肿瘤相关中性粒细胞(TAN)诱导食管癌“放疗抵抗”及机制研究

Radiation resistance has become an urgent problem in tumor therapy. Tumor microenvironment can alter radiosensitivity. Neutrophils are recruited to the irradiated tumor site at an early stage following radiation, while the roles they plays have not been fully explored. Epithelial-mesenchymal transdifferen-tiation (EMT) is one of the mechanisms of radiation resistance. Our previous studies demonstrated that neutrophils could promote the EMT of tumor cells. Preliminary studies found that radiation could not induce EMT of esophogeal cancer (EC) cells in vitro shortly after fractionated ionizing radiation. However, EMT was observed in vivo receiving the same treatment, with more infiltrated tumor associated neutrophils (TAN), increased MIP-2 level, and activiated PI3K-AKT-Snail signaling pathway. We hypothesize that TAN play key roles in inducing radiation resistance shortly after fractionated radiation by promoting EMT, and MIP-2/CXCR2-PI3K-AKT-Snail- E-cadherin signaling pathway may work as the key pathway. To test the hypothesis, we established cell co-culture model as well as subcutaneous transplatation tumor model of EC cells in nude mice. We will investigate the crucial roles of TAN in inducing radiation resistance of EC cells following radiotherapy, and definite the relevant key signaling pathway. Our study will lay the foundation to reveal the protumoral effect of TAN in the tumor microinviroment after radiation.

“放疗抗拒”是肿瘤治疗过程中亟待解决的问题。肿瘤微环境能调控放疗敏感性，中性粒细胞是放疗后早期被募集的免疫细胞，然而其发挥的作用尚未阐明。上皮间质转化（EMT）是“放疗抵抗”的重要机制之一。我们的前期研究证实，肿瘤相关中性粒细胞（TAN）能诱导肿瘤细胞发生EMT。预实验发现：人食管癌细胞系体外短程分次放疗后不能发生典型的EMT，而体内实验肿瘤细胞放疗后早期发生EMT，TAN明显浸润，MIP-2表达增多，肿瘤细胞PI3K-AKT-Snail通路明显激活。我们设想，TAN在放疗早期能诱导食管癌发生EMT进而导致“放疗抵抗”；其中MIP-2/CXCR2-PI3K-AKT-Snail-E-cadherin通路是关键通路。我们拟通过细胞共培养、裸鼠皮下成瘤模型等从分子、细胞、组织水平探讨TAN在诱导“放疗抵抗”中发挥的重要作用，明确其中的关键通路。为阐明放疗后微环境中TAN的促肿瘤作用奠定理论基础。

肿瘤微环境能调控放疗敏感性，中性粒细胞是放疗后早期被募集的免疫细胞，然而其发挥的作用尚未阐明。上皮间质转化（EMT）是“放疗抵抗”的重要机制之一。我们的前期研究证实，肿瘤相关中性粒细胞（TAN）与肿瘤细胞EMT状态相关。在该研究中，我们通过构建荷瘤鼠模型，从分子、细胞、组织以及动物水平多层次探讨了TAN在放疗后微环境的调控作用，我们发现，TAN是放疗后一线募集的免疫细胞；尽管多数研究认为肿瘤组织中已浸润的TAN表现为N2促肿瘤表型，我们发现，放疗能重塑新募集TAN的功能表型。放疗产生持续性DNA损伤，诱导炎症趋化因子释放，其中CXCL1、CXCL2和CCL5表达水平显著上调；放疗通过趋化因子依赖的病原相关分子模式（PAMP）促进TAN募集；放疗后新募集的TAN能够抑制EMT过程，促进放疗后TAN募集，能加强对EMT的抑制作用，进一步机制研究表明，放疗后新募集的TAN表达高水平的活性氧簇（ROS），ROS通过抑制PI3K-AKT-Snail通路，上调E-cadherin，抑制EMT过程，影响放疗敏感性和荷瘤鼠生存。ROS-PI3K-AKT-Snail通路是其中关键的信号通路。外源性给予G-CSF能显著促进放疗后TAN的募集，是调控放疗后TAN浸润的有效手段。同时，我们通过开展相应的临床研究，在胸部肿瘤中构建了基于中性粒细胞的放疗敏感性预测模型，并通过临床资料分析进一步验证了放疗联合使用G-CSF可能通过促进新的中性粒细胞生成及募集，影响放疗敏感性和预后，这为进一步明确TAN在放疗后肿瘤微环境中发挥的作用，提供了理论依据。同时新的预测模型的建立，有助于高危患者的筛查、预后判断以及个体化医疗的实施。在阐明TAN对放疗后微环境调控的相关机制的基础上实现科研成果向临床应用的转换，后续通过开展临床试验，探讨通过调控TAN的联合治疗手段，将为肿瘤的治疗提供新的思路。