研究背景
胰腺癌是一种恶性程度极高、预后差的消化系统恶性肿瘤。胰腺导管腺癌（pancreatic ductal adenocarcinoma，PDAC）为最常见的病理类型。流行病学资料显示，胰腺癌位居我国肿瘤死亡原因第六位，发病率呈上升趋势。多数患者初诊时因肿瘤局部侵犯或远处转移失去手术机会。胰腺癌肿瘤微环境（tumor microenvironment，TME）以大量炎性细胞浸润和广泛纤维化为主要特点。针对胰腺癌TME探索胰腺癌进展机制是目前的研究热点。TME中肿瘤相关巨噬细胞（tumor associated macrophages，TAMs）对胰腺癌发生发展和补体免疫逃逸具有促进作用。细胞外囊泡（extracellular vesicles，EV）参与TME中不同细胞间信号传递和物质交换，是胰腺癌TME中调控TAM分化和功能改变的关键介质。迁移体是近几年新发现的一类依赖细胞运动的EV样结构，其中富集众多信号介导因子、趋化因子和细胞因子等调控蛋白，对生物体发育、信号转导等具有重要意义。胰腺癌细胞产生的迁移体（pancreatic cancer cell-derived migrasome, PCCDM）对TME中免疫细胞尤其是TAM的表型及功能的影响，目前缺乏相关研究。

研究目的
明确PCCDM中调控胰腺癌TME的关键蛋白成分，分析PCCDM对TME中免疫细胞比例变化的影响以及该变化对胰腺癌生长的影响；探究PCCDM对TAM表型及功能的调控作用；探讨PCCDM诱导后的TAM对胰腺癌细胞功能的影响；探究PCCDM诱导后的TAM对T淋巴细胞的影响；探讨PCCDM诱导的TAM对胰腺癌补体免疫逃逸的影响；筛选PCCDM中富集的一些关键蛋白，初步探索PCCDM中对免疫微环境具有调控功能的重要蛋白成分在胰腺癌组织中的表达情况，探究其与患者临床病理特征以及组织中免疫细胞浸润的关系。

研究方法
1. 通过差速离心和密度梯度离心的方法提纯人和小鼠PCCDM，利用电镜和共聚焦显微镜对PCCDM进行超微结构和形态学观察，通过蛋白质谱和蛋白芯片检测PCCDM中富集的细胞因子和趋化因子等调控蛋白，利用共聚焦显微镜观察关键蛋白富集情况。构建免疫健全小鼠胰腺癌腹腔种植瘤模型，通过注射PCCDM构建免疫微环境，探究PCCDM诱导的免疫微环境中免疫细胞比例变化以及该变化对胰腺癌生长的影响。
2. 利用小鼠巨噬细胞系RAW264.7和骨髓来源巨噬细胞（bone marrow-derived macrophages，BMDMs）作为巨噬细胞模型，通过共培养的方法探究PCCDM对巨噬细胞形态及趋化能力的影响以及巨噬细胞吞噬PCCDM后表型及功能的变化；通过蛋白芯片和转录组测序对PCCDM诱导后巨噬细胞功能表型进行鉴定和分析。
3. 利用PCCDM诱导的巨噬细胞和对照组巨噬细胞的培养上清制作条件培养基，通过条件培养基刺激胰腺癌细胞，利用CCK8法检测条件培养基对胰腺癌增殖能力的影响，利用Traswell法检测条件培养基对胰腺癌细胞侵袭和迁移能力的影响。
4. 通过ELISA检测经PCCDM诱导后巨噬细胞RAW264.7和BMDM培养上清以及小鼠腹腔灌洗液中影响肿瘤免疫关键蛋白的分泌情况；通过小鼠脾脏提纯CD4⁺和CD8⁺T淋巴细胞，利用巨噬细胞条件培养基的方法探究PCCDM诱导后巨噬细胞对T细胞增殖能力的影响，应用蛋白抑制剂探究巨噬细胞上清中该调控蛋白对T细胞增殖能力的影响。
5. 利用人巨噬细胞系THP-1作为巨噬细胞模型，通过胰腺癌条件培养基刺激的方法探究胰腺癌对THP-1表型的影响；利用人新鲜血清构建补体微环境，通过流式细胞术检测M2型巨噬细胞对胰腺癌补体免疫逃逸的影响；通过转录组学测序探究胰腺癌和PCCDM诱导的巨噬细胞基因表达差异以及对补体免疫逃逸的影响机制。
6. 筛选PCCDM中的对肿瘤免疫具有调控作用的关键蛋白，通过免疫组化染色和数据库的方法探究胰腺癌组织中该蛋白表达情况与患者临床病例特征及预后的关系以及与免疫细胞浸润的关系。

