基于单细胞膜蛋白双模态分析的循环乳腺癌细胞精准鉴定与分型研究

Breast cancer is a malignant tumor that is highly heterogeneous and aggressive, making it difficult to be diagnosed and treated. Developing technologies for early detection and accurate identification of metastatic subgroups is the key to promote personalized treatment. Circulating tumor cells (CTCs), presented in the breast cancer patient, can serve as a “substitute” of the tumor tissue. Its molecular phenotype provides real-time, dynamic, and noninvasive “liquid biopsy” to assess metastatic process. However, recent CTC technologies mostly focus on genotype analysis, although the phenotype changes (e.g. membrane proteins) can better reflect the process of cancer metastasis, the ability of technology for protein-based phenotyping is limited. So, this project is proposed to address the challenge. In the project, signal amplification strategies will be first developed to make the sensitivity suitable for protein profiling at the single-cell level. Then, with the dual-mode analysis method, the localizations and expression states of several metastasis-associated membrane proteins will be analyzed. By integrating the two dimensions of information (localization and quantification) of these proteins, correlation between molecular phenotype and metastatic ability of CTC can be established, which will ultimately provide technical support for the accurate typing and metastatic assessment of breast cancer. Therefore, the implementation of this project is anticipated to give a multi-dimensional analytical tool for membrane proteins at the single-cell level, and also provide technical support for accurate assessment of cancer metastasis.

乳腺癌的易转移性和高异质性使其临床诊断和治疗面临巨大挑战。乳腺癌循环肿瘤细胞（CTC）作为癌症早期转移实时、动态、无创的“液体活检”指标，其分子表型亦可用来评估肿瘤的异质性。发展CTC监测技术，并对其进行精准鉴定与分型，是促进乳腺癌转移早期发现和实施个性化治疗的关键。目前已发展多种基于单细胞核酸分析的CTC技术，然而，比核酸分析更能体现癌转移过程实效性的膜蛋白动态变化的深度分析尚处于发展初期。基于此，本项目拟开发基于核酸扩增的信号放大策略，针对乳腺癌CTC关键膜蛋白分子，发展单细胞膜蛋白的双模态分析技术(定位和定量)，并综合利用这两个维度的信息，精准分析CTC的远端转移与其分子表型特征的关联性，最终为乳腺癌CTC的精准分型和转移潜能评估提供重要的技术支撑。该项目的实施一方面将为单细胞膜蛋白研究提供一种多维度的分析工具，另一方面可为癌转移的评估提供准确的临床依据。

细胞膜蛋白的多维分析可以精准鉴定循环肿瘤细胞（CTC）的分子表型特征，有助于侵袭性肿瘤的临床评估；此外，单细胞水平的CTC膜蛋白分析无法采用常规的蛋白质生化分析方法，不利于CTC在侵袭性肿瘤临床诊断中的应用。由此，该项目发展了单细胞水平的膜蛋白分析方法，提高细胞膜蛋白分析的灵敏性、准确性、便捷性，实现在不破坏细胞结构与功能情况下的膜蛋白原位双模态分析（定量和成像）。随后，该项目利用多种癌转移标志蛋白的组合作为肿瘤细胞的“身份标签”，以获得丰富的分子表型信息，用来判断循环肿瘤细胞的侵袭特征。最后，使用上述精准的分型技术和信号放大策略，构建了能够识别上述增强特征的电化学传感界面和光学检测体系，以实现对侵袭性乳腺癌细胞的检测，并在实验和临床环境下进行样本分析。综上所述，该项目实现了单细胞膜蛋白的双模态分析方法的构建，对乳腺癌膜标志蛋白的定量和成像分析，进而对转移型乳腺癌细胞的分子表型进行精准地分型和鉴定，为乳腺癌转移的诊断和预后监测提供了重要的临床判断依据和技术支撑。