PIPAAm修饰的ThermoRelease系统在肺腺癌侵袭转移、预后评估及探索耐药分子机制中的研究

Along with the proposal of precision medicine, circulating cancer cell (CTC) detection is increasingly becoming a hot field in cancer research, for its minimal invasiveness, convenient specimen obtaining and dynamic disease monitoring. Based on our previous studies, two generations of CTC chips have been developed to meet different clinical need: the first-gen NanoVelcro chip recruited CTC efficiently and the second-gen PLGA chip achieved the molecular analysis of CTC. However, both of them failed to accomplish CTC enumeration, molecular analysis and in vitro cultivation synchronously. ThermoRelease system established in this project introduced a thermo-sensitive material PIPAAm, which could specifically capture CTCs under 37℃ and then rapidly release them under 4℃ with super viability. We will further finely adjust some parameters, such as microfluid, temperature-controlled cycle and so on, to optimize our system in order to attain accurate CTC number from patients. According to above CTC numbers, we will set cut-off value to predict the prognosis, recurrence and metastasis for lung adenocarcinoma. Then, by comparing the gene differentiation between CTC and primary tumor and analyzing the relationship between gene changes and functions in in vitro cultures CTCs, we will explore the molecule mechanism of lung adenocarcinoma invasion and metastasis. ARMS PCR and NGS technologies were used to dynamically detect multi-gene change and to evaluate the link of these changes with therapeutic effect. Our study aims to illuminate the molecular mechanism of drug-resistance in lung adenocarcinoma and to provide a more reliable detection method for the precision treatment of lung adenocarcinoma.

随着精准医学概念的提出，循环肿瘤细胞（CTC）检测因其具有微创、获取便利、可动态监控病情等优点日益成为肿瘤邻域的研究热点。我们前期研制的第一代NanoVelcro芯片可高效捕获CTC；第二代PLGA芯片实现了CTC的分子检测；然而，他们无法同时进行CTC计数、分子检测和体外培养。本课题拟研发的ThermoRelease系统以热敏材料PIPAAm为基底，通过微流控、温控循环等优化实现37℃特异捕获CTC，4℃快速释放CTC并达到90%以上良好的细胞活性；进一步利用CTC计数预测肺腺癌的预后、复发和转移；通过比较CTC与原发肿瘤的基因差异，分析体外培养的CTC功能与基因改变之间的关系，深入探明肺腺癌侵袭转移的机制；通过ARMS PCR和NGS等技术动态分析CTC多基因改变与药物疗效的相关性，阐明肺腺癌靶向药物的耐药机制。该课题的顺利实施将为肺腺癌的精准治疗提供更加可靠的检测手段。

随着精准医疗时代的到来，循环肿瘤细胞（CTC）检测因其具有微创、获取便利、可动态监控病情等特点在肿瘤筛查、疗效评估、个体化治疗、预后监测等方面具有独特优势。为了更好地检测CTC，本课题组将Thermoresponsive聚合物刷子PIPAAm移植到NanoVelcro基底Thermoresponsive NanoVelcro芯片上构建了ThermoRelease系统，利用人工CTC和肺腺癌病人CTC样品验证了该系统在在37°C和4°C捕获和释放CTC的有效性；并且利用Thermoresponsive NanoVelcro系统纯化的CTC样本，建立了早期诊断和预测肺腺癌转移的CTC Cut-off值；通过纯化的肺腺癌 CTC体外培养及裸鼠成瘤实验，研究了药物敏感性；通过ARMS PCR和NGS等技术动态分析CTC EGFR突变， 揭示了T790M突变与吉非替尼诱导获得性耐药的相关性，为早期预后提供了可能；通过体外实验和动物模型，证明了CD155和CD96之间的直接相互作用促进了免疫抑制，以及HIF-1ɑ-调控的miR-1275通过同时激活Wnt/β-catenin和Notch信号通路，来维持肺腺癌细胞的干细胞样表型并促进癌症进展。该课题的成果为肺腺癌的精准治疗提供了更加可靠的检测手段，进一步利用了CTC计数预测肺腺癌的预后、复发和转移，深入探明了肺腺癌侵袭转移的机制，并且为寻找肺腺癌靶向药物提供了新的研究方向。