研究结果
1. 胰腺癌细胞在迁移运动中大量产生迁移体，经鉴定发现PCCDM富含CXCL5、TGF-β1、integrin通路蛋白和Rab家族蛋白等多种调控免疫微环境的因子。经PCCDM诱导的小鼠腹腔免疫微环境中免疫细胞浸润增加，其中M2型巨噬细胞比例增加、T细胞比例明显减少，这种变化对小鼠胰腺癌生长具有促进作用。
2. PCCDM可被巨噬细胞主动吞噬，并诱导巨噬细胞向PCCDM趋化。PCCDM诱导后的巨噬细胞中CD206+巨噬细胞比例增加，M2型巨噬细胞标记ARG1、IL-10和TGF-β1表达明显升高。蛋白芯片分析显示，在PCCDM诱导后的巨噬细胞中免疫抑制因子（TGF-β1, IL-4, IL-10, CSF1, CSF3, CTLA4和MMP9）以及对肿瘤具有重要作用的趋化因子和受体（CXCL5, CXCL9, CXCL12, CCL4, CCL7, CCL21, CCR3和CCR7）的表达水平较对照组巨噬细胞明显升高。转录组学测序结果显示，PCCDM诱导的巨噬细胞中表达上调的基因与促进肿瘤或免疫抑制相关的，包括IL-6、MMP9、MET、MYC、EGFR、CCL7、CSF3、CCL12、CXCL5、ITGA9、CDH5和CD274（即PD-L1）；表达下调的基因中有众多调控免疫细胞增殖和活化的基因，包括CCL24、CX3CL1和MHC-II类蛋白（H2-OA、H2-OB和H2-Q6）。
3. 在RAW264.7和BMDM巨噬细胞模型中，PCCDM诱导后的巨噬细胞对胰腺癌细胞增殖能力和侵袭迁移能力具有促进作用。
4. PCCDM诱导后的RAW264.7和BMDM培养上清以及小鼠腹腔灌洗液中ARG1含量显著上升；PCCDM诱导后的巨噬细胞对T细胞增殖能力具有抑制作用，ARG1抑制剂可部分逆转该作用。
5. 胰腺癌诱导THP-1向M2型巨噬细胞分化，分化后的巨噬细胞通过分泌IL-6上调胰腺癌细胞CD59表达并促进胰腺癌发生补体免疫逃逸；PCCDM诱导后巨噬细胞分泌IL-6；PCCDM参与巨噬细胞对胰腺癌CD59表达和补体免疫的调控过程。
6. PCCDM中富含CXCL5，胰腺癌组织和细胞系高表达CXCL5，胰腺癌组织中CXCL5高表达与患者T3分期、N2分期和病理低分化级别具有相关性；高表达CXCL5的患者预后相对较差；在CA242高的患者中CXCL5表达水平为独立危险因素；高表达CXCL5的胰腺癌组织中M2型巨噬细胞、中性粒细胞和IgG+浆细胞浸润增多。

研究结论
PCCDM中富含CXCL5和TGF-β1等调控蛋白，PCCDM可诱导抑制型免疫微环境促进肿瘤生长；PCCDM诱导的微环境中M2型巨噬细胞增多、T细胞减少；PCCDM可促进巨噬细胞趋化并诱导其转化为M2型巨噬细胞，促进胰腺癌增殖、侵袭和迁移，并通过分泌ARG1抑制T细胞增殖；PCCDM诱导的巨噬细胞通过分泌IL-6促进胰腺癌细胞高表达CD59，进而发生补体免疫逃逸；PCCDM中富含CXCL5，CXCL5高表达的胰腺癌患者预后差，组织中呈现抑制型免疫环境，表现为M2型巨噬细胞、中性粒细胞和IgG⁺浆细胞浸润增加。针对迁移体的治疗策略研究有可能为胰腺癌综合治疗提供新的思路。

