靶向CCL21/CCR7轴治疗肿瘤转移的研究进展

转移是肿瘤患者死亡最常见的原因,而淋巴转移是大多数肿瘤转移的主要途径之一。近年来,CC趋化因子配体21 (CC chemokine ligand 21,CCL21) 及其受体CC趋化因子受体7型 (CC chemokine receptor type 7,CCR7) 在淋巴转移中的作用逐渐受到关注。CCL21主要由淋巴内皮细胞产生,其与树突状细胞 (Dendritic cells,DCs) 和T细胞等表面CCR7的相互作用是免疫细胞淋巴迁移及淋巴结归巢的主要决定因素。然而,表达CCR7的肿瘤细胞也可以利用类似的机制进入淋巴管进行淋巴转移。如何靶向CCL21/CCR7轴,既能抑制淋巴转移,又不影响抗肿瘤免疫反应已成为肿瘤免疫治疗研究的重要议题。文中将对CCL21/CCR7轴在淋巴转移中的作用及其作为靶点治疗肿瘤转移的临床前和临床试验研究进行综述,为靶向CCL21/CCR7信号轴治疗肿瘤转移的相关研究提供参考。     

CCL21/CCR7轴与乳腺癌转移及治疗的相关研究进展

乳腺癌是发生在乳腺腺上皮组织的恶性肿瘤,是全世界女性死亡的最常见原因之一。趋化因子CCL21是一种具有趋化细胞迁移功能的低分子量蛋白质,CCL21可以与淋巴细胞表面表达的CCR7受体结合,促使其迁移至乳腺肿瘤相关部位;同时还能与乳腺肿瘤细胞上表达的CCR7配体结合,使其不断向正常部位转移和侵袭,从而导致癌细胞恶性发展。CCL21/CCR7轴与乳腺癌的治疗、转移、侵袭与预后有着密切联系。我们简要综述近年来CCL21/CCR7轴与乳腺癌的转移及治疗相关研究进展,以期为乳腺癌的临床治疗提供参考。     

CC趋化因子配体2与肝疾病

近年来CC趋化因子配体2(chemokine(C-C motif)ligand 2,CCL2)在肝脏疾病发病机制中的作用越来越受到重视.大量研究表明,CCL2在各种肝损伤中表达上调.CCL2是炎症反应的主要调节因子,通过与其受体CCR2相互作用,使血液中的单核细胞穿过血管内皮向炎症部位迁移.白色脂肪组织分泌的CCL2能直接诱导肝细胞的脂肪聚集,与非酒精性肝病的发病机理密切相关.肝实质细胞分泌的CCL2能激活并募集肝星形细胞,参与肝纤维化甚至肝硬化的形成.CCL2能介导肝癌细胞的转移和浸润,刺激肿瘤血管生成,其与肿瘤的关系也成为研究的热点.本文将阐述CCL2与病毒性肝炎、酒精性肝炎、非酒精性脂肪性肝炎、肝纤维化、肝硬化和肝癌的研究进展.     

芍药苷对哮喘模型小鼠气道炎症趋化因子及受体的干预作用

目的观察芍药苷给药后对小鼠哮喘模型气道炎症趋化因子及受体的影响。方法用卵蛋白(OVA)致敏和激发建立小鼠哮喘模型;ELISA法检测血清IL-6、TNF-α水平及支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中卵蛋白特异性Ig E(OVA-Ig E)和趋化因子CCL19、CCL21水平;RT-PCR法检测肺组织中趋化因子受体CCR7mRNA表达;Western blot检测肺组织CCR7及核转录因子-κB(nuclear factor-κB,NF-κB)蛋白表达。结果芍药苷干预组小鼠血清IL-6、TNF-α水平显著下降;BALF中OVA-Ig E和CCL19、CCL21水平显著降低;肺组织CCR7mRNA、CCR7及NF-κB蛋白表达明显减少。结论芍药苷对哮喘模型小鼠气道炎症趋化因子CCL19/CCL21及其受体CCR7具有显著的抑制作用。     

