肿瘤细胞治疗研究
英文原题：Blockade of exosome release alters HER2 trafficking to the plasma membrane and gives a boost to Trastuzumab.
Blockade of exosome release alters HER2 trafficking to the plasma membrane and gives a boost to Trastuzumab.
基于我们的发现，HER2+外泌体可能通过双重抑制Trastuzumab诱导的肿瘤生长抑制和NK细胞的细胞毒性，从而促进肿瘤进展。同时阻断外泌体释放似乎是提高Trastuzumab以及其他潜在HER2靶向mAbs疗效的有效方法。此外，外泌体分泌途径可能有助于HER2向质膜的转运，因为阻断外泌体分泌降低了表面HER2水平。
来自BT-474、SK-BR3和SK-OV3（HER2过表达肿瘤细胞）及MDA-MB-231细胞（HER2阴性）的外泌体通过二辛可宁酸（BCA）测定、western blotting和透射电子显微镜（TEM）进行纯化和表征。通过中性鞘磷脂酶-2（nSMase-2）抑制剂GW4869实现外泌体释放的抑制。使用MTT、流式细胞术和LDH释放测定检测外泌体阻断对Trastuzumab的抗增殖作用、凋亡诱导和抗体介导的细胞毒性（ADCC）活性的影响。此外，通过流式细胞术研究了外泌体抑制对HER2表面表达和内吞/内化的影响。
从HER2过表达癌细胞中纯化的外泌体对HER2蛋白呈阳性。阻断外泌体释放能够以剂量依赖性方式显著改善Trastuzumab诱导的凋亡、抗增殖和ADCC反应。用HER2+外泌体预处理Trastuzumab/纯化的NK细胞，而非PBMCs，也能降低Trastuzumab的ADCC效应。外泌体抑制还以时间依赖性方式显著下调表面HER2水平，但不影响其内吞/内化。
OBJECTIVE(S): Exosomal HER2 has been evidenced to interfere with antibody-induced anti-tumor effects. However, whether the blockade of HER2+ exosomes release would affect antibody-mediated tumor inhibition has yet to be investigated. METHODS: Exosomes derived from BT-474, SK-BR3 and SK-OV3 (HER2-overexpressing tumor cells) and MDA-MB-231 cells (HER2 negative) were purified and characterized by bicinchoninic acid (BCA) assay, western blotting and Transmission electron microscopy (TEM). Inhibition of exosome release was achieved by neutral sphingomyelinase-2 (nSMase-2) inhibitor, GW4869. The effects of exosome blockade on the anti-proliferative effects, apoptosis induction, and antibody-mediated cellular cytotoxicity (ADCC) activity of Trastuzumab were examined using MTT, flow cytometry, and LDH release assays. Also, the effects of exosome inhibition on the surface expression and endocytosis/internalization of HER2 were studied by flow cytometry. RESULTS: Purified exosomes derived from HER2 overexpressing cancer cells were positive for HER2 protein. Blockade of exosome release was able to significantly improve apoptosis induction, anti-proliferative and ADCC responses of Trastuzumab dose dependently. The pretreatment of Trastuzumab/purified NK cells, but not PBMCs, with HER2+ exosomes could also decrease the ADCC effects of Trastuzumab. Exosome inhibition also remarkably downregulated surface HER2 levels in a time-dependent manner, but does not affect its endocytosis/internalization. CONCLUSION: Based on our findings, HER2+ exosomes may benefit tumor progression by dually suppressing Trastuzumab-induced tumor growth inhibition and cytotoxicity of NK cells. It seems that concomitant blocking of exosome release might be an effective approach for improving the therapeutic effects of Trastuzumab, and potentially other HER2-directed mAbs. In addition, the exosome secretion pathway possibly contributes to the HER2 trafficking to plasma membrane, since the blockade of exosome secretion decreased surface HER2 levels.
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