花生四烯酸通过5-脂氧合酶调控结直肠癌肿瘤免疫微环境的研究

The efficacy of immune checkpoint blockade in the treatment of advanced colorectal cancer (CRC) is limited to 5% of the population with microsatellite instability-high (MSI-H). Looking for a combined target is the key point to expand its application. The patient’s tumor burden is high in advanced stage. In order to imitate the situation in human body, we adopted mouse model with a high tumor burden of mouse CRC cells. The results showed that the therapeutic effect of PD-1 antibody was poor in high tumor burden model, while short-term high-fat diet could significantly enhance the therapeutic effect of PD-1 antibody treatment. To clarify the mechanism of this phenomenon, we compared the serum metabolites of high fat diet group with normal diet group after treatment with PD-1 antibody under high tumor load models by metabonomics. We found that the arachidonic acid (ARA) metabolic pathway changed most obviously, and the up-regulation of 5-lipoxygenase (5-LOX) gene was the highest in ARA metabolic pathway. By down-regulating 5-LOX in cancer cells, T cells infiltration in tumor tissues was significantly reduced. Therefore, the objectives of the current project are to clarify the effect of ARA on the immune microenvironment of CRC tumors through 5-LOX, explore the mechanism of 5-LOX in promoting T cell recruitment and cytotoxic function, and reverse the drug resistance of immunotherapy through targeting ARA. Completion of this study will not only reveal the mechanism of ARA regulating tumor immunity, but also clarify the synergistic effect of ARA and PD-1 antibody, thus providing a new idea for the combined regimens of CRC immunotherapy.

免疫检查点阻断在晚期结直肠癌（CRC）治疗中疗效仅限于5%的微卫星高度不稳定人群，寻找联合靶点是拓展其应用的关键。为模拟晚期患者肿瘤负荷高的情况，我们采用肿瘤负荷高的小鼠CRC细胞皮下成瘤模型。结果显示PD-1抗体在高肿瘤负荷模型中疗效欠佳，而PD-1抗体治疗时给予短期高脂饮食可显著增强其疗效。为明确其机制，我们采用代谢组学比较高肿瘤负荷下PD-1抗体治疗后高脂与正常饮食组的血清代谢物，发现花生四烯酸（ARA）代谢通路改变最明显，其中5-脂氧合酶（5-LOX）基因上调最高。下调肿瘤细胞的5-LOX显著减少肿瘤组织中T细胞浸润。因此，本项目拟明确ARA通过5-LOX对CRC肿瘤免疫微环境产生影响，探索5-LOX促进T细胞招募和杀伤功能的机制，并通过靶向ARA逆转免疫治疗耐药。该问题的解决将揭示ARA调控肿瘤免疫的机制，明确ARA和PD-1抗体的协同作用，为CRC免疫治疗联合方案提供新思路。

以免疫检查点抑制剂为代表的免疫疗法在实体瘤的治疗中具有里程碑式的意义，极大地提升了实体瘤患者的生存。但在结直肠癌（CRC）中，免疫治疗目前仍面临有效率低、缺乏有效联合治疗方案的问题。本研究针对一种Omega-6多不饱和脂肪酸，花生四烯酸（ARA），开展相关研究。ARA作为一种常见的食品添加剂，可促进生长发育，在保健品及医药领域具有广泛的应用。在本研究中，我们在C57BL/6J小鼠中构建了鼠源肠癌肿瘤细胞系MC38皮下成瘤模型，发现外源性给予ARA治疗能够显著促进PD-1抗体的疗效。接下来我们对给予ARA及PD-1抗体单独或联合处理的小鼠肠道内容物进行宏基因组测序，分析结果表明ARA能够改善肠道微生态，增加肠道中益生菌的比例，改变肠道菌群的代谢。进一步我们收集了ARA及PD-1抗体单独或联合处理的小鼠的肠道内容物及血清，针对ARA代谢通路进行靶向代谢组学分析。结果表明ARA能够经肠道菌群代谢产生12(S)-HETE，并进入血液循环。我们的结果首次发现ARA通过调节肠道菌群发挥促进免疫治疗疗效的功能，并首次提出肠道菌群代谢产物12(S)-HETE在免疫治疗中发挥关键作用。本项目的发现将为ARA联合肠菌制剂在CRC免疫治疗中的临床转化应用提供理论基础。