Abstract 2453: Neuroblastoma is bi-phasic and includes classical neuro-epithelial cells and chemo-resistant mesenchymal cells
Authors: Versteeg Rogier , Groningen Tim van , Valentijn Linda J. , Westerman Bart A. , Molenaar Jan J. , Westerhout Ellen M. , Hamdi Mohamed , Tytgat Godelieve A. , Koster Jan , Nes. Johan van 
Published: July 2016

Abstract
Introduction
Most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. The mechanisms driving relapse and resistance remain elusive. We investigated whether neuroblastoma tumors include divergent cell types that may underlie this plasticity.
Experimental procedures
Fresh tumor cells cultured in neural stem cell medium were analyzed by FACS, whole genome sequencing, Chip-seq, mRNA profiling, and motility and chemo-sensitivity assays. Inducible transgenes were used to test state-transitions. Tumors were analyzed by immunohistochemistry.
Results
New neuroblastoma cell lines always included two cell types, which share the same genetic defects but have highly divergent phenotypes. One type has a neuro-epithelial (NE) phenotype and expresses all classical neuroblastoma markers. The other type has a mesenchymal (MES) character, is motile and lacks all neuroblastoma markers. Immunohistochemistry (IHC) detected a small fraction of MES cells in most primary neuroblastoma.
In four isogenic cell line pairs, we found that MES cells were more chemo-resistant than their NE-type counterparts. Indeed, comparison of primary neuroblastoma lesions before and after chemotherapy showed an accumulation of viable MES-type cells in post treatment samples. Moreover, comparison of primary, pre-treatment tumors with relapses emerging 4-5 years later in the same patients showed a strong enrichment for MES cells in the latter.
As these data suggest a role for MES-type cells in relapse development, we analyzed their key regulatory pathways. The isogenic MES-NE cell line pairs showed consistent mRNA expression differences between both phenotypes, activating major signaling routes and transcription factors. Chip-seq identified divergent histone modifications. MES cells had high NOTCH pathway activity and PRRX1 expression. Induced expression of NOTCH or PRRX1 converted multiple NE-type cell lines into MES-type cells, including chemo-resistance. Further analysis of these routes reconstructed molecular wiring of MES-type cells. This identified key-players like MEK and PDGFRβ, which were successfully targeted by small molecules to specifically kill MES cells in vitro.
Conclusions
Our data suggest that neuroblastoma is a bi-phasic tumor. MES and NE cells differ in many characteristics, but can transdifferentiate into each other. MES and NE cells may correspond to developmental stages, i.e. mesenchymal migratory cells delaminated from the neural crest and more differentiated cells of the adrenergic lineage. MES cells accumulate after chemo-therapy and in relapses. They may survive classical therapy and over time seed relapses, that ultimately become heterogeneous again. Elimination of MES cells with small molecule inhibitors shows how cells with a potential key role in relapse development are amenable to therapy.
Citation Format: Rogier Versteeg, Tim van Groningen, Linda J. Valentijn, Bart A. Westerman, Jan J. Molenaar, Ellen M. Westerhout, Mohamed Hamdi, Godelieve A. Tytgat, Jan Koster, Johan van Nes. Neuroblastoma is bi-phasic and includes classical neuro-epithelial cells and chemo-resistant mesenchymal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2453.