A major question in neuroblastoma research is why most high stage neuroblastoma initially respond to chemotherapy, but ultimately relapse as therapy-resistant tumor. We detected in new neuroblastoma cell lines two cell types with shared genetic defects but highly divergent phenotypes. One type expresses all classical neuroblastoma markers and has a neuro-epithelial (NE) phenotype. The other type lacks all neuroblastoma markers, is motile and has a mesenchymal (MES) character. Immunohistochemistry detected a small fraction of MES cells in most primary neuroblastoma. In four isogenic cell line pairs, MES cells were more chemo-resistant than their NE-type counterparts. Indeed, in primary tumors, viable MES-type cells accumulated after chemo-therapy. Moreover, relapses emerging 5 years after diagnosis were highly enriched for MES cells as compared to the primary pre-treatment samples of the same patients.
As these data suggest a role for MES-type cells in relapse development, we analyzed their key regulatory pathways. In isogenic cell line pairs, MES and NE-type cells widely differed in major signaling routes, transcription factors and histone modifications. MES cells had high NOTCH and PRRX1 pathway activity. Induced expression of NOTCH or PRRX1 converted multiple NE-type cell lines into MES-type cells, including chemo-resistance. These pathways activated 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 seed relapses, that ultimately become heterogeneous again. Elimination of MES cells with small molecule inhibitors shows how they are amenable to therapy.