Background
Pancreatic cancer is one of the malignant digestive tumors, with a high degree of malignancy and a poor prognosis. Pancreatic ductal adenocarcinoma is the most common pathological type. Epidemiological data show that pancreatic cancer is the sixth leading cause of cancer-related death in China with rising incidence. Most patients lose the opportunity of surgery due to local invasion or distant metastasis of the tumor at the initial diagnosis. Tumor microenvironment (TME) of pancreatic cancer is characterized by massive infiltrations of inflammatory cells and extensive fibrosis. It is a hot topic to explore the effects of TME on pancreatic cancer progression. Tumor associated macrophages (TAMs) in TME promoted the development of pancreatic cancer and the immune escape of complement. Extracellular vesicles (EV) are involved in the signal transmission and material exchange between different cells in TME, and are a key medium for regulating TAM differentiation and functional changes in pancreatic cancer. Migrasome is a kind of EV-like structure that is newly discovered in recent years and dependent on cell movement, in which many signal mediators, chemokines, cytokines and other regulatory proteins are enriched, which is of great significance for organism development and signal transduction. There is a lack of research of the effect of pancreatic cancer cell-derived migrasomes on the phenotype and functions of TAM in TME in pancreatic cancer.

Objective
To identify the protein components in PCCDM that could regulate the tumor immune microenvironment, and to explore the effects of these migrasomes on the proportion of immune cells in TME and the influence of such changes on the growth of pancreatic cancer. To explore the effects of PCCDM on the regulation of phenotype and function of TAMs. To investigate the effects of TAMs induced by PCCDM on pancreatic cancer cell function. To investigate the effects of TAMs induced by PCCDM on T lymphocytes. To investigate the effects of TAMs induced by PCCDM on immune escape of pancreatic cancer complement. Some key proteins enriched in PCCDM were screened to preliminarily explore the expression of the key protein in PCCDM that regulate the immune microenvironment in pancreatic cancer tissues and explore their relationship with immune cell infiltration and prognosis.

Methods
1. PCCDMs were purified by differential centrifugation and density gradient centrifugation, and the ultrastructure and morphology observation of migrasomes was identified by electron microscopy. The cytokines and chemokines and other regulation proteins enriched in migrasomes were detected by protein spectrum and protein chip, and the production process of migrasomes and the concentration of the key proteins were observed by confocal microscopy. A peritoneal model of pancreatic cancer in immunologically mice was constructed, and the immune microenvironment was constructed by intraperitoneal injection of migrasomes. Then explore the changes in the proportion of immune cells in the immune microenvironment induced by migrasome and the influence of such changes on the growth of pancreatic cancer.
2. Using murine macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMDMs) as macrophage models, the effects of PCCDM on the morphology and chemotaxis of macrophages and the changes of phenotype and function of the migrasome-induced macrophages were investigated by the co-culture system. The functional phenotypes of macrophages induced by migrasomes derived from pancreatic cancer cells were identified and analyzed by protein chip and transcriptome sequencing.
3. The cultured supernatant of macrophages induced by migrasomes and macrophages in the control group was used to prepare the conditioned medium, and the pancreatic cancer cells were stimulated by the conditioned medium. The effects of the conditioned medium on the proliferation ability of pancreatic cancer cells were detected by CCK8 method, and the effects of the conditioned medium on the invasion and migration ability of pancreatic cancer cells were detected by Traswell method.
4. ELISA was used to detect the secretion of the key protein that could regulate the tumor immunity in RAW264.7 and BMDM macrophage models and mice peritoneal lavage fluid after PCCDM education. CD4⁺ and CD8⁺ T lymphocytes were purified from the spleen of mice, and the effects of macrophages induced by PCCDM on the proliferation ability of T cells was investigated by using the method of conditioned medium. The effects of the key protein inhibitor on the proliferation ability of T cells in the macrophage cultured supernatant was investigated.
5. Using human macrophage line THP-1 as the macrophage model, the effects of pancreatic cancer on THP-1 phenotype was investigated by the stimulation of conditioned medium from pancreatic cancer. Using human fresh serum to construct complement microenvironment, flow cytometry was used to detect the effects of M2-type macrophages on the complement immune escape of pancreatic cancer cells. Transcriptome sequencing was used to investigate the gene expression differences in macrophages induced by PCCDM and pancreatic cancer cells, as well as the mechanism on complement immune escape.
6. Key proteins in PCCDM that regulate tumor immunity were screened. Immunohistochemical staining and database methods were used to explore the relationship between the expression of this protein in pancreatic cancer tissues and the clinical characteristics and prognosis of patients, as well as the relationship between the expression of this protein and the infiltration of immune cells.

