南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (12): 1791-1798.doi: 10.12122/j.issn.1673-4254.2022.12.06

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三种商品化光敏剂诱导小鼠免疫原性细胞死亡的效果比较

林 萱，毛 铎，白瑞霞   

  1. 内蒙古医科大学，内蒙古 呼和浩特 010110；内蒙古自治区人民医院检验科，内蒙古 呼和浩特 010017；中山大学附属第一医院精准医学研究院，广东 广州 510080
  • 出版日期:2022-12-20 发布日期:2023-01-12

Comparison of three commercial photosensitizers for efficiency of inducing immunogenic cell death in anti-tumor immunotherapy

LIN Xuan, MAO Duo, BAI Ruixia   

  1. Inner Mongolia Medical University, Hohhot 010110, China; Department of Clinical Laboratory, People's Hospital of Inner Mongolia Autonomous Region, Hohhot 010017, China; Precision Medicine Research Institute, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
  • Online:2022-12-20 Published:2023-01-12

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摘要： 目的 研究二氢卟吩 e6（Ce6）、中性红（NR）以及虎红钠盐（RB）3种商品化光敏剂的光敏性质、光动力治疗（PDT）效果及诱导免疫原性细胞死亡（ICD）能力，筛选出最高效的ICD诱导剂用于抗肿瘤免疫治疗。方法 溶液实验：利用不同的活性氧（ROS）荧光探针评估溶液中3种光敏剂的光敏能力。细胞实验：探针检测肿瘤细胞内ROS生成能力；CCK-8细胞毒性试验比较3种光敏剂的细胞杀伤效果以及生物相容性；检测细胞表面暴露的钙网蛋白（ecto-CRT），高迁移率族蛋白1（HMGB1）以及培养基上清三磷酸腺苷（ATP）浓度评估3种药物的ICD诱导能力。动物实验：建立BALB/c小鼠4T1皮下肿瘤模型，10 d后分别在瘤内注射Ce6和NR（30 μL，5 mg/mL），2 h后白光光照处理（10 min，400 mW/cm2），监测小鼠体质量及肿瘤组织体积变化。14 d后取材，通过流式细胞技术分析肿瘤组织中CD8+ T细胞及脾脏组织CD8+ T的IFN-γ因子分泌量；通过HE染色和TUNEL组织染色分析肿瘤组织中细胞凋亡情况。结果 在3种光敏剂中，Ce6的ROS产生能力最高，且显示出最强的肿瘤细胞杀伤效果；ecto-CRT、HMGB1荧光成像及胞外ATP含量显示Ce6相对于NR和RB具有更好的ICD诱导能力。动物实验显示，Ce6和NR组小鼠在光动力治疗后肿瘤体积显著变小，其中Ce6肿瘤治疗效果明显好于NR及PBS组。流式结果显示Ce6与NR组瘤内CD8+ T细胞占比分别为12.7%和6.1%，脾脏组织IFN-γ+ CD8+ T细胞占比分别为7.1%和2.8%，两组之间差异有统计学意义（P<0.05）。HE染色和TUNEL染色显示相对于PBS组，两个治疗组肿瘤组织有明显核凋亡情况，且Ce6的治疗效果明显强于NR。结论 3种光敏剂中Ce6的ROS生成效率最高，光动力效果最好，诱导ICD能力最强，并且在肿瘤治疗中能明显提升CD8+ T细胞的数量及功能，能相对高效的启动适应性免疫应答，抑制肿瘤生长。

关键词: 二氢卟吩 e6；中性红；虎红钠盐；光动力治疗；免疫原性细胞死亡；抗肿瘤免疫治疗

Abstract: 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 cell-derived 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 ecto-CRT, 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. Conclusion 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.

Key words: chlorin e6; Neutral Red; Rose Bengal sodium salt; photodynamic therapy; immunogenic cell death; anti-tumor immunotherapy