Unveiling heterogeneity in neuroblastoma cell-line using single-cell RNA- sequencing

Neuroblastoma is one of the most common pediatric cancers, accounting for approximately 15% of all childhood cancers. This tumor type exhibits significant heterogeneity; in some patients, the disease undergoes spontaneous regression, while in others, it metastasizes, often leading to a fatal outcome. Pharmacological treatments for neuroblastoma are currently not curative, highlighting the urgent need to identify new therapeutic targets. However, the absence of recurrent somatic mutations in these tumors complicates the development of targeted therapies. NDM29, a long non-coding RNA, has been identified as a crucial player in neuroblastoma biology. Previous studies have demonstrated that increasing NDM29 levels in a neuroblastoma cell model (S1.1), derived from the immortalized SKNBE2 cell line, leads to a reduction in the tumor's malignancy and an enhancement of neuronal characteristics within the cell culture. Gene ontology analysis of a panel of genes associated with tumor malignancy revealed a notable decrease in their expression when compared to the same cell line with basal NDM29 levels. This suggests that NDM29 may be influencing pathways related to malignancy. Moreover, our immunofluorescence analysis of this cell model uncovered the presence of several distinct subpopulations, revealing a surprising degree of heterogeneity within the culture. This was particularly unexpected, considering the clonal origin of the SKNBE2-S1.1 cell line. The observed heterogeneity is likely due to transdifferentiation processes occurring within the cell culture— phenomena commonly observed in tumors and which complicate therapeutic strategies. To further investigate this, single-cell transcriptome sequencing was performed, revealing the presence of nine distinct subpopulations. This finding confirmed the hypothesis generated from the immunofluorescence analysis. The aim of this thesis was to identify and isolate these subpopulations, allowing for individual study and a deeper phenotypic characterization. Understanding the properties and behaviors of these subpopulations may provide valuable insights into the underlying mechanisms of neuroblastoma and potentially support the development of more effective therapeutic approaches.