Suthe Sreedhar Reddy, Yao Hang-Ping, Weng Tian-Hao, Wang Ming-Hai
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases and National Clinical Research Center for Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Department of Pharmaceutical Sciences and Cancer Biology Research Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas, USA.
Curr Cancer Drug Targets. 2023;23(2):103-117. doi: 10.2174/1568009622666220825115528.
Cancer stem-like cells in triple-negative breast cancer (TNBC-SLCs) are the tumorigenic core for malignancy. Aberrant expression of the RON receptor tyrosine kinase has implications in TNBC tumorigenesis and malignancy.
In this study, we identified the RON receptor as a pathogenic factor contributing to TNBC cell stemness and validated anti-RON antibody-drug conjugate Zt/g4-MMAE for eradication of RONexpressing TNBC-SLCs.
Immunofluorescence and Western blotting were used for analyzing cellular marker expression. TNBC-SLCs were isolated by magnetic-immunofluorescence cell-sorting techniques. Spheroids were generated using the ultralow adhesion culture methods. Levels of TNBC-SLC chemosensitivity were determined by MTS assays. TNBC-SLC mediated tumor growth was determined in athymic nude mice. The effectiveness of Zt/g4-induced RON internalization was measured by immunofluorescence analysis. Efficacies of Zt/g4-MMAE in killing TNBC-SLCs in vitro and in eradicating TNBC-SLCmediated tumors were determined in mouse models. All data were statistically analyzed using the GraphPad Prism 7 software.
Increased RON expression existed in TNBC-SLCs with CD44+/CD24- phenotypes and ALDH activities and facilitated epithelial to mesenchymal transition. RON-positive TNBC-SLCs enhanced spheroid-formatting capability compared to RON-negative TNBC-SLCs, which were sensitive to small molecule kinase inhibitor BMS-777607. Increased RON expression also promoted TNBC-SLC chemoresistance and facilitated tumor growth at an accelerated rate. In vitro, Zt/g4-MMAE caused massive TNBC-SLC death with an average IC50 value of ~1.56 μg per/ml and impaired TNBC cell spheroid formation. In mice, Zt/g4-MMAE effectively inhibited and/or eradicated TNBC-SLC mediated tumors in a single agent regimen.
Sustained RON expression contributes to TNBC-SLC tumorigenesis. Zt/g4-MMAE is found to be effective in vivo in killing TNBC-SLC-mediated xenograft tumors. Our findings highlight the feasibility of Zt/g4-MMAE for the eradication of TNBC-SLCs in the future.
三阴性乳腺癌中的癌干细胞（TNBC - SLCs）是恶性肿瘤的致瘤核心。RON受体酪氨酸激酶的异常表达与TNBC的肿瘤发生和恶性程度有关。
在本研究中，我们确定RON受体是导致TNBC细胞干性的致病因素，并验证了抗RON抗体 - 药物偶联物Zt/g4 - MMAE对根除表达RON的TNBC - SLCs的作用。
采用免疫荧光和蛋白质印迹法分析细胞标志物表达。通过磁免疫荧光细胞分选技术分离TNBC - SLCs。使用超低附着培养方法生成球体。通过MTS试验测定TNBC - SLCs的化学敏感性水平。在无胸腺裸鼠中测定TNBC - SLC介导的肿瘤生长。通过免疫荧光分析测量Zt/g4诱导的RON内化的有效性。在小鼠模型中确定Zt/g4 - MMAE在体外杀死TNBC - SLCs以及根除TNBC - SLC介导的肿瘤的疗效。所有数据均使用GraphPad Prism 7软件进行统计分析。
在具有CD44 + /CD24 - 表型和ALDH活性的TNBC - SLCs中，RON表达增加，并促进上皮 - 间质转化。与RON阴性的TNBC - SLCs相比，RON阳性的TNBC - SLCs增强了球体形成能力，后者对小分子激酶抑制剂BMS - 777607敏感。RON表达增加还促进了TNBC - SLCs的化学抗性，并加速了肿瘤生长。在体外，Zt/g4 - MMAE导致大量TNBC - SLCs死亡，平均IC50值约为每毫升1.56μg，并损害TNBC细胞球体形成。在小鼠中，Zt/g4 - MMAE在单药治疗方案中有效抑制和/或根除TNBC - SLC介导的肿瘤。
持续的RON表达有助于TNBC - SLCs的肿瘤发生。发现Zt/g4 - MMAE在体内对杀死TNBC - SLC介导的异种移植肿瘤有效。我们的研究结果突出了Zt/g4 - MMAE在未来根除TNBC - SLCs方面的可行性。
