Departments of1Neurosurgery and.
4Department of Neurosurgery, Washoukai Sadamoto Hospital, Matsuyama, Ehime, Japan.
J Neurosurg. 2018 Dec 1;129(6):1416-1428. doi: 10.3171/2017.6.JNS162398. Epub 2018 Jan 19.
OBJECTIVEHigh invasiveness of malignant gliomas frequently causes early local recurrence of the tumor, resulting in extremely poor outcome. To control such recurrence, novel therapies targeted toward infiltrating glioma cells around the tumor border are required. Here, the authors investigated the antitumor activity of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas to explore the possibility for clinical use of 5-ALA-mediated SDT (5-ALA-SDT).METHODSIn vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry and TUNEL staining. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined by analysis of the effect of pretreatment with the radical scavenger edaravone. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated.RESULTSThe 5-ALA-SDT inhibited cell growth and changed cell morphology, inducing cell shrinkage, vacuolization, and swelling. Flow cytometric analysis and TUNEL staining indicated that 5-ALA-SDT induced apoptotic cell death in all gliomas. The 5-ALA-SDT generated significantly higher ROS than in the control group, and inhibition of ROS generation by edaravone completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. The proliferative activity of the entire tumor was markedly decreased. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact.CONCLUSIONSThe 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field, whereas the surrounding brain tissue remained normal, resulting in longer survival of the HIFU-treated mice compared with that of untreated mice. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.
目的恶性脑胶质瘤侵袭性强，常导致肿瘤早期局部复发，预后极差。为控制这种复发，需要针对肿瘤边缘浸润性胶质瘤细胞的新型治疗方法。作者研究了声动力学疗法（SDT）联合声敏剂 5-氨基酮戊酸（5-ALA）对恶性脑胶质瘤的抗肿瘤活性，以探讨 5-ALA 介导的 SDT（5-ALA-SDT）的临床应用可能性。
方法 在 U87 和 U251 胶质瘤细胞和 U251Oct-3/4 胶质瘤干细胞中评估 5-ALA-SDT 的体外细胞毒性。通过流式细胞术和 TUNEL 染色分析治疗相关的细胞凋亡。测量细胞内活性氧（ROS），并通过分析自由基清除剂依达拉奉预处理对治疗相关细胞毒性的影响来研究 ROS 在治疗相关细胞毒性中的作用。研究高强度聚焦超声（HIFU）联合 5-ALA-SDT 对肿瘤生长、荷瘤小鼠存活和小鼠脑组织学特征的影响。
结果 5-ALA-SDT 抑制细胞生长并改变细胞形态，诱导细胞收缩、空泡化和肿胀。流式细胞术分析和 TUNEL 染色表明，5-ALA-SDT 在所有胶质瘤中均诱导凋亡性细胞死亡。与对照组相比，5-ALA-SDT 产生的 ROS 明显更高，依达拉奉抑制 ROS 生成完全消除了 5-ALA-SDT 的细胞毒性作用。在体内研究中，与对照组相比，HIFU 联合 5-ALA-SDT 可显著延长荷瘤小鼠的存活时间（p < 0.05）。组织学上，5-ALA-SDT 在聚焦区域主要产生肿瘤组织坏死，并诱导 HIFU 辐照场靶区周围肿瘤细胞凋亡。整个肿瘤的增殖活性明显降低。HIFU 超声照射场周围的正常脑组织保持完整。
结论 5-ALA-SDT 对恶性脑胶质瘤具有细胞毒性。SDT 产生的 ROS 被认为可促进胶质瘤细胞凋亡。HIFU 联合 5-ALA-SDT 在聚焦区域诱导肿瘤坏死，在 HIFU 辐照场的周边区域诱导细胞凋亡，而周围脑组织保持正常，与未治疗组相比，HIFU 治疗组小鼠的存活时间更长。这些结果表明，HIFU 联合 5-ALA-SDT 可能提供一种侵袭性较小且具有肿瘤特异性的治疗方法，不仅针对肿瘤肿块，还针对恶性脑胶质瘤中的浸润性肿瘤细胞。
