State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 17 Panjiayuan Subdistrict, Chaoyang, Beijing, 100021, People's Republic of China.
National Center for Orthopaedics, Beijing Research Institute of Traumatology and Orthopaedics, Beijing Jishuitan Hospital, Capital Medical University, No. 31 Xinjiekou E Road, Xicheng, Beijing, 100035, People's Republic of China.
Stem Cell Res Ther. 2023 Sep 27;14(1):273. doi: 10.1186/s13287-023-03453-x.
Identification of promising targeted antigens that exhibited cancer-specific expression is a crucial step in the development of novel antibody-targeted therapies. We here aimed to investigate the anti-tumor activity of a novel monoclonal antibody (mAb) 11C9 and identify the antibody tractable target in the hepatocellular cancer stem cells (HCSCs).
The identification of the targeted antigen was conducted using SDS-PAGE, western blot, mass spectrometry, and co-immunoprecipitation. Silence of HSP90 was induced by siRNA interference. Positive cells were sorted by fluorescence-activated cell sorting. Double-immunofluorescent (IF) staining and two-color flow cytometry detected the co-expression. Self-renewal, invasion, and drug resistance were assessed by sphere formation, matrigel-coated Transwell assay, and CCK-8 assay, respectively. Tumorigenicity was evaluated in mouse xenograft models. RNA-seq and bioinformatics analysis were performed to explore the mechanism of mAb 11C9 and potential targets.
MAb 11C9 inhibited invasion and self-renewal abilities of HCC cell lines and reversed the cisplatin resistance. HSP90 (~ 95 kDa) was identified as a targeted antigen of mAb 11C9. Tissue microarrays and online databases revealed that HSP90 was overexpressed in HCC and associated with a poor prognosis. FACS and double-IF staining showed the co-expression of HSP90 and CSCs markers (CD90 and ESA). In vitro and in vivo demonstrated the tumorigenic potentials of HSP90. The inhibition of HSP90 by siRNA interference or 17-AAG inhibitor both decreased the number of invasion, sphere cells, and CD90 or ESA cells, as well as reversed the resistance. Bioinformatics analysis and western blot verified that HSP90 activated Wnt/β-catenin signaling.
The study preliminarily revealed the anti-tumor activity of mAb 11C9. More importantly, we identified HSP90 as a targeted antigen of mAb 11C9, which functions as an oncogene in phenotype shaping, stemness maintenance, and therapeutic resistance by activating Wnt/β-catenin signaling.
鉴定具有癌症特异性表达的有前途的靶向抗原是开发新型抗体靶向治疗的关键步骤。我们旨在研究新型单克隆抗体（mAb）11C9 的抗肿瘤活性，并鉴定肝癌干细胞（HCSC）中的抗体可靶向目标。
使用 SDS-PAGE、western blot、质谱和免疫共沉淀鉴定靶向抗原。通过 siRNA 干扰诱导 HSP90 沉默。通过荧光激活细胞分选对阳性细胞进行分选。双免疫荧光（IF）染色和双色流式细胞术检测共表达。通过球体形成、基质胶包被 Transwell 测定和 CCK-8 测定分别评估自我更新、侵袭和耐药性。通过小鼠异种移植模型评估致瘤性。进行 RNA-seq 和生物信息学分析以探讨 mAb 11C9 的作用机制和潜在靶点。
mAb 11C9 抑制 HCC 细胞系的侵袭和自我更新能力，并逆转顺铂耐药性。HSP90（~95kDa）被鉴定为 mAb 11C9 的靶向抗原。组织微阵列和在线数据库显示 HSP90 在 HCC 中过度表达，并与预后不良相关。FACS 和双 IF 染色显示 HSP90 与 CSCs 标志物（CD90 和 ESA）的共表达。体内外研究均显示 HSP90 的致瘤潜力。siRNA 干扰或 17-AAG 抑制剂抑制 HSP90 均可减少侵袭、球体细胞以及 CD90 或 ESA 细胞的数量，并逆转耐药性。生物信息学分析和 western blot 验证 HSP90 激活 Wnt/β-catenin 信号通路。
该研究初步揭示了 mAb 11C9 的抗肿瘤活性。更重要的是，我们鉴定 HSP90 为 mAb 11C9 的靶向抗原，该抗原通过激活 Wnt/β-catenin 信号通路在表型形成、干细胞维持和治疗耐药性中发挥癌基因作用。
