Deciphering heterogeneity and plasticity in neuroblastoma

Abstract Through the analysis of neuroblastoma super-enhancer landscape, we could recently identy the core regulatory circuitries (CRC) that drive neuroblastoma identity, revealing two main types of identity: a noradrenergic identity defined by a CRC module including PHOX2B, HAND2, and GATA3, and a neural crest cell (NCC)-like identity driven by a CRC module containing AP-1 transcription factors (Boeva et al., Nature Genet 2017). We observed that the heterogeneous SK-N-SH cell line exhibited a mixed identity whereas the SH-SY5Y and SH-EP cell lines, subcloned from the parental SK-N-SH cell line, presented with a noradrenergic and NCC-like identity, respectively. We documented that treatment of SK-N-SH cells with chemotherapeutic agents resulted in enrichment of NCC-like cells. Here, we used several neuroblastoma cell lines, chemotherapeutic treatment, FACS sorting, and single-cell analysis using Chromium Single Cell 3 Solution (10X Genomics) to further investigate cell identity and plasticity in neuroblastoma. We showed that treatment of the noradrenergic SH-SY5Y cell line with chemotherapy resulted in a high proportion of cells exhibiting a mesenchymal phenotype, associated with a decreased expression of the noradrenergic module and increased expression of the NCC-like module. Interestingly, cells reacquired their neuroblastic morphology after treatment withdrawal. Differential analysis of RNA-seq data comparing group I (noradrenergic) and group II (NCC-like/mesenchymal) cell lines identified one membrane marker expressed in group II but not in group I cell lines. Interestingly, FACS analysis of the SK-N-SH cell line identified two distinct populations of negative and positive cells for this membrane marker, as well as few cells with a moderate expression. Further RNA-seq analysis indicated that sorted negative cells had highly similar expression profiles compared to noradrenergic SH-SY5Y cells, whereas sorted positive cells were close to the NCC-like/mesenchymal SH-EP cells, indicating that this membrane marker may be used as a surrogate marker of cell NCC-like/mesenchymal identity. Our recent data show that culturing SK-N-SH sorted cells presenting with high or low level of the surrogate marker for few weeks is sufficient to reconstitute cell heterogeneity, since FACS analysis after culture identifies again a heterogeneous population of cells with low, intermediate, or high level of expression of the surrogate marker. This observation is also true for the SH-SY5Y cell line presenting with a minor population of positive/NCC-like cells. However, in a subset of neuroblastoma cell lines our FACS analysis with the surrogate marker of NCC-like identified only a homogeneous population of cells, either negative or positive. These data highly suggest that some neuroblastoma cells exhibit plasticity and are able to shift between the NCC-like and noradrenergic identities. To further unravel cell identity heterogeneity in the SK-N-SH model, we performed single-cell RNA-seq analysis. We expect to be able to identify different populations of cells expressing specific gene signatures. The understanding of cell identity, heterogeneity, and plasticity in neuroblastoma is of upmost importance with respect to the development of new therapeutic strategies to eradicate tumor cells in neuroblastoma patients and improve treatment and outcome of this childhood cancer that continues to have very poor prognosis in its aggressive forms. Citation Format: Simon Durand, Valentina Boeva, Caroline Louis-Brennetot, Agathe Peltier, Cécile Pierre-Eugène, Sylvain Baulande, Olivier Delattre, Isabelle Janoueix-Lerosey. Deciphering heterogeneity and plasticity in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B20.