趋化因子CCL11-糖胺聚糖-趋化因子受体CCR3的相互作用研究

目的 探究趋化因子CCL11与受体CCR3、糖胺聚糖（glycosaminoglycans，GAGs）相互作用过程及机制，为深入阐明CCL11-GAGs-CCR3相互作用关系提供理论参考。方法 利用基因工程技术，构建筛选了CCR3-EGFP单分子表达水平的CHO稳转细胞系，利用全内反射荧光成像（total internal reflection fluorescence，TIRF）与等温滴定量热（isothermal titration calorimetry，ITC）技术研究了不同体外溶液条件下GAGs与CCL11的相互作用，并利用趋化实验及活细胞单分子成像实验考察了GAGs-CCL11对CCR3-EGFP稳转细胞趋化行为的调控及CCR3-EGFP在细胞膜上聚集状态的影响。结果 随着硫酸软骨素链长度的增加，其与CCL11结合放热增多，表明其相互作用力增强，其促进CCL11聚集作用增强。单分子荧光成像技术结合趋化试验研究发现，不同种类及不同比例的GAGs均会影响CCL11与CCR3的相互作用，GAGs的加入，抑制了CCL11对CCR3-EGFP稳转细胞的趋化效应及促CCR3-EGFP聚集的能力，且随着硫酸软骨素分子质量的增加，抑制作用显著增强。结论 GAGs的存在可以显著调控CCL11的聚集状态，进而影响其与受体CCR3的相互作用，本研究为进一步阐明CCL11-GAGs-CCR3相互作用关系提供了一定的实验基础。     

CCL5/CCR5生物学轴在肿瘤中作用的研究进展

肿瘤因其易转移、易复发的特性成为一大难以治愈的疾病,已严重威胁到人类的生命健康。肿瘤微环境在肿瘤的生长、迁移、免疫逃逸、血管生成等过程中具有明显的促进作用。肿瘤微环境中细胞分泌的CCL5发挥的作用已受到越来越多的关注,且许多研究表明抑制CCL5/CCR5生物学轴可抑制肿瘤迁移、血管生成等,预示着这可能成为一个新的肿瘤治疗策略。本文总结了近年来关于CCL5/CCR5生物学轴的研究,包括CCL5/CCR5生物学轴介导的肿瘤生长迁移、血管生成、免疫逃逸等作用,及CCR5抑制剂在肿瘤治疗中的广阔前景。     

CCL25和CCL28及其相应受体CCR9和CCR10

CCL25和CCL28是cc趋化因子家族成员,主要表达于胸腺树突状细胞和黏膜上皮细胞,CCL25和CCL28相应的受体分别是CCR9和CCR10,表达于T淋巴细胞和B淋巴细胞。CCL25和CCL28及其相应受体对循环IgA浆母细胞和T淋巴细胞的归巢起重要作用,而且CCL28还具有广谱的抗微生物活性。     

CCL27及其受体在抗病毒免疫中的作用

趋化因子是一类结构相关、对白细胞具有趋化活性、相对分子质量为(8～10)×103的小分子蛋白质,在炎症和免疫反应中可诱导白细胞和淋巴细胞迁移.CCL27是一种CC型趋化因子,其配体为CCR10.CCL27与接触性软疣病毒编码产生的蛋白质MC148有较高的同源性,但却与人疱疹病毒8型编码产生病毒巨噬细胞炎症蛋白-Ⅱ竞争结合受体CCR10.     

CCL22在人类免疫缺陷病毒感染中的作用

CCL22是CC型的趋化因子,其受体为CCR4.CCL22抑制巨噬细胞中R5人类免疫缺陷病毒1型的复制,其影响的不是病毒侵入或反转录等早期过程,而是作用于病毒侵入细胞后阶段.对CCL22独特功能的深入研究和CCR4这一病毒新共受体的发现将为人类免疫缺陷病毒的防治提供新的途径.     

