目的:比较3种商业免疫原性细胞死亡(ICD)诱导剂,即氯化石蜡e6(Ce6),中性红(NR),和玫瑰红钠盐(RB),因为它们的光敏特性,光动力疗法(PDT)和ICD诱导效率在抗肿瘤免疫治疗。
方法:使用活性氧(ROS)探针评估3种ICD诱导剂的光敏性,并且使用DCFH-DA探针评估它们诱导细胞内ROS产生的能力。使用CCK-8试剂盒比较了3种光敏剂的细胞毒性和生物相容性,并通过检测表面暴露钙网蛋白(ecto-CRT)的水平来评估其ICD诱导效率,高迁移率族蛋白1(HMGB1)和三磷酸腺苷(ATP)。在动物实验中,携带4T1细胞源性皮下肿瘤的BALB/c小鼠模型瘤内注射Ce6或NR溶液(30μL,5mg/mL),随后2小时后进行白光照射10分钟(400mW/cm2)。监测小鼠体重和肿瘤大小变化,治疗后14天,通过流式细胞术分析肿瘤中CD8T细胞和脾脏中IFN-γCD8T细胞的百分比。HE和TUNEL染色用于分析小鼠的肿瘤细胞凋亡。
结果:在3种光敏剂中,Ce6对肿瘤细胞表现出最强的ROS诱导能力和杀伤作用。ectoCRT的结果,HMGB1和ATP水平检测均显示了Ce6更强的ICD诱导能力。在荷瘤小鼠中,Ce6和NR组治疗后肿瘤生长明显受到抑制。Ce6组CD8+T细胞和IFN-γ+CD8+T细胞百分比分别为12.7%和7.1%,分别,显著高于NR组(6.1%和2.8%,分别;P<0.05)。HE和TUNEL染色显示Ce6和NR组肿瘤组织中明显的肿瘤细胞凋亡,Ce6组治疗效果更为显著。
结论:在3种光敏剂中,Ce6具有诱导ROS产生的最高效率,具有最强的PDT功效和ICD诱导能力。Ce6还可以在抗肿瘤免疫疗法中增加CD8+T细胞的数量和功能,以启动强大的适应性免疫应答。
OBJECTIVE: To compare 3 commercial immunogenic cell death (ICD) inducers, namely Chlorin e6 (Ce6), Neutral Red (NR), and Rose Bengal Sodium salt (RB), for their photosensitive properties, efficacy for photodynamic therapy (PDT) and ICD induction efficiency in antitumor immunotherapy.
METHODS: Reactive oxygen species (ROS) probes were used to evaluate the photosensitivity of the 3 ICD inducers, and their capacity for inducing intracellular ROS production was evaluated using a DCFH-DA probe. The cytotoxicity and biocompatibility of the 3 photosensitizers were compared using a CCK-8 kit, and their ICD-inducing efficiency was assessed by detecting the levels of surface-exposed calreticulin (ecto-CRT), high mobility group protein 1 (HMGB1) and adenosine triphosphate (ATP). In the animal experiment, BALB/c mouse models bearing 4T1 cellderived subcutaneous tumor were given intratumoral injection of Ce6 or NR solution (30 μL, 5 mg/mL), followed 2 h later by white light irradiation for 10 min (400 mW/cm2). Body weight and tumor size changes of the mice were monitored, and the percentage of CD8+ T cells in the tumor and IFN-γ+ CD8+ T cells in the spleen were analyzed by flow cytometry 14 days after the treatment. HE and TUNEL staining was used to analyze tumor cell apoptosis in the mice.
RESULTS: Among the 3 photosensitizers, Ce6 exhibited the strongest ROS-inducing capability and killing effect on the tumor cells. The results of ectoCRT, HMGB1 and ATP level detection all demonstrated a stronger ICD induction ability of Ce6. In the tumor-bearing mice, the tumor growth in Ce6 and NR groups was significantly inhibited after the treatment. The percentages of CD8+ T cells and IFN-γ+ CD8+ T cells were 12.7% and 7.1% in Ce6 group, respectively, significantly higher than those in NR group (6.1% and 2.8%, respectively; P < 0.05). HE and TUNEL staining revealed obvious tumor cell apoptosis in the tumor tissues in both Ce6 and NR groups, but the therapeutic effect was more prominent in Ce6 group.
CONCLUSIONS: Among the 3 photosensitizers, Ce6 has the highest efficiency for inducing ROS production with the strongest PDT efficacy and ICD induction capability. Ce6 can also increase the number and function of CD8+ T cells in anti-tumor immunotherapy to initiate robust adaptive immune response.