Single-cell sequencing of neuroblastoma CHP134 cells treated with doxazosin, isotretinoin (13cRA) and their combination.. 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 α1-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 α1B adrenergic receptor as a novel pharmacological target in NB. We propose evaluating the addition of α1-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, α-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

神经母细胞瘤（Neuroblastoma, NB）是一种侵袭性儿童肿瘤，高危病例的5年总生存率仅约50%。神经母细胞瘤的多模式治疗方案包含异维A酸（isotretinoin，13cRA），该药物于巩固后治疗阶段作为抗增殖、促分化剂使用，以最小化残留病灶并预防肿瘤复发。多沙唑嗪（doxazosin）是一款可安全用于儿科患者的α1受体拮抗剂（α1-antagonist），当其与13cRA联合应用于携带神经母细胞瘤异种移植瘤的小鼠时，可显著抑制肿瘤生长，而两种药物单药使用时均未表现出抗肿瘤效果。为揭示该协同作用背后的分子机制，研究团队对CHP134对照细胞，以及分别经13cRA（5 μM）、多沙唑嗪（10 μM）及其联合给药处理的细胞开展了单细胞RNA测序（single-cell RNA-seq）。测序数据显示，当细胞暴露于13cRA或多沙唑嗪与13cRA的联合给药方案时，单细胞转录组会发生显著重编程，而单独使用多沙唑嗪时则无此现象。具体而言，研究团队在307株接受联合给药处理的细胞中鉴定出91个特异性标志物基因，其中41个为协同下调基因，50个为协同上调基因。对上述协同表达基因的功能富集分析结果显示，其显著富集于细胞凋亡、细胞周期调控以及神经元细胞身份相关的基因通路。因此，本研究在临床前模型中鉴定出α1B肾上腺素能受体（α1B adrenergic receptor）作为神经母细胞瘤的新型药理学靶点。研究团队建议在神经母细胞瘤的巩固后治疗中加入α1受体拮抗剂，以更高效地控制残留病灶。关键词：异维A酸、13cRA、13-顺式维甲酸、多沙唑嗪、神经母细胞瘤、单细胞RNA测序、iCELL8、肾上腺素能受体、α受体阻滞剂、联合治疗、协同作用 总体实验设计：对1287个单个细胞开展单细胞RNA测序（scRNA-seq），这些细胞经单药（多沙唑嗪或13cRA）或联合给药（多沙唑嗪与13cRA）孵育24小时后进行样本制备与测序