生物肽SP对NK-92MI细胞迁移及趋化因子受体表达的影响

探讨生物肽P物质(substance P,SP)对NK92-MI细胞迁移力和细胞表面趋化因子受体表达的影响,能更好地解释SP调控NK细胞迁移的作用机制,为NK细胞的功能研究及潜在的免疫疗法提供补充依据。Transwell法检测SP对NK92-MI细胞迁移能力的影响及SP对趋化因子CCL21和CXCL12对NK92-MI细胞趋化作用的影响;Real-time PCR检测SP对CCR7和CXCR4 mRNA表达水平的影响;流式细胞术检测SP对CCR7和CXCR4膜表达水平的影响。结果显示:①SP促进NK92-MI细胞的迁移,是在低浓度范围(10~(-12)～10~(-10)mol/L)随SP浓度增加,促进作用逐渐增强,高浓度范围(10~(-8)～10~(-6) mol/L)随SP浓度增加,促进作用又有所减弱,SP浓度在10~(-10) mol/L时,趋化指数达峰值;SP增强趋化因子CCL21和CXCL12对NK92-MI细胞的趋化作用,这种增强作用在10~(-10) mol/L浓度最显著。②SP在10~(-12)～10~(-6) mol/L浓度范围内均能明显促进CCR7 mRNA的表达,且CCR7 mRNA表达水平随着SP浓度增加而增高;SP在10~(-10 )～10~(-6 ) mol/L浓度范围内能明显促进CXCR4 mRNA的表达。③CCR7的膜表达水平随着SP浓度的增加具有逐渐增高的趋势,在10~(-8) mol/L和10~(-6) mol/L浓度组,CCR7的表达有明显增加;而CXCR4的膜表达则随SP浓度的增加,具有先增高后回降的趋势,在10~(-10) mol/L和10~(-8) mol/L浓度组,CXCR4的表达有明显增加。SP能直接促进NK92-MI细胞的迁移,说明SP对NK细胞具有直接趋化作用;SP通过上调趋化因子受体CCR7和CXCR4的表达水平,协同趋化因子,间接发挥对NK-92MI细胞的趋化作用。     

Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 +/- 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 +/- 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2(-/-)/arrestin 3(-/-) murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.     

Solution structure of CCL21 and identification of a putative CCR7 binding site

CCL21 is a human chemokine that recruits normal immune cells and metastasizing tumor cells to lymph nodes through activation of the G protein-coupled receptor CCR7. The CCL21 structure solved by NMR contains a conserved chemokine domain followed by an extended, unstructured C-terminus that is not typical of most other chemokines. A sedimentation equilibrium study showed CCL21 to be monomeric. Chemical shift mapping indicates that the CCR7 N-terminus binds to the N-loop and third β-strand of CCL21's chemokine domain. Details of CCL21-receptor recognition may enable structure-based drug discovery of novel antimetastatic agents.     

Interaction of Polysialic Acid with CCL21 Regulates the Migratory Capacity of Human Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). Immature DCs (iDCs) are situated in the periphery where they capture pathogen. Subsequently, they migrate as mature DCs (mDCs) to draining lymph nodes to activate T cells. CCR7 and CCL21 contribute to the migratory capacity of the DC, but it is not completely understood what molecular requirements are involved. Here we demonstrate that monocyte-derived DCs dramatically change ST8Sia IV expression during maturation, leading to the generation of polysialic acid (polySia). PolySia expression is highly upregulated after 2 days Toll-like receptor-4 (TLR4) triggering. Surprisingly, only immunogenic and not tolerogenic mDCs upregulated polySia expression. Furthermore, we show that polySia expression on DCs is required for CCL21-directed migration, whereby polySia directly captures CCL21. Corresponding to polySia, the expression level of CCR7 is maximal two days after TLR4 triggering. In contrast, although TLR agonists other than LPS induce upregulation of CCR7, they achieve only a moderate polySia expression. In situ we could detect polySia-expressing APCs in the T cell zone of the lymph node and in the deep dermis. Together our results indicate that prolonged TLR4 engagement is required for the generation of polySia-expressing DCs that facilitate CCL21 capture and subsequent CCL21-directed migration.     

