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Abstract：
Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a high mortality rate. Direct reprogramming of glial cells to different cell lineages, such as induced neural stem cells (iNSCs) and induced neurons (iNeurons), provides genetic tools to manipulate a cell\'s fate as a potential therapy for neurological diseases. NeuroD1 (ND1) is a master transcriptional factor for neurogenesis and it promotes neuronal differentiation. In the present study, we tested the hypothesis that the expression of ND1 in GBM cells can force them to differentiate toward post-mitotic neurons and halt GBM tumor progression. In cultured human GBM cell lines, including LN229, U87, and U373 as temozolomide (TMZ)-sensitive and T98G as TMZ-resistant cells, the neuronal lineage conversion was induced by an adeno-associated virus (AAV) package carrying ND1. Twenty-one days after AAV-ND1 transduction, ND1-expressing cells displayed neuronal markers MAP2, TUJ1, and NeuN. The ND1-induced transdifferentiation was regulated by Wnt signaling and markedly enhanced under a hypoxic condition (2% O2 vs. 21% O2). ND1-expressing GBM cultures had fewer BrdU-positive proliferating cells compared to vector control cultures. Increased cell death was visualized by TUNEL staining, and reduced migrative activity was demonstrated in the wound-healing test after ND1 reprogramming in both TMZ-sensitive and -resistant GBM cells. In a striking contrast to cancer cells, converted cells expressed the anti-tumor gene p53. In an orthotopical GBM mouse model, AAV-ND1-reprogrammed U373 cells were transplanted into the fornix of the cyclosporine-immunocompromised C57BL/6 mouse brain. Compared to control GBM cell-formed tumors, cells from ND1-reprogrammed cultures formed smaller tumors and expressed neuronal markers such as TUJ1 in the brain. Thus, reprogramming using a single-factor ND1 overcame drug resistance, converting malignant cells of heterogeneous GBM cells to normal neuron-like cells in vitro and in vivo. These novel observations warrant further research using patient-derived GBM cells and patient-derived xenograft (PDX) models as a potentially effective treatment for a deadly brain cancer and likely other astrocytoma tumors.
摘要：
多形性胶质母细胞瘤（GBM）是一种侵袭性脑肿瘤，死亡率高。直接将神经胶质细胞重编程为不同的细胞谱系，如诱导神经干细胞（iNSC）和诱导神经元（iNeurons），提供遗传工具来操纵细胞的命运，作为神经疾病的潜在疗法。NeuroD1（ND1）是神经发生的主要转录因子，它促进神经元分化。在本研究中,我们检验了GBM细胞中ND1的表达可以迫使它们向有丝分裂后神经元分化并阻止GBM肿瘤进展的假设。在培养的人GBM细胞系中，包括LN229，U87和U373作为替莫唑胺（TMZ）敏感细胞和T98G作为TMZ抗性细胞，神经元谱系转化由携带ND1的腺相关病毒(AAV)包诱导。AAV-ND1转导后21天，表达ND1的细胞展示神经元标志物MAP2、TUJ1和NeuN。ND1诱导的转分化受Wnt信号调节，在低氧条件下显着增强（2％O2vs.21%O2)。与载体对照培养物相比,表达ND1的GBM培养物具有更少的BrdU阳性增殖细胞。通过TUNEL染色观察到细胞死亡增加，在TMZ敏感性和抗性GBM细胞中ND1重编程后，伤口愈合试验证明了迁移活性降低。与癌细胞形成鲜明对比的是，转化细胞表达抗肿瘤基因p53。在正畸GBM小鼠模型中，将AAV-ND1重编程的U373细胞移植到环孢菌素免疫受损的C57BL/6小鼠脑的穹窿中。与对照GBM细胞形成的肿瘤相比，来自ND1重编程培养物的细胞形成较小的肿瘤，并在大脑中表达神经元标志物，如TUJ1。因此，使用单因素ND1重编程克服了耐药性，在体外和体内将异质GBM细胞的恶性细胞转化为正常的神经元样细胞。这些新颖的观察结果需要使用患者来源的GBM细胞和患者来源的异种移植（PDX）模型作为致命脑癌和其他星形细胞瘤肿瘤的潜在有效治疗方法进行进一步的研究。
