摘要
目的进行CD44^(+)CD24^(-)三阴性乳腺癌(TNBC)干细胞样细胞富集及生物学行为研究, 并构建^(68)Ga标记的CD44多肽[^(68)Ga-1, 4, 7-三氮杂环壬烷-1, 4, 7-三乙酸(NOTA)-CD44p], 评价其对TNBC干细胞样细胞表面标志物CD44的靶向能力。方法采用悬浮球培养法, 进行TNBC细胞株MDA-MB-231与非TNBC细胞株MCF-7中的CD44^(+)CD24^(-)细胞亚群的富集培养。采用流式细胞仪检测不同组干细胞标志物的表达情况, 用细胞划痕实验检测CD44^(+)CD24^(-)细胞亚群迁移能力, 通过Transwell侵袭实验评价CD44^(+)CD24^(-)细胞亚群的侵袭能力。制备^(68)Ga-NOTA-CD44p, 并用高效液相色谱(HPLC)法对其进行纯化和鉴定。通过细胞摄取和阻断实验评价^(68)Ga-NOTA-CD44p对CD44^(+) TNBC细胞的靶向能力。采用两独立样本t检验和单因素方差分析(最小显著差异t检验)进行数据分析。结果悬浮球培养法成功富集CD44^(+)CD24^(-) TNBC干细胞样细胞球。与非TNBC细胞MCF-7细胞相比, TNBC细胞MDA-MB-231细胞表现出更好的成球能力[(18.50±3.73)与(31.83±4.92)个;t=5.29, P<0.001]以及更高的CD44^(+)CD24^(-)细胞亚群占比[(24.97±8.12)%与(90.93±4.46)%;F=170.10, t=14.93, 均P<0.001]。CD44^(+)CD24^(-) MDA-MB-231组较CD44^(+)CD24^(-) MCF-7组细胞的伤口愈合率[(71.00±11.00)%与(28.33±4.16)%;F=42.91, t=8.02, 均P<0.001]、侵袭率[(60.60±16.87)%与(24.16±8.15)%;F=11.83, t=4.40, 均P<0.01]明显升高。MDA-MB-231对^(68)Ga-NOTA-CD44p具有较强的摄取能力, CD44p阻断后摄取能力下降。结论与CD44^(+)CD24^(-) MCF-7相比, CD44^(+)CD24^(-) MDA-MB-231表现出更高的恶性生物学行为;^(68)Ga-NOTA-CD44p可靶向TNBC干细胞样细胞表面标志物CD44, 研究为以肿瘤干细胞为靶点的TNBC靶向治疗手段的建立奠定了基础。
Objective:To conduct enrichment and biological behavior studies on CD44^(+)CD24^(-)triple-negative breast cancer(TNBC)stem-like cells,and to construct ^(68)Ga-labeled CD44 peptide(^(68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid(NOTA)-CD44p)and evaluate its targeting ability towards the surface marker CD44 of TNBC stem-like cells.Methods:Suspension sphere culture method was utilized to enrich and cultivate CD44^(+)CD24^(-)cell subpopulations from TNBC cell line MDA-MB-231 and non-TNBC cell line MCF-7.Flow cytometry was used to detect the expression of stem cell markers of different groups,cell scratch assay was performed to assess the migration ability of CD44^(+)CD24^(-)cell subpopulations,and Transwell invasion assay was performed to evaluate the invasion ability of CD44^(+)CD24^(-)cell subpopulations.^(68)Ga-NOTA-CD44p was prepared,followed by purification and identification with high-performance liquid chromatography(HPLC).The targeting ability of ^(68)Ga-NOTA-CD44p towards CD44^(+)TNBC cells was evaluated through cellular uptake and blocking experiments.Data were analyzed by independent-sample t test,one-way analysis of variance and the least significant difference t test.Results:Suspension sphere culture successfully enriched CD44^(+)CD24^(-)TNBC stem-like cell spheres.Compared to the non-TNBC cell line MCF-7,TNBC cell line MDA-MB-231 exhibited better sphere-forming ability(18.50±3.73 vs 31.83±4.92;t=5.29,P<0.001)and a higher proportion of CD44^(+)CD24^(-)cell subset((24.97±8.12)%vs(90.93±4.46)%;F=170.10,t=14.93,both P<0.001).The wound healing rate((71.00±11.00)%vs(28.33±4.16)%;F=42.91,t=8.02,both P<0.001)and invasion rate((60.60±16.87)%vs(24.16±8.15)%;F=11.83,t=4.40,both P<0.01)of CD44^(+)CD24^(-)MDA-MB-231 group cells were significantly increased compared to the CD44^(+)CD24^(-)MCF-7 group.MDA-MB-231 cells showed strong uptake ability of ^(68)Ga-NOTA-CD44p,which decreased after CD44p blocking.Conclusions:Compared to CD44^(+)CD24^(-)MCF-7 cells,CD44^(+)CD24^(-)MDA-MB-231 cells exhibit higher malignant biological behavior.^(68)Ga-NOTA-CD44p targets the surface marker CD44 of TNBC stem-like cells,laying the research foundation for targeted therapy against TNBC with tumor stem cells as targets.
作者
杨蕊
朱锐颖
宿晨
程凯
周洁
贾贞
达梦婷
赵久达
陈道桢
Yang Rui;Zhu Ruiying;Su Chen;Cheng Kai;Zhou Jie;Jia Zhen;Da Mengting;Zhao Jiuda;Chen Daozhen(Department of Eugenic Genetics Institute,Jiangnan University Maternity Hospital,the Affiliated Wuxi Maternity and Child Health Care Hospital of Nanjing Medical University,Wuxi 214002,China;Clinical Laboratory,the Second People's Hospital of Haidong City,Haidong 810600,China;Breast Disease Treatment Center,Qinghai University Affiliated Hospital,Xining 810000,China)
出处
《中华核医学与分子影像杂志》
北大核心
2025年第5期294-299,共6页
Chinese Journal of Nuclear Medicine and Molecular Imaging
基金
青海省基础研究计划(2022-ZJ-912)。
