The combination therapy using tyrosine kinase receptors inhibitors and repurposed drugs to target patient-derived glioblastoma stem cells

The lesson from many studies investigating the efficacy of targeted therapy in glioblastoma (GBM) showed that a future perspective should be focused on combining multiple target treatments. Our research aimed to assess the efficacy of drug combinations against glioblastoma stem cells (GSCs). Patient-derived cells U3042, U3009, and U3039 were obtained from the Human Glioblastoma Cell Culture resource. Additionally, the study was conducted on a GBM commercial U251 cell line. Gene expression analysis related to receptor tyrosine kinases (RTKs), stem cell markers and genes associated with significant molecular targets was performed, and selected proteins encoded by these genes were assessed using the immunofluorescence and flow cytometry methods. The cytotoxicity studies were preceded by analyzing the expression of specific proteins that serve as targets for selected drugs. The cytotoxicity study using the MTS assay was conducted to evaluate the effects of selected drugs/candidates in monotherapy and combinations. The most cytotoxic compounds for U3042 cells were Disulfiram combined with Copper gluconate (DSF/Cu), Dacomitinib, and Foretinib with IC50 values of 52.37 nM, 4.38 µM, and 4.54 µM after 24 h incubation, respectively. Interactions were assessed using SynergyFinder Plus software. The analysis enabled the identification of the most effective drug combinations against patient-derived GSCs. Our findings indicate that the most promising drug combinations are Dacomitinib and Foretinib, Dacomitinib and DSF/Cu, and Foretinib and AZD3759. Since most tested combinations have not been previously examined against glioblastoma stem-like cells, these results can shed new light on designing the therapeutic approach to target the GSC population.

多项针对胶质母细胞瘤（glioblastoma, GBM）靶向治疗疗效的研究均表明，未来的研究方向应聚焦于多靶点联合治疗方案。本研究旨在评估药物联合方案对胶质母细胞瘤干细胞（glioblastoma stem cells, GSCs）的疗效。研究者从人胶质母细胞瘤细胞培养库（Human Glioblastoma Cell Culture resource）中获取了患者来源的U3042、U3009及U3039细胞株。此外，本研究还使用了商用GBM细胞系U251开展实验。研究针对受体酪氨酸激酶（receptor tyrosine kinases, RTKs）、干细胞标志物以及与重要分子靶点相关的基因开展了基因表达分析，并采用免疫荧光（immunofluorescence）和流式细胞术（flow cytometry）对上述基因编码的目标蛋白进行了检测。细胞毒性实验前，研究者先分析了作为所选药物靶点的特定蛋白的表达水平。本研究采用MTS比色检测法（MTS assay）开展细胞毒性实验，以评估所选药物/候选化合物单药及联合使用的效果。针对U3042细胞，细胞毒性最强的化合物为双硫仑联合葡萄糖酸铜（Disulfiram combined with Copper gluconate, DSF/Cu）、达可替尼（Dacomitinib）以及福替尼（Foretinib），经24小时孵育后，其半数抑制浓度（half maximal inhibitory concentration, IC50）分别为52.37 nM、4.38 µM及4.54 µM。药物相互作用采用SynergyFinder Plus软件进行评估。通过该分析，研究者筛选出了针对患者来源胶质母细胞瘤干细胞的最优药物联合方案。本研究结果显示，最具开发前景的药物联合方案为达可替尼与福替尼、达可替尼与双硫仑/葡萄糖酸铜，以及福替尼与AZD3759。鉴于多数受试联合方案此前尚未针对胶质母细胞瘤干细胞样细胞开展相关研究，本研究结果可为靶向胶质母细胞瘤干细胞群体的治疗策略设计提供全新的研究思路。