Radiation-Induced Cellular Plasticity Primes Glioblastoma for Forskolin-Mediated Differentiation [Day21]. Radiation-Induced Cellular Plasticity Primes Glioblastoma for Forskolin-Mediated Differentiation [Day21]

Glioblastoma is the deadliest brain cancer in adults and all patients succumb to the tumor. While surgery followed by chemo-radiotherapy delays disease progression, these treatments do not lead to tumor control and targeted therapies or biologics have failed to further improve survival. Utilizing a transient radiation-induced state of multipotency, we used the adenylcyclase activator forskolin to alter the fate of irradiated glioma cells. The effects of the combined treatment on neuronal marker expression, cell cycle distribution and proliferation were studied. Gene expression profiling was conducted using bulk RNA-seq. Changes in cell populations were investigated using single cell RNA-seq. Effects on glioma stem cells were studied in extreme limiting dilution assays and the effects on median survival were studied in both syngeneic and PDOX mouse models of glioblastoma. The combined treatment induced the expression of neuronal markers in glioma cells, reduced proliferation and led to a distinct gene expression profile. scRNA-seq revealed that the combined treatment forced glioma cells into a microglia- and neuron-like phenotype. In vivo, this treatment led to a loss of glioma stem cells and prolonged median survival. Collectively, our data suggest that revisiting a differentiation therapy with forskolin in combination with radiation could lead to clinical benefit. Overall design: HK-374 gliomaspheres were plated onto the Poly-D-Lysine/Laminin 6-well plate and irradiated with 4 Gy the next day. 48 hours after the irradiation, the cells were treated with forskolin (10 µM) for 21 consecutive days.

胶质母细胞瘤（Glioblastoma）是成人中致死性最高的脑部恶性肿瘤，所有患者最终均会因该肿瘤死亡。尽管手术联合放化疗可延缓疾病进展，但此类治疗无法实现肿瘤控制，且靶向治疗或生物制剂未能进一步改善患者生存。本研究利用辐射诱导的瞬时多能状态，采用腺苷酸环化酶（adenylcyclase）激活剂佛司可林（forskolin）改变受照射胶质瘤细胞的细胞命运。本研究探讨了联合治疗对神经元标志物表达、细胞周期分布及细胞增殖的影响。采用批量RNA测序（bulk RNA-seq）开展基因表达谱分析；通过单细胞RNA测序（single cell RNA-seq）探究细胞群体的变化。通过极限稀释分析法研究其对胶质瘤干细胞的影响，并在胶质母细胞瘤的同基因（syngeneic）及PDOX小鼠模型中评估其对中位生存期的作用。联合治疗可诱导胶质瘤细胞表达神经元标志物，抑制细胞增殖，并产生独特的基因表达谱。单细胞RNA测序结果显示，联合治疗可促使胶质瘤细胞转化为小胶质细胞及神经元样表型。体内实验表明，该治疗可减少胶质瘤干细胞数量并延长中位生存期。综上，本研究数据提示，重新探索佛司可林联合辐射的分化治疗策略或可带来临床获益。实验设计：将HK-374胶质瘤球接种至多聚-D-赖氨酸/层粘连蛋白包被的6孔板中，次日给予4 Gy辐射。照射后48小时，用10 µM佛司可林连续处理细胞21天。