肺癌细胞及浸润性免疫抑制细胞表达II型甲酰肽受体构建肺癌微环境的机制研究

The infiltration of immune cells is a characteristic of the tumor microenvironment. Immune suppressive cells recruited into tumor microenvironment subvert the host defense and create a microenvironment favoring tumor escape. These cells include myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs). Chemoattractant receptors are a family of G protein coupled seven trans-membrane cell surface receptors (GPCRs). The formyl peptide receptors(FPRs) and its variants are categorized into classical GPCRs. Functions.of GPCRs have been discovered to not only regulate leukocyte trafficking and promote immune responses, but also play important roles in angiogenesis and tumor progression. Accumulating evidence indicates that chemoattractant GPCRs and their ligands promote the progression of malignant tumors based on their capacity to orchestrate the infiltration of the tumor microenvironment by immune cells. These functions and expression of FPRs in human lung cancer cells has not been known well. The expression of FPR2 in human non-samll cell lung cancer(NSCLC) specimens was examined by immunohistochemistry (IHC).We found the high level expression of FPR2 in NSCLC cancer tissue may be correlated with pathological type and clinical stages of NSCLC patients thus predicted the poor survival of patients. Our group found that some selected human lung cancer cell lines also expressed functional FPR2, which mediated cell proliferation, invasion and activated the Erk1/2 signaling pathway stimulated by FPR2 agonists. Our group generated myeloid specific mFPR2-deletion mice by crossing lysozyme-Cre (LysMCre) mice with mFPR2-floxed mice. We found that myeloid-specific mFpr2-KO mice bearing subcutaneously implanted Lewis lung carcinoma (LLC) cells exhibited significantly shortened survival than normal mice due to more rapidly growing tumors.Macrophages derived from myeloid-Fpr2-KO mice showed a more potent chemotactic response to LLC-derived supernatant. The tumor volume and growth were reduced by treatment of FPRs agonist W-peptide in Lewis lung carcinoma tumor-bearing mice compared to the PBS group. These preliminary study results suggest Fpr2 may play an important role in host defense against implanted LLC by recruiting TAMS into tumor microenvironment and regulating the polarization of macrophages in tumor bearing host. We therefore hypothesize that FPR2 may contribute to the malignant features of human lung cancer, but also may play a role in regulating the infiltration of immune suppressive cells to shape the lung cancer microenvironment in favor of host defense. Our group had been generated four kinds of mouse strain, including FPR single (Fpr1-/-, Fpr2-/-, myeloid-specific-Fpr2-/-) and double knock out mice (FPR1-2-/-). We will establish these LLC tumor-bearing animal models and use some experimental methods, such as confocal immunofluorescence staining, chemotaxis assay, western blotting, flow cytometry, to research the role and mechanism of FPRs expressed by lung cancer cells and infiltrating tumor-associated cells into tumor in shaping the tumor microenvironment of lung cancer. The study will promote further understanding of the mechanisms of host-tumor interaction for the development of novel anti-tumor therapeutic strategies.

调控肿瘤微环境是肿瘤免疫耐受及治疗的研究热点和重要方向，肿瘤相关巨噬细胞 (TAMs)在构建促瘤或抑瘤微环境中起重要作用。甲酰肽受体(FPRs)在肺癌微环境领域相关报道极少。申请人前期研究发现人类肺癌组织FPR2表达与肺癌分期、病理类型可能相关；肺癌细胞表达FPR2可能与癌细胞增殖、转移相关；Lewis肺癌(LLC)皮下种植瘤在髓系特异性Fpr2基因敲除鼠比野生鼠增殖快，生存短；髓系巨噬细胞对LLC培养上清有趋化效应。用Fprs配体w-pep腹腔注射治疗LLC荷瘤小鼠，治疗组肿瘤大小和增殖速度明显低于对照组。据此提出“肺癌细胞和浸润性免疫抑制细胞表达的FPR2是构建肿瘤微环境的重要环节”假说。本项目拟运用流式细胞术、免疫共聚焦等技术，利用FPRs基因缺陷鼠模型，首次以独特视角研究FPRs在肺癌微环境中是否兼具促瘤和抑瘤的双向作用，为肺癌免疫防御机制奠定新的理论基础，寻找免疫治疗新靶点。

研究背景：调控肿瘤浸润性免疫细胞，改变肿瘤微环境是肿瘤免疫耐受机制及免疫治疗策略的研究热点和重要方向。肿瘤微环境中G蛋白偶联跨细胞膜表面受体家族（G Protein Coupled transmembrane cell surface Receptors ，GPCRs）不仅介导免疫细胞趋化和活化，且与肿瘤迁移、侵袭相关。但是经典型GPCRs中甲酰肽受体(FPRs)与恶性肿瘤的发生、发展、侵袭、转移的相关研究报道很少见。.研究内容和重要结果：研究发现FPR2的表达与非小细胞肺癌不同病理类型、临床分期相关；人类非小细胞肺癌细胞表达FPR2具有普遍性。FPR2受体在外源性刺激剂或内源性配体激活下可促进A549肺癌细胞增殖、迁移、趋化等恶性生物学行为，机制与FPR2受体活化后介导MAPK通路（ERK1/2）的磷酸化增高相关。动物试验和细胞学试验同样证明肺癌细胞的FPR2受体对于肺癌移植瘤生长、EMT发生、VEGF分泌、血管形成具有重要促瘤意义。研究发现髓系细胞Fpr2功能缺陷鼠的移植瘤生长速度较正常鼠快，提示髓系细胞Fpr2功能缺陷导致宿主对小鼠Lewis 肺癌（LLC）细胞的防御能力明显降低。Fpr2功能下调/缺失小鼠的骨髓来源巨噬细胞对LLC培养上清的趋化效应明显增强。功能正常的骨髓巨噬细胞Fpr2受体可以同时抑制CCR2、CCR4，进而抑制巨噬细胞对肿瘤微环境中CCL2的趋化反应，减少了巨噬细胞瘤内浸润，从而调控肺癌微环境向抑瘤环境构建和转换。myeloid Fpr2-/-小鼠骨髓来源细胞和脾脏细胞中免疫抑制细胞CD11b+Gr-1+细胞亚群（MDSC）均较野生型鼠增多；且Fpr2功能缺陷鼠对LLC肺癌细胞的宿主防御能力比Fpr2功能正常鼠明显降低。应用W-peptide活化荷瘤鼠Fprs功能可以降低髓外淋巴器官（脾脏）内的MDSC及亚群数量，抑制LLC肿瘤增殖，且活化强度和启动时间与抗肿瘤防御反应的强弱相关。.科学意义和应用价值：肺癌细胞表达FPR2具有促进肿瘤生长、增殖、侵袭的作用；而宿主免疫细胞表达FPR2功能则与肿瘤免疫防御机制相关；活化宿主FPR2功能可能提高抗肿瘤疗效。本项目首次以独特视角研究发现FPRs在肺癌微环境中兼具促瘤和抑瘤的双向作用，为肺癌免疫防御机制奠定了新的理论基础，为肺癌免疫治疗提供了新靶点，具有重要转化临床应用的价值.