肿瘤微环境与前列腺癌骨转移研究进展

前列腺癌（prostate cancer, PCa）转移中大约80%是发生骨转移,是造成患者死亡的主要原因。前列腺癌骨转移主要呈现成骨和破骨混合性损伤。然而,诱发肿瘤细胞转移到骨以及引起混合样病变的机制仍不十分清楚。宿主微环境与前列腺癌细胞间的相互作用是其中一个重要原因。本文主要讨论宿主微环境如何影响前列腺癌骨转移各个阶段的发生及其中的机制,探讨多种细胞因子、分泌蛋白、微环境中的免疫细胞、成骨细胞、破骨细胞等分泌的因子在前列腺癌骨转移中的重要作用。     

Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis but whether it has additional effects on the tumor microenvironment is largely unexplored. We show that overexpression of PEDF in orthotopic MatLyLu rat prostate tumors increased tumor macrophage recruitment. The fraction of macrophages expressing inducible nitric oxide synthase, a marker of cytotoxic M1 macrophages, was increased, suggesting that PEDF could enhance antitumor immunity. In addition, PEDF overexpression reduced vascular growth both in the tumor and in the surrounding normal tissue, slowed tumor growth, and decreased lymph node metastasis. Contrary, extratumoral lymphangiogenesis was increased. PEDF expression is, for reasons unknown, often decreased or lost during prostate tumor progression. When AT-1 rat prostate tumor cells, expressing high levels of PEDF messenger RNA (mRNA) and protein, were injected into the prostate, PEDF is markedly downregulated, suggesting that factors in the microenvironment suppressed its expression. One such factor could be macrophage-derived tumor necrosis factor α (TNFα). A fraction of the accumulating macrophages expressed TNFα, and TNFα treatment downregulated the expression of PEDF protein and mRNA in prostate AT-1 tumor cells in vitro and in the rat ventral prostate in vivo. PEDF apparently has multiple effects in prostate tumors: it suppresses angiogenesis and metastasis, but it also causes macrophage accumulation. Accumulating macrophages may inhibit tumor growth, but they may also suppress PEDF and enhance lymph angiogenesis and, in this way, eventually enhance tumor growth.     

CCL2-CCR2 Chemokine Signaling Facilitates Tumor Cell Extravasation

Tumor-derived CCL2 activates CCR2-expressing endothelium to enhance vascular permeability.     

骨存储生长因子在肿瘤骨转移中的作用

摘 要：骨存储生长因子又称骨源性生长因子,是大量存在于骨中的一系列生长因子。这些生长因子由存在于骨中的细胞分泌,正常情况下维持稳态,发生骨转移时,一方面癌细胞分泌骨存储生长因子,另一方面癌细胞影响骨中其他细胞分泌骨存储生长因子,都会造成骨存储生长因子分泌量失衡。其中一些骨存储生长因子失衡,如转化生长因子-β和胰岛素样生长因子分泌量增加,其可加速溶骨性损害。近年来,随着对肿瘤转移微环境研究的加深,骨存储生长因子成为研究热点,为了加深对这些生长因子作用机制的了解,本文就目前骨存储生长因子与肿瘤骨转移的研究进展作一综述。     

Targeting CC Chemokine Ligand 2 (CCL2) for Cancer Skeletal Metastasis

Prostate, breast, and lung cancer preferentially metastasize to bone resulting in bone lesions and thus high mortality. However, the mechanisms     

Targeting of CCL2-CCR2-Glycosaminoglycan Axis Using a CCL2 Decoy Protein Attenuates Metastasis through Inhibition of Tumor Cell Seeding

The CCL2-CCR2 chemokine axis has an important role in cancer progression where it contributes to metastatic dissemination of several cancer types (e.g., colon, breast, prostate). Tumor cell–derived CCL2 was shown to promote the recruitment of CCR2⁺/Ly6C^hi monocytes and to induce vascular permeability of CCR2⁺ endothelial cells in the lungs. Here we describe a novel decoy protein consisting of a CCL2 mutant protein fused to human serum albumin (dnCCL2-HSA chimera) with enhanced binding affinity to glycosaminoglycans that was tested in vivo. The monocyte-mediated tumor cell transendothelial migration was strongly reduced upon unfused dnCCL2 mutant treatment in vitro. dnCCL2-HSA chimera had an extended serum half-life and thus a prolonged exposure in vivo compared with the dnCCL2 mutant. dnCCL2-HSA chimera bound to the lung vasculature but caused minimal alterations in the leukocyte recruitment to the lungs. However, dnCCL2-HSA chimera treatment strongly reduced both lung vascular permeability and tumor cell seeding. Metastasis of MC-38GFP, 3LL, and LLC1 cells was significantly attenuated upon dnCCL2-HSA chimera treatment. Tumor cell seeding to the lungs resulted in enhanced expression of a proteoglycan syndecan-4 by endothelial cells that correlated with accumulation of the dnCCL2-HSA chimera in the vicinity of tumor cells. These findings demonstrate that the CCL2-based decoy protein effectively binds to the activated endothelium in lungs and blocks tumor cell extravasation through inhibition of vascular permeability.     

