M2型肿瘤相关巨噬细胞通过p53途径下调肝癌化疗敏感性的机制研究

目的 通过研究M2肿瘤相关巨噬细胞调控p53表达来下调肝癌化疗敏感性的机制,以此拓宽建立抗肿瘤耐药的措施.方法 构建小鼠肝癌模型及分离肝癌组织,研磨组织提取巨噬细胞,通过RT-PCR方法检测巨噬细胞表面CD206及CD163分子表达情况.培养人单核巨噬细胞(THP-1),PMA (100ng/mL)和IL-4 (100ng/mL)作用诱导分化成肿瘤相关巨噬细胞后,与肝癌细胞(HepG2、SMMC7721,Hep 3B)共培养,加入奥沙利铂(20μ g/mL)作用24小时,通过Western blot方法检测凋亡相关蛋白Caspase 3、抗凋亡蛋白Bcl-2及p53的表达,通过MTT方法检测化疗对肝癌细胞增殖情况.结果 成功构建了肝癌模型,分离出了肝癌组织巨噬细胞,RT-PCR检测CD206和CD163的表达显著升高;人单核细胞(THP-1)经加入PMA(100ng/mL)和IL-4 (100ng/mL)分化诱导48h后其细胞的表面CD206和CD163的表达明显高于THP1分化诱导的细胞表面表达量;肝癌细胞SMMC7721和HepG2与M2型肿瘤相关巨噬细胞共培养24h,经奥沙利铂作用后,肿瘤细胞Bcl-2表达明显升高,Caspase 3和p53的表达显著降低;肿瘤细胞的增殖抑制率明显降低,而Hep 3B细胞的凋亡则明显降低.结论 M2型肿瘤相关巨噬细胞调控肝癌细胞中p53的表达,进而抑制了肝癌化疗的敏感性.     

4T1荷瘤小鼠肿瘤相关巨噬细胞（TAM）的表型及吞噬功能的研究

目的:研究小鼠乳腺癌实验动物模型中,荷瘤晚期肿瘤相关巨噬细胞的表型和功能,并探讨其与M2型巨噬细胞的关系.方法:从4T1荷瘤4周的雌性BALB/c小鼠中获取肿瘤相关巨噬细胞,用RT-PCR方法检测其巨噬细胞相关分子CCL3、CCL22、iNOS和Arg Ⅰ的表达水平,用FACS检测巨噬细胞中CDl6/32+和CD206+细胞所占比例,并通过酵母菌吞噬试验评估其功能.结果:肿瘤相关巨噬细胞与荷瘤小鼠的脾脏巨噬细胞相比,表达较高水平的CCL22和CD206,并且对酵母菌的吞噬能力显著下降.结论:4T1荷瘤4周小鼠肿瘤相关巨噬细胞在表型和功能上倾向于替代性活化的巨噬细胞.     

来那度胺可抑制单核细胞向M2型巨噬细胞分化及相关细胞因子的分泌

目的 探索来那度胺在肿瘤微环境中对于单核细胞向M2型巨噬细胞分化及IL-10、VEGF和TGF-β1水平的影响.方法 密度梯度离心法分离初治淋巴瘤患者及健康志愿者外周血单个核细胞(PBMCs).用Transwel124孔板共培养PBMCs与淋巴瘤细胞系HUT-78,并于共培养体系中加入来那度胺.流式细胞术分析肿瘤相关巨噬细胞(TAM)特征性表型CD68和CD163的表达,ELISA法检测共培养体系细胞因子IL-10、VEGF和TGF-β1的含量.结果 在PBMCs与HUT-78构成的体外共培养体系中加入来那度胺后,CD68+、CD163+、CD68+ CD163+细胞及CD68+ CD163+在CD68+细胞中所占比例均有显著的下降(P＜0.05).加入来那度胺后患者组CD68+ CD163+/CD68+下降程度明显高于健康志愿者(P＜0.05).加入来那度胺的患者共培养组相对于未加来那度胺的共培养组IL-10和VEGF水平均有显著下降(P＜0.01,P＜0.05).结论 来那度胺在PBMCs与淋巴瘤细胞构建的体外共培养体系中能够有效抑制单核细胞向M2型TAM的分化,且能够抑制患者组共培养体系中IL-10、VEGF分泌.     