Results
1. Pancreatic cancer cells could produce migrasome in large quantities during migration, and it was identified that migrasome is rich in CXCL5, TGF-β1, integrin pathway proteins, Rab family proteins and other factors that could regulate immune microenvironment. The infiltration of immune cells increased in the immune microenvironment of mice induced by PCCDM, in which the proportion of M2-type macrophages increased and the proportion of T cells significantly decreased, which had a promoting effect on the growth of pancreatic cancer cells in mice.
2. PCCDM could be actively swallowed by macrophages and induced macrophages to migrate to migrasomes. The proportion of CD206⁺ macrophages in macrophages induced by migrasome was increased, and the expression of ARG1, IL-10, and TGF-β1 in M2-type macrophages was significantly increased. Protein-chip analysis showed that the expression levels of immunosuppressive factors (TGF-β1, IL-4, IL-10, CSF1, CSF3, CTLA4 and MMP9) as well as chemokines and receptors (CXCL5, CXCL9, CXCL12, CCL4, CCL7, CCL21, CCR3 and CCR7) that play important roles in tumor were significantly higher in the migrasome-induced macrophages than that in the control group. Transcriptome sequencing results showed that the genes up-regulated in migrasome-induced macrophages were associated with tumor promotion or immunosuppression, including IL-6, MMP9, MET, MYC, EGFR, CCL7, CSF3, CCL12, CXCL5, ITGA9, CDH5, and CD274 (namely PD-L1). Among the down-regulated genes are numerous genes that regulate the proliferation and activation of immune cells, including CCL24, CX3CL1, and MHC-II proteins (H2-OA, H2-OB, and H2-Q6).
3. In the RAW264.7 and BMDM macrophage models, the macrophages induced by PCCDM exerted a promoting effect on the proliferation, invasion and migration of pancreatic cancer cells.
4. After the induction of PCCDM, the contents of ARG1 in RAW264.7 and BMDM culture supernatant and peritoneal perfusion fluid in mice were increased significantly. Macrophages induced by PCCDM had an inhibitory effect on T cell proliferation, which was partially reversed by ARG1 inhibitor.
5. Pancreatic cancer induced THP-1 differentiation into M2-type macrophages, and the differentiated macrophages up-regulated the expression of CD59 in pancreatic cancer cells by secreting IL-6 and promoted the occurrence of complement immune escape in pancreatic cancer cells. IL-6 was secreted by macrophages after induction of PCCDM. PCCDM is involved in the regulation of CD59 expression and complement immunity by macrophages in pancreatic cancer.
6. High expression of CXCL5 was observed both in pancreatic cancer tissue and cell lines. High expression of CXCL5 in pancreatic cancer tissues was correlated with T3 stage, N2 stage and poor differentiation status. Patients with high expression of CXCL5 had relatively a poor prognosis. The CXCL5 expression level was an independent risk factor in patients with high CA242. The infiltration of M2-type macrophages, neutrophils and IgG⁺ plasma cells were increased in the pancreatic cancer tissues with high expression of CXCL5.

Conclusion
Pancreatic cancer cells produced migrasomes rich in regulatory proteins such as CXCL5 and TGF-β1. Migrasome induced immunesuppressive microenvironment to promote tumor growth. The proportion of M2-type macrophages was increased and that of T cells was decreased in the microenvironment induced by migrasomes derived from pancreatic cancer. Migrasome could promote the chemotaxis of macrophages and induced their differentiation into M2-type macrophages, and these macrophages could promote the proliferation, invasion and migration of pancreatic cancer, and inhibit the proliferation of T cells by secreting ARG1. The macrophages induced by pancreatic cancer migrasome secreted IL-6 to promote the high expression of CD59 in pancreatic cancer cells, which led to the complement immune escape. Migrasomes derived from pancreatic cancer were rich in CXCL5. Patients with high expression of CXCL5 had a poor prognosis, and their tissues present an immunosuppressive environment, with increased infiltration of M2-type macrophages, neutrophils and IgG⁺ plasma cells. The study of the treatment strategy for migrasomes may provide a new idea for the comprehensive treatment of pancreatic cancer.