Oncostatin M enhances CCL21 expression by microvascular endothelial cells and increases the efficiency of dendritic cell trafficking to lymph nodes

CCL21, a lymphatic endothelial cell (LEC)-derived chemokine, and its receptor CCR7 regulate dendritic cell (DC) trafficking to lymph nodes (LN), but it is unclear how CCL21 expression is regulated. Oncostatin M (OSM) is an IL-6-like cytokine synthesized by activated DC and other leukocytes. In vitro, OSM (but not TNF-alpha) stimulated CCL21 mRNA and protein expression by human dermal microvascular EC (DMEC) in an ERK1/2-dependent fashion. Conditioned medium from OSM-treated DMEC stimulated CCL21-dependent chemotaxis of mouse bone marrow-derived DC (BMDC). Cultured BMDC expressed OSM, which was increased with the addition of LPS. Topical application of the contact-sensitizing hapten, trinitrochlorobenzene, resulted in enhanced OSM expression in the skin, whereas cutaneous injection of TNF-alpha did not. Injection of OSM into the footpad increased CCL21 mRNA expression in the draining LN by approximately 10-fold and in mouse skin by approximately 4-fold without increasing CCR7 mRNA. In vitro, OSM increased the permeability of DMEC and lung microvascular EC monolayers to FITC-dextran beads, and, in vivo, it enhanced accumulation of Evans blue dye in draining LN by approximately 3-fold (p = 0.0291). Of note, OSM increased trafficking of BMDC injected in footpads to draining LN by 2-fold (p = 0.016). In summary, OSM up-regulates CCL21 expression in skin and draining regional LN. We propose that OSM is a regulator of CCL21 expression and endothelial permeability in skin, contributing to efficient migration of DC to regional LN.     

Roles of SLC/CCL21 and CCR7 in human kidney for mesangial proliferation, migration, apoptosis, and tissue homeostasis

The release of chemokines by intrinsic renal cells is an important mechanism for the regulation of leukocyte trafficking during renal inflammation. The expression of chemokine receptors by intrinsic renal cells such as mesangial cells (MC) suggests an expanded role for chemokine-chemokine receptor biology in local immunomodulation and potentially glomerular homeostasis. By immunohistochemistry we found the chemokine receptor CCR7 expressed in a mesangial pattern while the CCR7 ligand SLC/CCL21 showed a podocyte-specific expression. CCR7 expression was further characterized by RT-PCR, RNase protection assays, and FACS analysis of cultured human MC, and was found to be constitutively present. Real-time PCR of microdissected glomeruli confirmed the expression of SLC/CCL21. A functional role for CCR7 was demonstrated for human MC migration and proliferation. A protective effect of SLC/CCL21 was shown for MC survival in Fas Ab-induced apoptosis. Finally, "wound healing" was enhanced in the presence of SLC/CCL21 in an in vitro injury model. The constitutive glomerular expression of CCR7 and its ligand SLC/CCL21 in adjacent cell types of the human kidney suggests novel biological functions of this chemokine/chemokine receptor pair and a potential role in processes involved in glomerular homeostasis and regeneration.     

The lymphoid chemokine CCL21 costimulates naive T cell expansion and Th1 polarization of non-regulatory CD4+ T cells

CCL21 (SLC/6Ckine) is constitutively expressed by secondary lymphoid tissue and attracts CCR7-expressing mature dendritic cells and naive T cells. Recent studies demonstrated that intra-tumoral delivery of CCL21 induces tumor regression in a T cell dependent manner. CCL21 is known to mediate T cell trafficking but little is known about its function as a costimulatory molecule. Herein, we demonstrate that CCL21 costimulates expansion of CD4+ and CD8+ T cells and induces Th1 polarization. These effects were specific for naive T cells, and we show that CD4+CD25+ regulatory T cells were hyporesponsive to CCL21 induced migration, and unresponsive to CCL21 costimulation. These unique functions of CCL21 to both attract naive T cells as well as costimulate their proliferation and differentiation, suggests that CCL21 is a pivotal molecule for priming T cell responses and has therapeutic implications for local delivery of CCL21. The coordinated effects of CCL21 on T cell migration and activation may also represent a more comprehensive paradigm for the activity of other chemokines as well.     

Novel CCL21-vault nanocapsule intratumoral delivery inhibits lung cancer growth

Background

Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer.

Principal Findings

Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3⁺T, CCR7⁺T, IFNγ⁺T lymphocytes, DEC205⁺ DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells.

Significance

This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an “off the shelf” approach to deliver antitumor cytokines to treat a broad range of malignancies.     