单核细胞趋化蛋白—1对胃癌荷瘤鼠作用的研究

肿瘤相关巨噬细胞对恶性肿瘤的生长、转移和预后,可能有着相当程度的影响。巨噬细胞在肿瘤局部的浸润与其相关的趋化因子的关系十分密切。人单核细胞趋化蛋白-1(MCP-1)为趋化因子超家族的成员之一[1]。本实验利用原核表达的MCP-1和胃癌细胞系SGC7901建立的荷瘤裸鼠模型,对MCP-1在胃癌的成瘤性及导致肿瘤相关巨噬细胞聚集的作用进行了观察和测定。1 材料和方法1.1 材料含人MCP-1 cDNA的质粒pUC19-MCP,由本校分子生物学研究所王字玲博士惠赠。融合表达载体pGEX-4T-1由本校分子生物学研究所提供。胃癌细胞系SGC7901由本研究…     

Linc00514通过调控miR-378a对肝癌巨噬细胞M2型极化的影响北大核心CSCD

目的:探讨Linc00514调节肝癌巨噬细胞M2型极化的作用及分子机制。方法:RT-qPCR检测Linc00514和miR-378a在肝癌患者组织和细胞中的表达。生物信息学软件和双荧光素酶报告基因实验预测和验证Linc00514与miR-378a的相互作用。将Linc00514-siRNA或NC-siRNA及NC inhibitor、miR-378a inhibitor质粒共转染至HepG2细胞。人单核细胞THP-1与转染后的HepG2细胞上清共培养诱导巨噬细胞极化。RT-qPCR和Western blot检测M2型巨噬细胞标志物精氨酸酶-1(Arg-1)、CD163、CD206和M1型巨噬细胞标志物TNF-α、人类白细胞抗原(HLA)-DR mRNA和蛋白水平。结果:肝癌组织和细胞系中,Linc00514表达显著升高,miR-378a表达显著降低(P<0.05)。肝癌组织中M2型巨噬细胞标志物Arg-1、CD163、CD206 mRNA表达显著升高(P<0.05)。Linc00514靶向负调控miR-378a表达。沉默Linc00514显著抑制Arg-1、CD163和CD206 mRNA和蛋白表达,促进M1型巨噬细胞标志物TNF-α和HLA-DR mRNA和蛋白表达(P<0.05)。抑制miR-378a显著逆转Linc00514对巨噬细胞极化的作用。结论:沉默Linc00514通过靶向miR-378a抑制肝癌巨噬细胞M2型极化。     

针对肿瘤相关巨噬细胞治疗肿瘤的研究进展

虽然肿瘤具有自发生长的能力,但是它们的增殖还是与其局部微环境密切相关。肿瘤相关巨噬细胞也是促进肿瘤生长并决定肿瘤转归的重要因素。目前,巨噬细胞被认为有两种表型,即M1型和M2型。它们的受体表达、分泌的细胞因子,产生的效应分子及功能等都各不相同,而肿瘤相关巨噬细胞通常都具有M2表型。通过对肿瘤相关巨噬细胞（TAM）的调控引导其向利于机体抗肿瘤免疫的方向发育可能为癌症治疗提供一个新的策略。     

