Phototoxicity in near-infrared photoimmunotherapy is influenced by the subcellular localization of antibody-IR700
Phototoxicity in near-infrared photoimmunotherapy is influenced by the subcellular localization of antibody-IR700
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DOI：
10.1016/j.pdpdt.2020.101926
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发表时间：
2020-09-01
影响因子：
3.3
通讯作者：
Ogawa, Mikako
中科院分区：
文献类型：
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作者：
Nakajima, Kohei;Ogawa, Mikako
背景资料：近红外光免疫疗法（Near-infrared photoimmunotherapy，NIR-PIT）是一种利用单克隆抗体-IRDye 700DX偶联物（mAb-IR 700）和近红外光的新型肿瘤光疗法。我们先前报道了mAb-IR 700在质膜上聚集并诱导膜内的物理损伤，导致坏死/免疫原性癌细胞死亡。然而，由内化的mAb-IR 700引起的细胞毒性作用，其在内吞作用后定位于溶酶体中，仍然不清楚。因此，在这项研究中，我们研究了如何内化单克隆抗体-IR 700影响phototoxicity.Methods：依赖于单克隆抗体-IR 700的亚细胞定位的细胞毒性进行了检查，通过改变洗涤后的孵育时间。在O-1（2）猝灭剂存在下，通过细胞活力测定来检查单线态氧（O-1（2））的影响。通过流式细胞术用Annexin V/碘化丙啶分析细胞死亡的类型。结果：mAb-IR 700在溶酶体中诱导的细胞毒作用弱于mAb-IR 700在质膜上诱导的细胞毒作用。O-1（2）猝灭剂可完全抑制mAb-IR 700在溶酶体中引起的细胞损伤。质膜上和溶酶体中的mAb-IR 700诱导坏死，但不诱导凋亡，细胞死亡。IR 700是本地化的溶酶体光照射前，但随后扩散到cytosol后立即irradiation.Conclusions：虽然主要的细胞毒性触发在NIR-PIT是质膜损伤，如前所述，单克隆抗体-IR 700在溶酶体也诱导坏死细胞死亡。内化的mAb-IR 700引起O-1（2）介导的损伤，导致溶酶体内容物明显渗漏到胞质溶胶中。NIR-PIT的作用机制依赖于mAb-IR 700的亚细胞定位。
Background: Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer phototherapy that utilizes monoclonal antibody-IRDye700DX conjugates (mAb-IR700) and NIR light. We previously reported that mAb-IR700 aggregated on the plasma membrane and induced physical damage within the membrane, leading to necrotic/immunogenic cancer cell death. However, cytotoxic effects caused by internalized mAb-IR700, which is localized in lysosomes after endocytosis, remain unclear. Thus, in this study, we investigated how internalized mAb-IR700 influences phototoxicity.Methods: Cytotoxicity depending on the subcellular localization of mAb-IR700 was examined by varying the incubation time after washing. The influence of a singlet oxygen (O-1(2)) was examined by cell viability assay in the presence of O-1(2) quencher. The type of cell death was analyzed by flow cytometry with Annexin V/propidium iodide. Furthermore, IR700 fluorescence in cells was observed by fluorescence microscopy.Results: mAb-IR700 in lysosomes induced cytotoxicity, which was weaker than that induced by mAb-IR700 on plasma membranes. Cellular damage caused by mAb-IR700 in lysosomes was completely inhibited by an O-1(2) quencher. mAb-IR700 on plasma membranes and in lysosomes induced necrotic, but not apoptotic, cell death. IR700 was localized in lysosomes before light irradiation but then diffused into the cytosol immediately after irradiation.Conclusions: Although the main cytotoxic trigger in NIR-PIT is plasma membrane damage as previously reported, mAb-IR700 in lysosomes also induces necrotic cell death. The internalized mAb-IR700 caused O-1(2)-mediated damage, leading to the marked leakage of lysosomal contents into the cytosol. The mechanism of NIR-PIT depends on the subcellular localization of mAb-IR700.