基于3D-肺癌类器官模型探讨"清热"中药对吉非替尼的增敏作用及代谢影响

It is very important for Traditional Chinese Medicine (TCM) clinical practice to know which TCM treatment principle should be combined with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Our team has been working on this purpose for several years, and serial of researches have been carried out, including: 1) Clinical epidemiology study found that patients with EGFR gene mutation were more with Yin-cold TCM syndrome type. After EGFR-TKI treatment, resistant patients were more with Yang-heat syndrome type. Thus, gefitinib had kind of TCM “warming-yang” effect. According to TCM theory, it should be combined with “clearing-heat” TCM drugs. 2) Experiments in A549 and H1650 cell lines indicated that Qing-kai-ling (QKL), a kind of “clearing-heat” TCM injection, had synergistic effect with gefitinib, while Shen-fu, a kind of “warming-yang” TCM injection, had antagonism effect. This also agreed with our hypothesis. 3) Our previous experiments found that cells treated with QKL+gefitinib had less glycolysis and more oxidative phosphorylation (OXPHOS). Studies reported that gefetinib resistant cancer stem cells had more glycolysis, which is much different from cells treated with QKL+gefitinib. Interestingly, another study indicated that OXPHOS in stem cells increased ROS, and then induced differentiation. Therefore, the synergistic effect of QKL to gefitinib may result from the metabolism reprogramming. In this study, we will use kinds of TCM injections with “clearing-heat” effect to prove our hypothesis that EGFR-TKI should be combined with “clearing-heat” treatment principle. What’s more, we will use the lung cancer 3D-organoid models, the preferred model for stem cell researches because of the stem cell niches, to find out the metabolism and ROS mechanism of this synergistic effect. Our research will be the theoretical basis for TCM treatment principle combined with EGFR-TKIs.

明确表皮生长因子酪氨酸激酶抑制剂（EGFR-TKI）应该与哪种治则中药联合具有重要的临床意义。我们团队已经开展了系列研究：1）流行病学研究发现EGFR突变患者以寒证居多，EGFR-TKI耐药时热证患者显著增多，提示EGFR-TKI具有“温阳”作用，很可能应该与“清热”中药联合；2）清热的清开灵与吉非替尼在细胞模型中具有协同作用，温阳的参附具有拮抗作用，支持上述假说；3）预实验发现清开灵+吉非替尼组有氧呼吸增加、无氧酵解减少，与文献中吉非替尼耐药干细胞的糖酵解代谢特征相反，而有氧呼吸及活性氧簇（ROS）是干细胞分化的重要信号，因此清热中药很可能通过代谢重排与吉非替尼发挥协同作用。本研究一方面利用多种清热中成药注射液验证EGFR-TKI是否应该与清热治则联合；另一方面以3D-肺癌类器官为模型模拟干细胞憩室，从干细胞能量代谢及ROS角度探讨其机制，为中医药联合EGFR-TKI提供治则方面的指导

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）是治疗晚期EGFR敏感突变非小细胞肺癌的首选药物，但耐药问题仍然是限制其临床疗效的瓶颈问题。要寻找中医药逆转EGFR-TKI耐药的方法，首先应该根据其耐药核心病机，明确中医的治则治法，然后指导处方用药，即开展“理-法-方-药”的系列研究。我们前期已经明确了EGFR-TKI的“温阳”作用，即明确了“理”，据此提出假说：中药逆转EGFR-TKI耐药应该以“清热”为法，即探索“法”。为验证该假说，本研究利用2D细胞株、3D细胞球、3D肺癌类器官及荷瘤小鼠多个模型，开展以下研究：.1）成功构建了肺癌类器官模型并应用于药敏实验和机制研究，积累了肺癌类器官研究经验；.2）多次实验、多个模型中均发现，细胞氧化还原比（Redox）下调，导致活性氧簇（ROS）下调，促进肿瘤干细胞干性维持（“Redox/ROS/干细胞”轴）是吉非替尼耐药的重要机制之一；.3）在多个模型中证实了清热中药清开灵、痰热清、热毒宁逆转吉非替尼耐药的疗效，而温阳中药参附与吉非替尼在部分模型中甚至表现出拮抗作用，进一步验证了中药逆转EGFR-TKI耐药的“清热”治则；.4）在多个模型中证实“Redox/ROS/干细胞”轴是清热中药清开灵、痰热清、热毒宁逆转吉非替尼耐药的重要机制之一：上调氧化还原比，上调ROS，抑制肿瘤干细胞。.该项目一方面明确了中医药逆转EGFR-TKI耐药的“清热”治则，为临床解决EGFR-TKI耐药这一瓶颈问题提供了治则方面的指导；另一方面验证了“Redox/ROS/干细胞”轴是EGFR-TKI耐药的重要机制之一，清热中药靶向该通路可发挥显著的增敏作用，提示“Redox/ROS/干细胞”是有效的靶点，从而为逆转EGFR-TKI耐药的新药开发提供了新靶点，具有较好的应用开发前景。至此，我们项目组已完成了中医药逆转EGFR-TKI耐药的“理-法”研究，下一步以“清热”治则为指导，我们将开展“方-药”研究：清热经典方剂逆转EGFR-TKI耐药的疗效和机制研究。