肿瘤相关巨噬细胞在共培养体系中的免疫抗肿瘤作用研究进展

巨噬细胞是机体重要的免疫细胞, 肿瘤相关巨噬细胞是肿瘤细胞或组织附近富集的巨噬细胞, 其作用主要是促进构建肿瘤炎性微环境和抑制肿瘤免疫应答。细胞共培养体系是在体外模拟机体内在环境所形成的一种共生培养体系, 细胞在共培养条件下与体内环境状况相对一致, 这使细胞间能够更好进行信息交流和物质交换, 是对单层细胞培养和动物实验不足的补充。肿瘤微环境下肿瘤相关巨噬细胞与肿瘤细胞共同生存, 构建肿瘤相关巨噬细胞与肿瘤细胞共培养体系有利于研究肿瘤相关巨噬细胞的免疫抗肿瘤作用和研发不同以往的免疫抗肿瘤药物, 这为治疗恶性肿瘤提供了一条新的探索道路。本文主要综述了巨噬细胞共培养的方式及其不同表型在免疫抗肿瘤中的作用, 突出肿瘤微环境下运用免疫疗法治疗恶性肿瘤的重要性。     

肿瘤相关巨噬细胞研究进展

单核巨噬细胞是一种多功能细胞,对不同的微环境信号应答表现出不同的功能。而极化的MI和M2巨噬细胞是巨噬细胞功能表现的两个极端。其中侵润到肿瘤组织的巨噬细胞受肿瘤诱导产生的细胞因子的影响使巨噬细胞表现出巨噬细胞M2型表型,这些极化的巨噬细胞在破坏适应性免疫反应和促进肿瘤生长与进展方面具有重要作用。肿瘤相关巨噬细胞（TAM）可以促进肿瘤进展包括促进肿瘤生长、侵润、转移,促进血管生长和免疫抑制等,因而研究TAM具有重要意义。     

免疫补体调节蛋白C1 抑制物对巨噬细胞极化的作用

目的:研究免疫补体调节蛋白C1 抑制物(C1INH)调节巨噬细胞向经典活化巨噬细胞(M1)和选择性激活巨噬细胞(M2)的极化作用。方法:首先从人血中分离单核细胞后,单核细胞在巨噬细胞集落刺激因子(M-CSF)或粒细胞-巨噬细胞集落刺激因子(GM-CSF)作用下转变为静态巨噬细胞;静态巨噬细胞在干扰素-γ(IFN-γ)或白细胞介素4(IL-4)+IL-13 刺激作用下分别转化为M1 或M2。通过流式细胞仪观察C1INH 对巨噬细胞CD14、CD163、CD206 表型标志物的作用;应用RT-PCR 分析C1INH 对巨噬细胞细胞因子、趋化因子、相关酶基因表达影响;利用Western blot 方法,探讨在炎症条件下C1INH 对巨噬细胞CD14 结合Toll 样受体4(TLR4)的影响。结果:C1INH 抑制M-CSF 源性M1 的CD14、CD163 和GM-CSF 源性M2 的CD206 表型。C1INH 减少M1 的肿瘤坏死因子(TNF-α)和IL-6 表达,而增加M2 的15 脂氧合酶(ALOX15)和IL-10 表达。C1INH 抑制M1 的诱导型一氧化氮合酶(iNOS)mRNA,而提高M2 的精氨酸酶1(Arg1)mRNA 表达。C1INH 促进M1 的杀菌活性和M2 对菌的吞噬功能。C1INH 阻断CD14-TLR4 介导信号传导通路。结论:C1INH 调节巨噬细胞极化。     

原发中枢神经系统淋巴瘤免疫微环境特征北大核心CSCD

原发中枢神经系统淋巴瘤(PCNSL)是罕见的脑肿瘤之一,其最常见的病理类型为弥漫性大B细胞淋巴瘤(DLBCL)。随着目前免疫治疗的兴起,人们对肿瘤免疫微环境(TME)的研究越来越深入,但对于PCNSL免疫微环境的系统性研究并不多。中枢神经系统作为免疫豁免部位之一,PCNSL免疫微环境主要由肿瘤浸润淋巴细胞(TILs)和肿瘤相关巨噬细胞(TAMs)组成,其中CD8^(+)细胞毒性T细胞(CTLs)和CD163^(+)巨噬细胞(M2样巨噬细胞)最多。与全身性DLBCL相比,PCNSL免疫微环境中浸润的免疫抑制性细胞更多(如Treg细胞和MDSCs),通过释放免疫抑制细胞因子使PCNSL的免疫抑制性更强。     

