PKM2调控肿瘤相关巨噬细胞Warburg效应促进食管癌转移的机制研究

The incidence rate of esophageal squamous cell carcinoma (ESCC) were 5 times higher in Kazakh in Xinjiang, compared with the national average in China. Control the invasion and metastasis is essential for the patients' prognosis. It has been reported that tumor-associated macrophages (TAMs) in tumor microenvironment plays a critical role in tumorigenesis and invision. TAMs promotes tumor matastasis through warburg effect, but the mechnism was still unclear. Our previous studies have shown that TAMs induced EMT of ESCC cell via secrete TGF-β, promotes ESCC invasion and matestasis, casue poor prognosis. However, when knockdown the expression of PKM2 in TAMs before co-cultured with ESCC cells, the ability of invision of ESCC cells were decreased significantly. These results illustrute that PKM2 may be a key factor in TAMs regulates matastasis of ESCC. As the rate limiting enzyme of glycolysis, whether PKM2 can regulates the warburg effect in TAMs, then promotes the invision and matastasis of ESCC, need to be studied. Here, this study was designed to discuss expression of PKM2 in TAMs, and elucidate the mechanism of PKM2 promotes invision and matastasis of ESCC via regulate TAMs glycolysis. We designed this project from clinical samples from patients with ESCC, in vitro and animal models, using a variety of methods (Immunofluorescence, cell co-culture, Co-IP, etc.), in order to provide a scientific basis for revealing the invasion and metastasis mechanisms of Kazakh's ESCC.

新疆哈族食管癌发病高达全国平均水平的5倍且预后差，控制其浸润、转移是影响预后的关键。微环境中肿瘤相关巨噬细胞（TAMs）通过Warburg效应促进肿瘤发生及浸润、转移，但机制未明。课题组前期研究发现TAMs分泌大量TGF-β，诱导食管癌细胞发生上皮间质转化（EMT），促进其浸润、转移，导致预后差，且将敲低PKM2的TAMs与食管癌细胞共培养，显示癌细胞侵袭性显著下降，提示PKM2能够调控TAMs促进食管癌转移，该过程可能是微环境中间质细胞促进癌转移的关键。作为糖酵解的限速酶PKM2是否调控TAMs糖代谢进而促进食管癌转移亟待深入研究。本项目拟从临床表型、细胞功能和动物模型层面，通过免疫荧光、细胞共培养、Co-IP等方法，研究TAMs中PKM2表达变化对食管癌细胞恶性表型的影响，明确微环境中PKM2调控TAMs糖代谢，诱导EMT促进癌转移的机制。基此结果为评价预测食管癌的浸润转移提供新思路。

为探讨改变肿瘤细胞恶性增殖的微环境对食管癌浸润转移的影响，本项目通过在体外细胞模型、体内临床样本和动物模型水平，运用慢病毒稳定转染、qRT-PCR、Transwell实验、代谢组学、免疫荧光等方法，检测PKM2表达变化对肿瘤相关巨噬细胞（TAMs）的影响，以及微环境中TAMs对食管癌细胞恶性表型的调控作用。在细胞水平，通过稳定转染干扰或过表达PKM2的慢病毒至TAMs中，发现敲低PKM2后M1型巨噬细胞标记物NOS2表达上调，M2型巨噬细胞标记物CD163和肿瘤相关巨噬细胞标记物CCL5表达下调；过表达PKM2后NOS2表达下调，CD163和CCL5表达上调，即PKM2表达能够促进TAMs表型分化。基于液相色谱串联质谱（LC-MS/MS）技术平台，检测包括糖代谢相关代谢物在内的57种能量代谢物在PKM2表达改变前后的变化，发现PKM2的表达与TAMs细胞能量代谢相关，其中2,3-二磷酸甘油酸水平与PKM2表达呈显著负相关。将食管癌细胞与敲低或过表达PKM2的TAMs共培养，观察共培养对食管癌细胞迁移、侵袭能力的影响，发现与敲低PKM2的TAMs共培养后，食管癌细胞迁移和侵袭能力显著下降，与过表达PKM2的TAMs共培养后，食管癌细胞迁移和侵袭能力显著增强，说明TAMs细胞中PKM2的表达能够影响食管癌细胞的迁移和侵袭能力；裸鼠成瘤模型验证该结果，发现敲低PKM2的TAMs细胞与食管癌细胞混合接种后，皮下荷瘤体积较对照组减小，而过表达PKM2的TAMs细胞与食管癌细胞混合接种后，皮下荷瘤体积较对照组增大。在临床样本水平通过免疫荧光染色检测PKM2及CD163的表达，发现二者在食管癌临床样本中均呈显著高表达，但基于此样本，CD163、PKM2与食管癌患者临床病理参数及预后之间均无显著相关性。以上结果表明PKM2作为微环境中重要的调控因子，能够促进TAMs的表型分化，同时影响TAMs代谢水平变化，是影响食管癌细胞侵袭转移恶性表型的重要因素。本研究结果为微环境影响肿瘤细胞恶性表型的机制研究提供了新的思路。