肿瘤坏死因子介导的骨髓源性抑制细胞促进肺腺癌进展相关机制的研究

Inflammatory Cells in tumor micro-environment played a very important role in cancer promotion. Tumor necrosis factor (TNF) secreted by tumor or tumor stromal cells has been reported as an important regulator of tumor microenvironment and promoter of inflammation–associated cancer. In our previous studies, it is shown that chronic inflammation caused the increasing of TNF and recruitment of the Myloid suppress dived cells (MDSC), which resulted in lung carcinogenesis in a K-ras induced lung cancer mouse model (CC-LR). We hypothesis that high levels of TNF in the chronic inflammation could induce MDSC invading into the tumor sites and cause the progression of lung cancer. In order to verify our hypothesis, we use genitic mouse to control the expression levels of TNF in the CC-LR. We use flow cytometry, immunofluorescence techniques to analysis the quantity and subtype of MDSC around the tumor sites and the cancer promotion condition. Then we separat the MDSC and co-culture the MDSC with lung cancer cell lines in vitro to further study the possible potential inflammatory signaling pathways. We are hoping that we can illuninate the mechanisms of TNF through recruitment of MDSC in lung cancer promotion, which maybe provide a noval target for the future immunotherapy.

肿瘤炎症微环境中炎症细胞局部浸润对肿瘤进展具有重要促进作用。肿瘤坏死因子（TNF）由肿瘤细胞及间质细胞分泌产生并可调节肿瘤微环境。本实验前期研究发现，在K-ras突变的原发肺癌小鼠模型中诱发慢性炎症可导致支气管灌洗液中TNF表达水平增高，促进骨髓源性抑制细胞（MDSC）浸润，导致肺癌肿瘤细胞增殖，但具体调控机制尚不明确。本课题组拟用转基因小鼠调节TNF在小鼠体内表达水平，利用流式细胞术分析慢性炎症下MDSC细胞数目及其亚型变化规律；应用免疫组化、Western Blot及免疫荧光等技术分析MDSC对肺部肿瘤增殖影响。通过炎症细胞分选技术分离提纯MDSC并利用免疫荧光技术分析其炎症信号通路活化情况；将之与肺腺癌细胞在Boyden小室中体外共培养，检测肿瘤细胞行为学改变，明确MDSC促进肿瘤进展相关调控机制。从而阐明TNF招募MDSC浸润促进肺癌进展的分子机制，为肺癌免疫治疗提供新靶点。

肿瘤炎症微环境中炎症细胞局部浸润对肿瘤进展具有重要促进作用。肿瘤坏死因子（TNF）由肿瘤细胞及间质细胞分泌产生并可调节肿瘤微环境。本实验前期研究发现，在K-ras突变的原发肺癌小鼠模型（CC-LR）中诱发慢性炎症可导致支气管灌洗液中TNF表达水平增高，促进骨髓源性抑制细胞（MDSCs）浸润，导致肺癌肿瘤细胞增殖，但具体调控机制尚不明确。本课题组拟用转基因小鼠调节TNF在小鼠体内表达水平，利用流式细胞术分析慢性炎症下MDSCs数目及其亚型变化规律；结果显示过表达TNF后，MDSCs局部肺浸润增加，免疫组化结果表明MDSCs能够促进肺部肿瘤细胞增殖及血管生成。当敲除CC-LR小鼠中TNF表达后，应用NTHi处理该组小鼠从而建立炎症微环境，其肿瘤表面细胞数及肿瘤体积均减小，肿瘤的增殖能力、血管生成能力及侵袭能力明显受到抑制；且MDSCs细胞的比例及数量都明显降低，尤其是M-MDSCs亚型。同时过表达TNF后，P65在肿瘤细胞及正常细胞组织中表达都明显增高。Western Blot结果显示细胞核内的P65表达明显增加，同时NF-κB内源性抑制因子IκBα的表达在细胞浆中表达发生降低。免疫荧光双染色结果显示，肿瘤浸润性巨噬细胞中及MDSCs中的NF-kB信号通路表达明显增加。该结果证明TNF是通过激活NF-κB通路招募M-MDSCs亚型细胞。此外，与CC-LR小鼠相比，TNF-CC-LR小鼠支气管关系液中IL-6分泌量明显增高，TGF-β分泌量也显著增加。在TNF过表达后，STAT3信号通路活化增加，应用IL-6抗体抑制STAT3信号通路，MDSCs局部浸润明显降低。该研究结果表明TNF能够通过调节STAT3信号通路招募MDSCs从而促进肺癌进展。同时通过分析临床患者肺癌组织中MDSCs研究结果表明，肺癌组织周围MDSCS浸润明显增加，同时随着肿瘤直径增加其浸润比例也继续增加。综上所述，该研究结果阐明了TNF通过NF-κB及STAT3信号通路招募MDSCs浸润促进肺癌进展的分子机制，为肺癌免疫治疗提供新靶点；此外也为肺腺癌患者免疫分子分型提供理论支持。