the efficacy of these therapies at a personalized level is still an issue (75,83). Immunotherapy strategies rely both, on the ability of the immune system to kill malignant cells by recognizing specific tumor antigens and the ability of tumor cells to evade this physiological defense system.…”

Resolving Metabolic Heterogeneity in Experimental Models of the Tumor Microenvironment from a Stable Isotope Resolved Metabolomics Perspective

“…Since then, 3D cultures and organoids have been developed for a wide range of cell types [49,108,111,112,[119][120][121][122][123][124][125]. There are many types of support matrices now available, from the widely used Matrigel (a mouse sarcoma cell extracellular matrix) to other organic polymer supports such as alginates and hydrogels [126][127][128][129][130][131][132][133][134]. Three-dimensional cultures can also be induced without matrix support by concentrating "magnetized" cells via introduction of nano magnetic beads under a magnetic field [121,135].…”