A Novel Specific Anti-CD73 Antibody Inhibits Triple-Negative Breast Cancer Cell Motility by Regulating Autophagy
A Novel Specific Anti-CD73 Antibody Inhibits Triple-Negative Breast Cancer Cell Motility by Regulating Autophagy
复制标题
新型特异性抗 CD73 抗体通过调节自噬抑制三阴性乳腺癌细胞运动
DOI：
10.3390/ijms20051057
复制
发表时间：
2019-03-01
影响因子：
5.6
通讯作者：
Ji, Xuemei
中科院分区：
文献类型：
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作者：
Qiao, Zheng;Li, Xiaoping;Ji, Xuemei
越来越多的研究聚焦于肿瘤的转移和发展。胞外核糖核酸酶CD73是参与免疫抑制的最常见的细胞表面酶之一。本研究构建了重组表达载体pET28a-CD73，并在大肠杆菌中以包涵体形式高效表达了CD73蛋白，并进行了复性。用杂交瘤技术获得了抗CD73的单抗3F7。抗体亚型鉴定为IgG2a，亲和常数为5.75 nM。该抗体可用于免疫荧光和流式细胞术。结果表明，CD73蛋白不仅定位于细胞质，而且分布于三阴性乳腺癌细胞MDA-MB-231和MDA-MB-468的表面。此外，CD73蛋白水平与患者的生存率有关。抗CD73抗体虽然不能抑制肿瘤细胞的生长，但可以增强阿霉素对三阴性乳腺癌细胞的细胞毒作用。体外功能测定结果表明，抗CD73单抗能抑制人三阴性乳腺癌和小鼠4T1细胞系的细胞迁移和侵袭。在此过程中，LC3I/LC3II比值和p62蛋白水平均升高，提示封闭CD73可抑制细胞自噬，雷帕霉素可恢复细胞的迁移和侵袭。体内实验表明，抗CD73单抗能显著抑制4T1细胞移植瘤的肺转移。综上所述，这种新型的抗CD73抗体可以作为三阴性乳腺癌治疗的辅助药物，并可用于肿瘤的诊断。
Increasing researches have focused on cancer metastasis and development. The ectonucleotidase CD73 is one of the most common cell surface enzymes that are involved in immunosuppression. In this study, the recombinant plasmid pET28a-CD73 was constructed and the CD73 protein was overexpressed in E. coli as an inclusion body that was then subjected to refolding. The anti-CD73 monoclonal antibody (3F7) was obtained by hybridoma technology. The antibody subtype was identified as IgG2a with an affinity constant of 5.75 nM. This antibody could be applied to immunofluorescence and flow cytometry. The results showed that the CD73 protein was not only located in the cytoplasm but also distributed on the surface of triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468. Moreover, the level of CD73 protein was associated with the survival rate. Although the anti-CD73 antibody was not able to inhibit tumor cell growth, it could enhance the cytotoxic effect of Doxorubicin to triple-negative breast cancer cells. In vitro function assay results indicated that anti-CD73 mAb could inhibit cell migration and invasion in both human triple-negative breast cancer and mouse 4T1 cell lines. In this process, both the LC3I/LC3II ratio and p62 protein levels increased, which indicated that the blockage of CD73 could inhibit cell autophagy, and cell migration and invasion were restored by rapamycin. In vivo, anti-CD73 mAb could significantly inhibit lung metastasis of 4T1 cells in a mouse xenograft model. Taken together, this novel anti-CD73 antibody could be developed as an adjuvant drug for triple-negative breast cancer therapy and can be useful in tumor diagnosis.