PO.ET08.01 · 实验与分子治疗
溶瘤单纯疱疹病毒M002通过减少G2/M期阻滞增强小鼠横纹肌肉瘤原位同种异体移植细胞的抗肿瘤放射应答
Oncolytic herpes simplex virus, M002, increases the antitumor radiation response by decreasing the G2/M arrest in murine rhabdomyosarcoma orthotopic allograft cells
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摘要 Abstract
中文摘要
引言：横纹肌肉瘤（RMS）是儿童中最主要的软组织肉瘤。对于大多数RMS患者，采用多模式治疗以获得最佳结局，包括化疗、放疗（RT）和手术。RT虽然是局部控制的重要组成部分，但常导致显著的长期后遗症。此外，RT后复发的RMS患儿仍然经历最差的结局。这些问题凸显了开发策略以最大限度减少晚期毒性和改善治疗指数的必要性。研究表明，RT诱导的抗原释放放大了由溶瘤病毒（OVs）触发的免疫应答，而肿瘤细胞的病毒感染阻碍了RT诱导的DNA损伤的修复，二者均增强了RT疗效。我们此前表明，用溶瘤单纯疱疹病毒（oHSV）M002处理小鼠原位同种异体移植（OA）-RMS细胞可增强对RT的应答。这些发现促使我们研究M002增强OA-RMS细胞抗肿瘤放射应答的机制。
方法：用M002（0或5噬斑形成单位（PFU）/细胞）处理小鼠OA-RMS细胞24小时，并进行全细胞RNA测序。RT剂量以275 kV和10.0 mA的X射线递送。使用alamarBlue测量活力。通过流式细胞术评估细胞周期分布。
结果摘要：OA-RMS细胞表现出对RT的抗性，表现为治疗后第6天对照细胞与RT处理细胞之间的活力无显著变化。M002（5 PFU/细胞）处理的OA-RMS细胞的RNA测序数据和通路分析显示G2/M通路相关基因显著下调。2 Gy RT与M002联合处理消除了单独RT引起的G2/M期阻滞。2 Gy RT后阻滞于G2/M期的OA-RMS细胞百分比增加至24.4%，而对照细胞为8.3%。M002与RT联合处理将阻滞于G2/M期的OA-RMS细胞百分比降至2.5%。与对照组相比，M002与RT联合显著增加了sub-G1群体，提示细胞正在死亡和凋亡。
结论：我们证明溶瘤单纯疱疹病毒M002显著抑制OA-RMS细胞中调控细胞周期G2/M期的基因。RT与M002联合减少了RT引起的G2/M期阻滞。G2/M阻滞的丧失伴随着细胞死亡的增加。这些结果表明M002是一种有前景的策略，可破坏G2/M检查点——这是RT抗性RMS肿瘤发挥功能的一种机制。
查看英文原文 English abstract
Introduction: Rhabdomyosarcoma (RMS) represents the predominant soft tissue sarcoma in children. For most RMS patients, multimodality therapy is employed to achieve optimal outcomes, incorporating chemotherapy, radiotherapy (RT) and surgery. RT, although an integral component of local control, often results in significant long-term sequelae. Further, children with RMS who relapse after RT continue to experience the poorest outcomes. These issues underscore the need to develop strategies to minimize late toxicities and improve therapeutic index. Investigations have indicated that RT-induced antigen release amplifies the immune response triggered by oncolytic viruses (OVs), and viral infection of tumor cells impedes the repair of RT-induced DNA damage, both enhancing RT efficacy. We previously showed that treatment of murine orthotopic allograft (OA)-RMS cells with an oncolytic Herpes Simplex Virus (oHSV), M002, enhanced the response to RT. These findings led us to investigate the mechanism by which M002 enhances the antitumor response to RT in OA-RMS cells.
Methods: Murine OA-RMS cells were treated with M002 (0 or 5 plaque forming units (PFU)/cell) for 24 hours and whole cell RNA sequencing was performed. RT doses were delivered as X-rays at 275 kV and 10.0 mA. Viability was measured using almarBlue. Cell cycle distribution was assessed by flow cytometry.
Summary of results: OA-RMS cells demonstrated resistance to RT as measured by no significant change in viability between control cells and RT treated cells on post-treatment day 6. RNA sequence data and pathway analysis of M002 (5 PFU/cell) treated OA-RMS cells showed significant downregulation of genes involved in the G2/M pathway. Combined treatment with 2 Gy RT and M002 abrogated G2/M phase arrest caused by RT alone. The percentage of OA-RMS cells arrested at the G2/M phase increased after 2 Gy RT to 24.4% compared to 8.3% in the control cells. Combined treatment with M002 and RT decreased the percentage of OA-RMS cells arrested at G2/M phase to 2.5%. Combining M002 with RT significantly increased the sub-G1 population compared to the control group, indicating dying and apoptotic cells.
Conclusion: We demonstrate that the oHSV, M002, significantly suppresses the genes regulating the G2/M phase of the cell cycle in OA-RMS cells. Combining RT with M002 reduced the G2/M phase arrest caused by RT. The loss of G2/M arrest was accompanied by an increase in cell death. These results indicate that M002 is a promising strategy to disrupt the G2/M checkpoint, a mechanism through which RT-resistant RMS tumors function.
利益披露 Disclosure
M. G. Shaikh, None..
N. Nazam, None..
M. L. Brown, None..
P. Nande, None..
A. M. Eakes, None..
S. Manzoor, None..
J. C. Opara, None..
J. M. Markert, None..
J. M. Aye, None..
E. A. Beierle, None.