基于靶向组织蛋白酶的新型荧光智能探针实时评估乳腺癌保乳切缘的研究

Breast-conserving surgery is standard of care for patients with early breast cancer. Precise excision of the primary tumor is essential for reducing local recurrence and preserving cosmetic effects. However, currently, it is a lack of effective techniques for intraoperatively guiding precise tumor excision in the clinic. Cathepsins are highly expressed in breast cancer tissue and low expressed in normal breast tissue. This makes them potential targets for tumor-targeted probes. Based on this, we previously creatively developed an indocyanine green near-infrared smart probe, which can be exclusively activated by cathepsins. This probe demonstrated tumor-specific uptake in a preliminary experiment on a mouse breast tumor allograft model. Therefore, it is a potential candidate probe for fluorescent surgical navigation in real-time. In this project, we will clarify the characteristics of the novel probe, including its stability, safety, and specificity. Moreover, we will investigate the optimal dose of the probe and imaging time point for in vivo fluorescent molecular imaging in a mouse breast tumor model. Furthermore, we will investigate the feasibility and accuracy of using this probe for guiding surgical excision of the allograft tumors. Finally, we will test the specificity of the probe in identifying human breast cancer tissue using fresh surgical specimens from patients with breast cancer to assess its value of clinical translation. The aim of this project is to establish standard operation and evaluation frameworks of using fluorescent smart probes for fluorescent image-guided surgery. This will establish the foundation for further exploring the application of these probes in surgical navigation in breast-conserving surgery.

保乳手术是早期乳腺癌患者的标准治疗方案。肿瘤的精准切除是降低保乳术后局部复发率的关键，也是获得良好乳房美容效果的保障。然而目前临床缺乏有效手段用于术中实时指导肿瘤精准切除。利用组织蛋白酶在乳腺癌和瘤周正常组织中差异表达的特性，本团队前期创新性地合成了能被组织蛋白酶特异激活的吲哚菁绿近红外荧光智能探针。该探针在小鼠乳腺癌移植瘤模型上特异性识别肿瘤组织，具有用于实时荧光手术导航的潜能。本项目将首先明确该探针的稳定性、安全性和靶向特异性；然后，基于实时荧光分子成像技术，在小鼠移植瘤模型上研究该探针的最佳成像剂量和时间，并探索其用于移植瘤切除手术导航的可行性和精准性；最后在离体新鲜手术标本上评估该探针识别人体乳腺癌组织的特异性，以评价其临床转化应用价值。通过本项目，我们将建立基于荧光智能探针的肿瘤精准切除手术导航的操作标准和评估体系，为进一步探索该类型探针在乳腺癌保乳手术导航中的应用奠定基础。

基于我们前期研究发现，肿瘤靶向性光学分子影像技术在保乳手术导航领域仍然面临着由于探针半衰期较长导致的临床操作不方便、背景荧光干扰、假阳率高等问题。为此，本项目设计了新型的能被组织蛋白酶特异性激活的吲哚菁绿智能探针（VGT-309），并对以上科学问题进行系统而全面的研究。同时，本项目进一步通过利用蛋白组学和免疫组化等方法对乳腺癌预后标志物进行筛选鉴定，以便于后续相关分子影像探针的设计与应用研究，具体结果总结如下：.1）成功合成了新型能被组织蛋白酶特异性激活的吲哚菁绿智能探针VGT-309，该探针包含组织蛋白酶结合单元、荧光基团ICG及以及淬灭单元三个部分，未与组织蛋白酶结合时不被激活，因此不发光；而被组织蛋白酶激活后可失去淬灭单元而发光。.2）在小鼠巨噬细胞RAW 264.7中阐明了VGT-309的靶向特异性，VGT-309可特异性与组织蛋白酶B/L，X和S亚型相结合，并呈剂量依赖性，为后续动物实验提供了基础。.3）在小鼠4T1乳腺癌移植瘤模型中，探针注射1小时后，肿瘤部位即可观察到明显的荧光信号，且强度随着时间的延长而升高，而背景的肌肉组织的荧光信号则维持在很低的水平，证实了VGT-309在肿瘤组织中的特异性富集，以及可实现早期快速成像。.4）在小鼠4T1乳腺癌移植瘤模型中，VGT-309产生的荧光信号能准确指示肿瘤组织；在肿瘤切除后，术腔未观察到明显的荧光信号，对术腔进行取检，病理检查未见肿瘤组织残留。上述结果提示，利用VGT-309结合光学分子成像技术有望实现精准的恶性肿瘤切除手术导航。.5）蛋白组学结合生物信息学分析发现，ALDH3A2低表达与新辅助治疗获得pCR患者肿瘤复发风险升高相关。免疫组化染色及分析发现，HGF和CXCL12在乳腺癌中高表达与男性乳腺癌患者总生存改善具有相关性。筛选鉴定这些与乳腺癌预后相关的标志物有利于后续设计乳腺癌靶向性分子探针，进而有助于优化乳腺癌的诊治策略。.总之，组织蛋白酶特异性荧光探针VGT-309结合光学分子影像技术能够有效识别肿瘤并用于指导移植瘤切除手术。探索新型的乳腺癌预后相关标志物对后续分子探针研发具有重要的意义。