[三种用于抗肿瘤免疫治疗中诱导免疫原性细胞死亡效率的商用光敏剂的比较]

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

作者信息

Lin X, Mao D, Bai R

机构信息

Inner Mongolia Medical University, Hohhot 010110, China.

Precision Medicine Research Institute, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China.

出版信息

Nan Fang Yi Ke Da Xue Xue Bao. 2022 Dec 20;42(12):1791-1798. doi: 10.12122/j.issn.1673-4254.2022.12.06.

DOI:10.12122/j.issn.1673-4254.2022.12.06

PMID:36651246

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9878406/

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 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/cm). 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; < 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.

摘要

目的

比较3种商用免疫原性细胞死亡（ICD）诱导剂，即二氢卟吩e6（Ce6）、中性红（NR）和孟加拉玫瑰红钠盐（RB）的光敏特性、光动力疗法（PDT）疗效以及在抗肿瘤免疫治疗中的ICD诱导效率。

方法

使用活性氧（ROS）探针评估3种ICD诱导剂的光敏性，并使用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的钙网蛋白（ecto-CRT）、高迁移率族蛋白1（HMGB1）和三磷酸腺苷（ATP）水平来评估它们的ICD诱导效率。在动物实验中，对携带4T1细胞衍生皮下肿瘤的BALB/c小鼠模型进行瘤内注射Ce6或NR溶液（30 μL，5 mg/mL），2小时后进行10分钟的白光照射（400 mW/cm）。监测小鼠的体重和肿瘤大小变化，并在治疗14天后通过流式细胞术分析肿瘤中CD8⁺ T细胞和脾脏中IFN-γ⁺ CD8⁺ T细胞的百分比。使用苏木精-伊红（HE）和末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色分析小鼠肿瘤细胞凋亡情况。

结果

在3种光敏剂中，Ce6表现出最强的ROS诱导能力和对肿瘤细胞的杀伤作用。ecto-CRT、HMGB1和ATP水平检测结果均表明Ce6具有更强的ICD诱导能力。在荷瘤小鼠中,治疗后Ce6组和NR组的肿瘤生长均受到显著抑制。Ce6组中CD8⁺ T细胞和IFN-γ⁺ CD8⁺ T细胞的百分比分别为12.7%和7.1%，显著高于NR组（分别为6.1%和2.8%，P<0.05）。HE和TUNEL染色显示Ce6组和NR组肿瘤组织中均有明显的肿瘤细胞凋亡，但Ce6组的治疗效果更显著。