CCL25 and CCL28 promote alpha4 beta7-integrin-dependent adhesion of lymphocytes to MAdCAM-1 under shear flow

Inflammatory bowel disease is characterized by the recruitment of lymphocytes to the gut via mucosal vessels. Chemokines are believed to trigger alpha(4)beta(1)- and alpha(4)beta(7)-integrin-mediated adhesion to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on mucosal vessels, although the contribution of each pathway and the chemokines involved are not well characterized. These interactions occur under conditions of hemodynamic shear, which is critical in determining how lymphocytes integrate chemokine signals to promote transmigration. To define the role of specific chemokines in mediating lymphocyte adhesion to VCAM-1 and MAdCAM-1, we studied the ability of immobilized chemokines to activate adhesion of human lymphocytes in a flow-based adhesion assay. Adhesion to immobilized MAdCAM-1 was alpha(4)beta(7) dependent, with no contribution from alpha(4)beta(1), whereas alpha(4)beta(1) mediated rolling and static adhesion on VCAM-1. Immobilized CC-chemokine ligand (CCL) 25 and CCL28 were both able to trigger alpha(4)beta(7)-dependent lymphocyte arrest on MAdCAM-1 under shear, highlighting a potential role for these chemokines in the arrest of lymphocytes on postcapillary venules in the gut. Neither had any effect on adhesion to VCAM-1, suggesting that they selectively trigger alpha(4)beta(7)-mediated adhesion. Immobilized CCL21, CCL25, CCL28, and CXC-chemokine ligand (CXCL) 12 all converted rolling adhesion to static arrest on MAdCAM-1 by activating lymphocyte integrins, but only CCL21 and CXCL12 also triggered a motile phenotype characterized by lamelipodia and uropod formation. Thus alpha(4)beta(1)/VCAM-1 and alpha(4)beta(7)/MAdCAM-1 operate independently to support lymphocyte adhesion from flow, and chemokines may act in concert with one chemokine triggering integrin-mediated arrest and a second chemokine promoting motility and transendothelial migration.     

Polysialic acid is required for neuropilin-2a/b-mediated control of CCL21-driven chemotaxis of mature dendritic cells and for their migration in vivo

Migration of mature dendritic cells (mDCs) to secondary lymphoid organs is required for the development of immunity. Recently, we reported that polysialic acid (PSA) and the transmembrane glycoprotein neuropilin-2 (NRP2) control mDC chemotaxis to CCL21 and that this process is dependent on the C-terminal basic region of the chemokine. Herein, we provide further insight into the molecular components controlling PSA regulated chemotaxis in mDCs. In the present study, we demonstrate that human mDCs express the NRP2 isoforms NRP2a and NRP2b, that both of them are susceptible to polysialylation and that polysialylation is required to specifically enhance chemotaxis toward CCL21 in mDCs. The results presented suggest that PSA attached to NRP2 isoforms acts as a binding module for the CCL21 chemokine, thereby facilitating its presentation to the chemokine receptor CCR7. To investigate the relevance of polysialylation on mDC migration, a xenograft mouse model was used and the migration of human DCs to mouse lymph nodes analyzed. Here, we demonstrate that the depletion of PSA from mDCs results in a drastic reduction in the migration of the cells to draining popliteal lymph nodes. With this finding, we provide first evidence that PSA is a crucial factor for in vivo migration of mDCs to lymph nodes.     

MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20

MicroRNA 21 (miR-21) has been implicated in various aspects of carcinogenesis. However, its function and molecular mechanism in cervical squamous carcinoma have not been studied. Using TaqMan quantitative real-time PCR and Northern blot, we confirmed that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. Remarkably, we showed that the level of miR-21 correlates with the tumor differentiation and nodal status by ISH. Furthermore, we demonstrated that miR-21 regulates proliferation, apoptosis, and migration of HPV16-positive cervical squamous cells. In order to identify candidate target genes for miR-21, we used gene expression profiling. By luciferase reporter assays, we confirmed that CCL20 is one of its target genes, which is related to the HPV16 E6 and E7 oncogenes. Our results suggest that miR-21 may be involved in cervical squamous cell tumorigenesis.