靶向S1P环境响应型多模态显像监测乳腺癌内分泌治疗耐药性的实验研究

Estrogen receptor positive (ER+) Breast cancer is common in women, resulting in significantly compromised survival. For these patients, tamoxifen (Tam), an estrogen receptor (ER) modifier, is used in clinical routines, since it was proven with survival benefits. However, noninvasive meanings to monitor and/or even predict the treatment response of this strategy is still urgently needed, considering that around 1/4 of these patients gain no treatment benefits from this treatment due to drug resistance. Unfortunately, the reason why ER+ breast cancer develop the drug resistance is yet far away from a consensus, with only vague evidences reported. Previous literature reports that Tam resistance is correlated to the increase of sphingosine 1-phosphate (S1P) and its receptors in ER+ breast cancer. Matrix metalloproteinase-2 (MMP-2) is an important component of breast cancer microenvironment and has been confirmed as a biomarker to predict the invasiveness of breast cancer in vivo and in vitro. In light of above, we designed an environmental-response nanoparticles with TPE labeled by 99mTc to target S1P and MMP-2 simultaneously, expecting a feasibility to explore the correlation between the expression levels of these two biomarkers, and Tam-resistance in ER+ breast cancer in vivo using radionuclides and fluorescence imaging. In short term, we would set up an optimal imaging protocols, which will provide a new multi-modal molecular imaging technology in vivo for non-invasive, early detection and real-time monitoring of S1P receptors and MMP-2 levels in ER+ breast cancer animal models and the relationship between Tam resistance and these changes. Ultimately, we believe that it could help early diagnosing of Tam resistance in ER+ breast cancer, predicting the invasiveness of breast cancer, and achieving the purpose of effectively predicting the prognosis of patients with ER+ breast cancer.

乳腺癌是影响女性健康的恶性肿瘤，选择性ER调节剂他莫昔芬（Tam）在雌激素受体阳性（ER+）患者内分泌治疗中被常规使用。然而约1/4患者出现Tam耐药，其机制不甚明了。研究显示Tam耐药乳腺癌与鞘氨醇-1-磷酸（S1P）及其受体表达增高相关，而基质金属蛋白酶-2（MMP-2）是肿瘤微环境的重要因子，已证实其预测乳腺癌侵袭性的作用。因此，本项目选取S1P受体及MMP-2为双功能靶点，设计环境响应型多功能纳米探针，并在连接四苯基乙烯（TPE）的基础上应用99mTc进行标记，以用于核素-荧光多模态显像，实现早期、无创、实时监测乳腺癌模型体内S1P1受体与MMP-2表达水平，及其变化与Tam耐药间的关系，为深入探究Tam耐药原理、早期灵敏筛选和有效预测ER+乳腺癌内分泌治疗耐药提供有效监测手段，为乳腺癌的精准诊疗提供依据。

目的：合成99mTc-HYNIC-S1P1mAb作为新的核素探针，进行荷瘤鼠的SPECT显像及相关实验，探讨核素探针在高表达S1PR1肿瘤模型显像中的可行性。.方法：使用SHNH作为螯合剂，连接S1PR1后再用99mTc标记，最终合成新的核素探针99mTc-HYNIC-S1P1mAb。使用PD10柱子纯化分子探针，纯化前后分别使用放射性层析扫描仪测定分子探针的标记率及放射化学纯度。将99mTc-HYNIC-S1P1mAb与正常SK-HEP-1细胞、MCF-7细胞一同孵育，分别收集细胞上清液及细胞裂解液，测定不同细胞在分子探针摄取的改变。构建SK-HEP-1及MCF-7荷瘤鼠模型，使用SPECT进行静态显像及生物分布研究。.结果：99mTc-HYNIC-S1P1mAb的标记率为61.45±9.16%（n=4），经过PD-10柱纯化后放化纯为96.7±0.04%（n=4）。实验组SK-HEP-1细胞对99mTc-HYNIC-S1P1mAb的摄取率随着时间延长而出现明显的增加，6h摄取率为10.90±0.79%（n=4），阻断组细胞对标记探针的摄取率显著低于实验组，6h摄取率为4.51±0.36%（n=4），MCF-7组6h摄取率为3.96±0.26%（n=4）。注射99mTc-HYNIC-S1P1mAb 后SK-HEP-1实验组荷瘤鼠的显像剂早期主要分布于肝脏，肿瘤12h开始隐约显影，18h以及24h肿瘤显影清晰；阻断组荷瘤鼠以及阴性组荷瘤鼠两组肿瘤在5个时间点均无明显显像剂浓聚，24h时阻断组盒阴性组荷瘤鼠的肿瘤对显像剂摄取明显低于同时间点实验组肿瘤。生物分布结果与SPECT显像结果相符。WB证实SK-HEP-1实体瘤的S1PR1受体高表达，而MCF-7肿瘤组织和肌肉组织中的S1PR1受体表达很低。免疫组化证实肺、肝脏、脾脏、肾脏、肌肉及MCF-7肿瘤组织中见少量S1PR1受体表达于血管内皮细胞，SK-HEP-1肿瘤组织高表达S1PR1受体，且分布于肿瘤细胞膜中。.结论：99mTc标记S1P1mAb具有较高的放化纯及良好的稳定性。体外及体内实验均表明99mTc-HYNIC-S1P1mAb与S1PR1高表达的SK-HEP-1细胞具有特异性及亲和力，用于靶向示踪S1PR1高表达的细胞是可行且有效的，可以作为基础进一步拓展该分子探针更多的应用。