Transcriptomic analysis of Diffuse Intrinsic Pontine Glioma (DIPG) identifies a targetable ALDH-positive subset of highly tumorigenic cancer stem-like cells

Understanding the cancer stem-cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism and mTOR signaling in ALDH+ compared to ALDH- supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation and neurosphere formation and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacological MAPK/PI3K/mTOR targeting downregulated MYC, E2F and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. Characterization of DIPG CSCs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG. Eight SU-DIPG-XIII samples were analyzed. Cells were maintained under normal cell culture conditions in T-75 flasks. At the time of treatments, each T-75 flask (at ~75% confluency there are 8x10^6 million cells) was centrifuged, conditioned media was saved, and cells were resuspended in 4 mL of the conditioned media and transfered to a new T-25 flask for treatment (one T-25 flask for every T-75 flask). Samples were treated with 100 nM of PD0325901 (PD-901), 100 nM of GSK2126458 (GSK-458), 50 nM PD-901 + 50 nM GSK-458 (combination) or equimolar DMSO for two hours prior to ALDEFLUOR Assay staining and FACS sorting into ALDH+ and ALDH- conditions. ALDH+ DMSO treated and ALDH- DMSO treated samples were used as controls for comparison.

深入阐明弥漫内生型桥脑胶质瘤（diffuse intrinsic pontine glioma, DIPG）中的癌症干细胞（cancer stem-cell, CSC）图谱，对于解决治疗抵抗问题、发掘全新治疗策略而言至关迫切。研究通过流式细胞术发现，患者来源的DIPG细胞呈现乙醛脱氢酶（aldehyde dehydrogenase, ALDH）与CD133的异质性表达。转录组层面的表征分析显示，相较于ALDH阴性（ALDH-）细胞，ALDH阳性（ALDH+）细胞中MYC、E2F、DNA损伤修复（DNA damage repair, DDR）基因、糖酵解代谢以及mTOR信号通路的mRNA水平显著升高，这既支持了其干细胞样表型，也提示了可靶向的治疗位点。ALDH+细胞展现出更强的增殖能力与神经球形成能力，原位移植后可形成肿瘤并导致宿主生存期缩短。采用靶向MAPK/PI3K/mTOR的药物处理后，MYC、E2F与DDR的mRNA表达被下调，同时糖酵解代谢水平也被抑制。体内实验表明，靶向PI3K/mTOR可通过降低癌症干细胞干性，同时抑制皮下移植瘤与ALDH+原位移植瘤的生长。对DIPG癌症干细胞的表征结合MAPK/PI3K/mTOR信号通路靶向治疗，为针对DIPG中癌症干细胞表型的分子靶向治疗提供了研究动力。本研究共分析了8例SU-DIPG-XIII样本。细胞在T-75培养瓶中于常规细胞培养条件下传代培养。待处理时，每瓶汇合度约75%的细胞（含8×10^6个细胞）经离心后保留条件培养基，随后将细胞重悬于4 mL条件培养基中，并转移至新的T-25培养瓶中进行处理（每1个T-75培养瓶对应1个T-25培养瓶）。样本分别用100 nM PD0325901（PD-901）、100 nM GSK2126458（GSK-458）、50 nM PD-901联合50 nM GSK-458（联合组），或等摩尔浓度的二甲基亚砜（DMSO）处理2小时，随后进行ALDEFLUOR检测染色，并通过流式细胞术分选出ALDH+与ALDH-细胞群。其中ALDH+ DMSO处理组与ALDH- DMSO处理组作为对照用于后续比较。