Detection of M2 macrophages and colony-stimulating factor 1 expression in serous and mucinous ovarian epithelial tumors

Tumor-associated macrophages (TAM) are known to possess the immunosuppressive M2 macrophage phenotype. They contribute to tumor growth, invasion, and metastasis by producing various mediators. Macrophages, especially M2 polarized macrophages, preferentially express CD163 and CD204, but few studies have investigated macrophage phenotypes in human ovarian tumors. The purpose of the present study was therefore to present results on macrophage differentiation in human ovarian serous and mucinous epithelial tumors. The method focused on immunostaining of paraffin-embedded tumor samples. Almost all macrophages infiltrating tumor tissues expressed CD163 and CD204, indicating the phenotypic shift toward M2 macrophage. The numbers of CD68-positive macrophages as well as of CD163- and CD204-positive macrophages in borderline and malignant tumors were significantly higher than in benign tumors. They correlated well with histological gradient of malignancy. Macrophage colony-stimulating factor (also known as colony-stimulating factor; CSF-1), which is one of the cytokines considered to induce TAM to polarize toward an M2 phenotype, was then evaluated. CSF-1 expression in malignant tumor cells was significantly higher than that in benign tumor cells and correlated with histological malignancy. These results suggest that CSF-1 derived from tumor tissues induces macrophages to shift toward the M2 phenotype, which is considered to promote tumor growth.     

Tumor-associated macrophages promote tumor cell proliferation in nasopharyngeal NK/T-cell lymphoma

Objective: To explore the relationship between the number of tumor-associated macrophages (TAMs) and proliferative activity of tumor cells and the relationship between two macrophage biomarkers CD68 and CD163 in nasopharyngeal NK/T-cell lymphoma. Methods: Immunohistochemistry was used to reconfirm the diagnosis of nasal NK/T-cell lymphoma and detect the numbers of TAMs and the ki-67 label index of the tumor cells in all 31 cases. In addition, 12 cases of inflammatory cases were collected as controls, for which the immunostaining of CD68 and CD163 were done as well. Then staining results were analyzed with Pearson correlation and t test. Results: The number of TAMs was positively correlated with tumor proliferative activity (P = 0.024) in nasopharyngeal NK/T-cell lymphoma. The expression of CD68 and CD163 was closely related (P = 0.009), and the positive rate of CD68 was generally higher than CD163, however there is no statistical significance. Conclusion: The increase in numbers of TAMs in nasopharyngeal NK/T-cell lymphoma is related to higher proliferative index, indicating the TAMs play an important role in tumor proliferation. Meanwhile both CD68 and CD163 might be the markers for TAMs but CD163 would be the better one.     

Cross-linking of FcgammaR triggers shedding of the hemoglobin-haptoglobin scavenger receptor CD163

CD163, the hemoglobin (Hb)-haptoglobin scavenger receptor, is a monocyte/macrophage-restricted member of the scavenger receptor, cysteine-rich family of proteins. In addition to being expressed on the cell surface, a soluble form of CD163 has also been reported. Like tumor necrosis factor alpha (TNF-alpha), surface CD163 is proteolytically cleaved from the plasma membrane in response to lipopolysaccharide (LPS) stimulation. As cross-linking of the Fcgamma receptor (FcgammaR) is similarly known to induce TNF-alpha shedding, the effect of FcgammaR stimulation on CD163 shedding was investigated. We found that FcgammaR stimulation resulted in a rapid release of surface CD163 into the supernatant that was blocked by inhibitors of protein kinase C and tyrosine kinases. Although LPS and FcgammaR stimulation in short-term cultures suppressed CD163 mRNA expression, long-term cultures of monocytes treated with LPS-but not with a FcgammaR cross-linking reagent-resulted in an interleukin-10-dependent recovery of surface CD163 expression. These studies suggest that the presence of immune complexes in infection or autoimmunity may radically alter the nature of CD163-dependent monocyte/macrophage processes. This may be particularly important in disease states in which immune complexes and high levels of free Hb are present, such as in autoimmune hemolytic anemia, transfusion reactions, or infections by hemolytic bacteria.     

