Single-cell sequencing of neuroblastoma CHP134 cells treated with doxazosin, isotretinoin (13cRA) and their combination.

Neuroblastoma (NB) is an aggressive childhood tumor, with high-risk cases having a 5-year overall survival probability of about 50%. The multimodal therapeutic scheme of NB includes isotretinoin (13cRA), used in the post-consolidation phase as an anti-proliferative and pro-differentiative agent to minimize residual disease and prevent relapse. Administration of doxazosin, a safe a1-antagonist used in pediatric patients, with 13cRA in NB xenografted mice exerted a marked control of tumor growth, while each of the two drugs alone was ineffective. To unveil the molecular mechanisms underlying this synergism, single-cell RNA-seq was performed in CHP134 control cells, and cells treated with 13cRA (5 uM), doxazosin (10 uM), and their combination. The data revealed that the transcriptome of single cells was markedly reprogrammed when exposed to 13cRA and to the combination of doxazosin and 13cRA, but not when exposed to doxazosin alone. In detail, we identified 91 genes as specific markers of 307 cells treated with the drug combination: 41 synergistically downregulated and 50 upregulated. Functional analysis of these synergistically expressed genes reported significant enrichment of genes related to apoptosis, control of the cell cycle, and neuronal identity. We, therefore, preclinically identify the a1B adrenergic receptor as a novel pharmacological target in NB. We propose evaluating the addition of a1-antagonists in the post-consolidation therapy of NB to more efficiently control residual disease. Keywords: isotretinoin, 13cRA, 13-cis retinoic acid, doxazosin, neuroblastoma,single-cell RNA-seq, iCELL8, adrenergic receptors, a-blockers, combinatorial therapy, synergy Overall design: Single-cell RNA sequencing (scRNA-seq) of 1287 cells after 24h of single (doxazosin OR 13cRA) and combined (doxazosin AND 13cRA) drug incubation