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In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell–activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell–mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a–dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell–mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell–mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell–based immunotherapy for neuroblastoma.

The presence of tumor-infiltrating T cells is crucial for the development of immunotherapies and the prediction of clinical response for many human cancers. Recently, we have shown that tumor-infiltrating T cells have a prognostic value greater than, and independent of, the criteria currently used to stage neuroblastoma, the most common pediatric extra-cranial solid tumor accounting for 15% of childhood cancer-related death. We demonstrated that expression of PD-L1 in tumor cells is inversely correlated with that of HLA class I, and that when combined they represent a promising prognostic biomarker in neuroblastoma. To further dissect the immune heterogeneity of neuroblastoma microenviroment, we evaluated the density of infiltrating dendritic cells (iDC), which are crucial for drug-induced anticancer immune response, in the same cohort of NB samples characterized for type, density, and composition of tumor T lymphocytes and expression of tumor PD-L1 and HLA class I. As for T cells, high density of iDCs was correlated with the presence of infiltrating T cells, expression of tumor HLA class I and favorable clinical outcome, suggesting that iDCs may be critical for robust tumor control by improving prediction of patient survival. Citation Format: Ombretta Melaiu, Renata Boldrini, Marco Chierici, Cesare Furlanello, Doriana Fruci. Clinical relevance of tumor-infiltrating immune cells in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B37.