CSF1 expression in nongynecological leiomyosarcoma is associated with increased tumor angiogenesis

Leiomyosarcoma (LMS) is a malignant tumor of smooth muscle cells for which few effective therapies exist. A subset of LMS cases express macrophage colony-stimulating factor (CSF1) and the resultant tumor-associated macrophage (TAM) infiltration predicts poor clinical outcome. Further, TAMs have been shown to increase tumor angiogenesis. Here, we analyzed 149 LMS cases by immunohistochemistry for vascular marker CD34 and show that high microvessel density (MVD) in nongynecological LMS cases significantly predicts poor patient outcome. The majority of high MVD cases were also CSF1-positive, and when combining high MVD with CSF1 expression, an even stronger prognostic correlation with patient outcome was obtained. Gene expression profiling revealed that MVD has a stronger correlation with CSF1 expression than with expression of vascular endothelial growth factor isoforms, which have traditionally been used as markers of angiogenesis and as anti-angiogenic therapeutic targets. Finally, patterns of CSF1 expression and TAM recruitment remained consistent between primary tumors and their metastases, and between primary tumors and those grown as xenografts in mice, highlighting the stability of these features to the biology of LMS tumors. Together, these findings suggest an important role for CSF1 and the resulting TAM infiltration in the pathological neovascularization of LMS tumors and provide a rationale for CSF1-targeted therapies in LMS.     

The rat macrophage scavenger receptor CD163: expression, regulation and role in inflammatory mediator production

The monoclonal antibody ED2 is widely used to define macrophages (mphi) in the rat. We have recently identified the ED2 antigen as the rat CD163 glycoprotein. CD163 is a member of the scavenger receptor cysteine-rich group B (SRCR-B) family and functions as a scavenger receptor for hemoglobin-haptoglobin complexes. Moreover, CD163 has also been indicated as a marker for alternatively activated mphi. In the current study, we identify rat CD163/ED2-antigen as a marker for mature tissue mphi. Rat CD163 is constitutively expressed on most subpopulations of mature tissue mphi, including splenic red pulp mphi, thymic cortical mphi, Kupffer cells in the liver, resident bone marrow mphi and central nervous system perivascular and meningeal mphi, but is apparently absent from monocytes. Rat CD163 expression can be promoted by glucocorticoids, and this can be further enhanced by IL4. Finally, engagement of rat CD163 on peritoneal mphi induces the production of pro-inflammatory mediators, including NO, IL-1beta, IL-6 and TNF-alpha. Collectively, our findings identify rat CD163 as a broadly expressed macrophage scavenger receptor that may play a role in the activation of mphi during hemolytic and/or inflammatory conditions.     

Identification of genes expressed in tumor-associated macrophages

Most malignant tumors contain so-called tumor-associated macrophages (TAM) as a major component of their leukocytic infiltrate. To investigate the impact of the tumor microenvironment on activation and differentiation of macrophages, we established a 3-dimensional model system by culturing human monocytes within multicellular tumor spheroids. After 7 days, monocyte-derived TAM were isolated and analyzed for phenotypic alterations as compared to macrophages cultured without tumor cell contact. We found the known macrophage differentiation marker Carboxypeptidase M to be suppressed while CD14, HLA-DR, and CD16 were up-regulated. Using Differential Display, we identified several genes that were differentially expressed between TAM and control macrophages. Prolidase, a peptidase known to influence the chemoattraction of neutrophils and macrophage activity, was down-regulated in TAM. In contrast, the Toll-like receptor family-related molecules MD-1 and RP105 were up-regulated by tumor cell contact, both at the RNA and protein level. From our data we conclude that TAM represent a distict macrophage population characterized by low expression of differentiation-associated macrophage antigens but also by a constitutive state of activation.     

