Optical nanoscopy approach to unveil chromatin alterations implicated in neuroblastoma transformation

Chromatin organization is a well-orchestrated mechanism regulating cellular physiology and its changes are associated with the onset of many diseases, including cancer. Neuroblastoma (NB) is the most common extracranial tumor in childhood, and it shows remarkable heterogeneity in the phenotypes and evolutionary patterns. Although the molecular mechanisms of this heterogeneity are still unclear, the impairment of the “epigenetic machinery” and the consequent chromatin reorganization might play an important role. We aim to assess the possible alterations in chromatin nanoscale architecture correlated with NB transformation using NB cells genetically engineered to overexpress the non-coding RNA NDM29, which promotes differentiation of tumor cells into a neuronal lineage. In both malignant NB cells (Mock) and neuron-like cells (S1.1), we explored chromatin organization and nuclear architecture by optical nanoscale microscopy using both confocal and stimulation emission depletion (STED) imaging methods, in parallel with molecular biology analyses. We characterized the nuclear 3D morphology of Mock vs. S1.1 cells by measuring volume, shape, and circularity of the nuclei as well as chromatin compaction and distribution. We imaged the immunolabeled histone H3 modifications (H3K9 trimethylation and H3K9 acetylation) to characterize chromatin regions typically associated with highly and poorly condensed chromatin, respectively. In parallel, we quantified the global DNA methylation status by measuring the levels of 5-methylcytosine (5-mC) using the enzyme-linked immunosorbent assay (ELISA) MethylFlash Global DNA. Comparing Mock vs. S1.1 cells, we observed (i) significant remodeling of nuclear morphometry; (ii) marked rearrangement of chromatin domains rich in highly-acetylated and -methylated H3K9; (iii) changes in average chromatin compaction and DNA methylation. Altogether, the results point to an involvement of epigenetic mechanisms which may sustain the chromatin remodeling during NB malignant transformation, paving the way towards new prognostic and therapeutic approaches and understanding chromatin remodeling in physiological/pathological processes.