基于CD44和ESA介导的乳腺癌干细胞多靶向分子成像的实验研究

Breast cancer stem cells (BCSCs) are the chief culprit in development,invasion,and recurrence of breast cancers. The efficacy of single targeted therapy for breast cancer is poor due to multi-genetic variation,so targeted therapy for the BCSC based on multiple targets will be a new trend for breast cancer treatment. Therefore,in vivo molecular imaging based on multiple targets for the BCSCs is significant in clinic. In our earlier experiment,paramagnetic gadolinium (Gd3+) coupling of different size of the non-cadmium quantum dots (QDs) were successfully prepared for producing bi-mode nanoparticles (pQDs) of MR-fluorescence imaging,yielding different wavelengths due to the different diameters of QDs such as QD700 and QD800. BCSCs and models of BCSC-bearing mice were respectively and successfully isolated and established. On the basis of a new conjugated dual-mode nanoparticle (pQDs),the monoclonal antibodies of CD44 and ESA are respectively labeled with pQD700 and pQD800 in order to build a kind of bi-targeted and bi -mode molecular probe (pQD700-CD44mAb and pQD800-ESAmAb) with high sensitivity and strong targeting. With three-dimensional quantitative in vivo fluorescence imager and high-field MR scanner,BCSCs will be imaging and the different targets will be visually distinguished in vivo via dual-targeted probe. Thereby,the information of BCSCs in tumors and sentinel nodes including position,content,distribution and other results can be obtained and compared with the results of electron microscopy and immunohistochemistry. This project,through exploring BCSCs with multi-targeted imaging and quantitative analysis,aims at providing a new strategy for targeted diagnosis and therapy of breast cancer.

乳腺癌干细胞(BCSC)是乳腺癌发生、侵袭和复发的主要因素，目前单一靶标的肿瘤靶向治疗对多基因变异的乳腺癌疗效不佳，针对BCSC的多靶向治疗将是乳腺癌治疗的一个新方向。因此，对BCSC进行活体多靶向分子成像具有重要意义。本课题组前期将顺磁性钆(Gd3+)偶联不同粒径的非镉量子点(QDs)成功制备了不同波长的MR-荧光双模态纳米颗粒(pQDs)，并在成功分离BCSC和构建乳腺癌动物模型基础上，拟采用CD44和ESA单克隆抗体分别标记pQDs，构建两种敏感性高、靶向性强的双模态分子探针(pQD700-CD44mAb和pQD800-ESAmAb)。借助高场MR和三维定量荧光成像仪，对BCSC进行活体成像并对不同靶标进行可视化区分，将获得的乳腺癌及前哨淋巴结中BCSC含量、分布等定量信息与电镜、免疫组化进行对照印证。本项目通过研究BCSC活体多靶向成像及定量分析，旨在为乳腺癌多靶向诊治提供新策略。

本项目通过构建靶向于乳腺癌干细胞（BCSC）的磁共振-近红外荧光双模态分子探针，并制备能在同一激发光下呈现不同荧光的靶向于BCSC不同靶标的两种双模态探针，以达到对乳腺癌中BCSC靶向成像及可视化区分靶标的目的。主要结果：（1）采用相对无毒的非镉量子点，调控合成工艺参数、筛选合适配体，及惰性生物蛋白BSA表面修饰改性，并通过方法改良抑制了量子点制备过程中蓝移现象。制备出了单分散性好、结晶度高、荧光效率佳、发射波长可调的非镉亲水性核壳量子点(QDs)。（2）将QDs与Gd偶联，合成了具有更高T1驰豫率、同一激发光下产生不同颜色荧光的顺磁性非镉量子点pQDs，pQDs再偶联单克隆抗体，进而构建了能靶向结合于BCSC细胞膜上CD44与ESA的分子探针。经一系列实验证实，分子探针具有较高成像敏感性、靶向特异性及生物安全性。（3）成功分离出CD44＋/CD24－、CD44＋/ ESA＋/CD24－的BCSC，其具有在无血清培养液中成球生长并无限传代的能力。将该BCSC种植裸鼠皮下及乳腺原位，成功构建了富含BCSC的荷瘤裸鼠乳腺癌模型。（4）首次针对乳腺癌干细胞进行体外细胞及在体荷瘤乳腺癌的MR-荧光双模态靶向分子成像和定量分析。并利用同一激发光下产生不同颜色荧光的两种靶向探针对不同靶标进行可视化区分。.本项目研究结果的科学意义在于：(1)为肿瘤早期诊断和分级提供“新模式”。利用可视的影像学方法，以BCSC在瘤组织中所占比例进一步对肿瘤进行评价，可更科学全面地反映乳腺癌的生物学行为，评估转移概率、预后等；(2)为肿瘤治疗提供精确信息，为疗效提供新的评估方法。依据乳腺癌及前哨淋巴结中的BCSC，来制定治疗计划，以及依据BCSC灭活和残存情况，来早期无创性评价治疗疗效，及时调整并加强针对BCSC的治疗方案，将可能更有效预防乳腺癌复发和转移。