Heme Oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10 release

The shift between pro-inflammatory (M1) and anti-inflammatory (M2) states of macrophage polarization allows the resolution of inflammatory processes as well as the maintenance of a basal anti-inflammatory environment in tissues continuously exposed to harmless antigens (e.g., lung and gut). To identify markers for the anti-inflammatory state of macrophages, expression profiling was performed on human macrophages polarized by either GM-CSF or M-CSF, which lead to the generation of TNF-α and IL-12p40-producing pro-inflammatory macrophages [M1 (GM-CSF)] or IL-10-producing anti-inflammatory macrophages [M2 (M-CSF)] upon exposure to LPS, respectively. A different iron metabolism gene signature was detected in both macrophage types, with the heme regulatory molecules CD163 and Heme Oxygenase-1 (HO-1) being preferentially expressed by M2 (M-CSF) macrophages. M1-polarizing cytokines (GM-CSF, IFNγ) inhibited, while IL-4 enhanced, the M-CSF-driven HO-1 expression. In agreement with this in vitro data, HO-1 expression in metastatic melanoma was primarily detected in CD163⁺ tumor-associated macrophages, which are known to exhibit an M2-skewed polarization phenotype. In contrast to the HO-1 inhibitor tin protoporphyrin (SnPP), the administration of cobalt protoporphyrin (CoPP), a potent inducer of HO-1 resulted in increased LPS-triggered IL-10 release from M2 (M-CSF) macrophages. The data suggests that HO-1 is important for the anti-inflammatory activities of M-CSF-polarized M2 macrophages. Moreover, since M2 (M-CSF) macrophages also express higher levels of the CD163 scavenger receptor, the CD163/HO-1/IL-10 axis appears to contribute to the generation of an immunosuppressive environment within the tumor stroma.     

IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion

Innate immune cells can constitute a substantial proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer; however, the mechanisms by which they modulate cancer progression are incompletely understood. Here, we show that high levels of cathepsin protease activity are induced in the majority of macrophages in the microenvironment of pancreatic islet cancers, mammary tumors, and lung metastases during malignant progression. We further show that tumor-associated macrophage (TAM)-supplied cathepsins B and S are critical for promoting pancreatic tumor growth, angiogenesis, and invasion in vivo, and markedly enhance the invasiveness of cancer cells in culture. Finally, we demonstrate that interleukin-4 (IL-4) is responsible for inducing cathepsin activity in macrophages in vitro and in vivo. Together, these data establish IL-4 as an important regulator, and cathepsin proteases as critical mediators, of the cancer-promoting functions of TAMs.     

Ratio of M2 macrophage expression is closely associated with poor prognosis for Angioimmunoblastic T‐cell lymphoma (AITL)

Angioimmunoblastic T‐cell lymphoma (AITL) is a peripheral T‐cell lymphoma characterized by systemic disease with polymorphous infiltrate including macrophages. Although many studies of tumor‐associated macrophage (TAM) populations in various malignant tumors have been published, only a few have dealt with activation of macrophage phenotypes such as M1 and M2 in tumor tissue. Because M2 macrophages highly express CD163, we suspected that CD163 may be a useful marker for identification of activation of macrophage phenotypes in AITL. We performed a retrospective study of immunohistochemical expression using two markers for macrophages [CD68 (PG‐M1), CD163] and of the correlation of these expressions with overall survival of 42 AITL patients. The number of CD68‐positive cells in AITL tissues did not correlate with overall survival (P= 0.59), whereas the number of CD163‐positive cells and overall survival correlated to some extent (P= 0.08). Meanwhile, a higher ratio of CD163‐positive to CD68‐positive cells in AITL significantly correlated with worse overall survival (P= 0.036). Considering that this ratio reflects the proportion of macrophages polarized to the M2 phenotype, our findings indicate that activation of macrophages towards the M2 phenotype correlates with worse prognosis. Our findings indicate that the ratio of M2 macrophages expressed may be a useful marker for prognosis of AITL.