Prostate Cancer Cell-Derived Urokinase-Type Plasminogen Activator Contributes to Intraosseous Tumor Growth and Bone Turnover

A variety of proteases have been implicated in prostate cancer (PC) bone metastasis, but the individual contributions of these enzymes remain unclear. Urokinase-type plasminogen activator (uPA), a serine protease, can activate plasminogen and stimulate signaling events on binding its receptor uPAR. In the present study, we investigated the functional role of PC cell-associated uPA in intraosseous tumor growth and bone matrix degradation. Using a severe combined immunodeficient-human mouse model, we found that PC3 cells were the major source of uPA in the experimental bone tumor. Injection of uPA-silenced PC3 cells in bone xenografts resulted in significant reduction of bone tumor burdens and protection of trabecular bones from destruction. The suppressed tumor growth was associated with the level of uPA expression but not with its activity. An increase in the expression of PAI-1, the endogenous uPA inhibitor, was found during in vitro tumor-stromal interactions. Up-regulation of PAI-1 in bone stromal cells and preosteoclasts/osteoblasts was due to soluble factor(s) released by PC cells, and the enhanced PAI-1 expression in turn stimulated PC cell migration. Our results indicate that both tumor-derived uPA and tumor-stroma-induced PAI-1 play important roles in intraosseous metastatic PC growth through regulation of a uPA-uPAR-PAI-1 axis by autocrine/paracrine mechanisms.     

Decorin Suppresses Prostate Tumor Growth through Inhibition of Epidermal Growth Factor and Androgen Receptor Pathways

Epidermal growth factor receptor (EGFR) and androgen receptor (AR) pathways play pivotal roles in prostate cancer progression. Therefore, agents with dual-targeting ability may have important therapeutic potential. Decorin, a proteoglycan present in the tumor microenvironment, is known to regulate matrix assembly, growth factor binding, and receptor tyrosine kinase activity. Here, we show that in prostate-specific Pten^P-/- mice, a genetically defined, immune-competent mouse model of prostate cancer, systemic delivery of decorin inhibits tumor progression by targeting cell proliferation and survival pathways. Moreover, in human prostate cancer cells, we show that decorin specifically inhibits EGFR and AR phosphorylation and cross talk between these pathways. This prevents AR nuclear translocation and inhibits the production of prostate specific antigen. Further, the phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathway is suppressed leading to tumor cell apoptosis. Those findings highlight the effectiveness of decorin in the presence of a powerful genetic cancer risk and implicate decorin as a potential new agent for prostate cancer therapy by targeting EGFR/AR-PI3K-Akt pathways.     

pH值调控肿瘤细胞生长转移的机制

实体瘤存在一个显著的特征是肿瘤胞内呈碱性和胞外呈酸性的微环境,这种酸性微环境能促进肿瘤细胞的生长、侵袭转移.调节肿瘤酸性微环境的主要因子是膜离子交换体,这些离子交换体如NHE和VHA不仅是肿瘤酸性微环境的主要调节者,而且能促进肿瘤细胞的生长转移.通过调节肿瘤胞内外pH值变化可以抑制肿瘤细胞生长转移,但pH值的变化对肿瘤细胞生长转移的调控机制尚不清楚.全文对肿瘤细胞转移的发生机制、肿瘤酸性微环境的形成和膜离子交换体对其调节作用以及三者的关系进行阐述.     

108例恶性肿瘤骨转移的临床分析

目的 总结分析恶性肿瘤骨转移发病特点及临床表现,以提高骨转移瘤的诊治水平.方法 对108例恶性肿瘤骨转移的临床特点及近期疗效进行回顾性分析.结果 恶性肿瘤骨转移以40～80岁为发病高峰(78.7%),原发灶男性以肺癌,女性以乳腺癌最为多见.转移部位以脊柱、肋骨、骨盆等部位多见.多数患者(86.1%)表现为不同程度的疼痛,少数以局部肿块、功能障碍、病理性骨折甚至截瘫为主要临床表现,13.9%的患者仅以原发灶症状就诊时发.现骨转移.影像学以溶骨性多见(79.3%),16.7%的患者出现不同程度的碱性磷酸酶升高.治疗以全身治疗为主,采用化疗、内分泌治疗、生物治疗、双膦酸盐类药物、止痛药应用及放疗、姑息性手术等综合治疗手段.结论 恶性肿瘤骨转移发生率较高,临床表现复杂.应结合骨扫描、X线、CT等手段早期诊断、综合治疗,以提高生存质量,延长生存时间.     

PSA、FPSA检测和骨显像对前列腺癌骨转移的诊断价值

目的：探讨联合应用前列腺特异性抗原（PSA）、游离PSA（FPSA）检测和全身骨显像诊断前列腺癌骨转移的意义。方法：回顾性分析70例经临床确诊的前列腺癌患者，全部行血清PSA、FPSA测定，并作全身骨显像。结果：PSA〈4ng／ml在14例病人中，发生骨转移者7例，诊断阳性率为50％；PSA4ng／ml～20ng／ml共7例，发生骨转移者6例、诊断阳性率为87％；PSA〉20ng／ml组49例，发生骨转移45例，阳性率为92％。结论：PSA、FPSA检测结合全身骨显像，可尽早、全面地发现前列腺癌患者全身骨转移。     

PSA、FPSA检测和骨显像对前列腺癌骨转移的诊断价值

目的:探讨联合应用前列腺特异性抗原(PSA)、游离PSA(FPSA)检测和全身骨显像诊断前列腺癌骨转移的意义.方法:回顾性分析70例经临床确诊的前列腺癌患者,全部行血清PSA、FPSA测定,并作全身骨显像.结果:PSA<4ng/ml在14例病人中,发生骨转移者7例,诊断阳性率为50%;PSA 4ng/ml～20ng/ml共7例,发生骨转移者6例,诊断阳性率为87%;PSA＞20ng/ml组49例,发生骨转移45例,阳性率为92%.结论:PSA、FPSA检测结合全身骨显像,可尽早、全面地发现前列腺癌患者全身骨转移.     

巴曲酶抑制肿瘤生长和转移的实验研究

[目的]探讨来源于蛇毒的脑梗死治疗药--巴曲酶对肿瘤生长和转移的抑制作用及其相关的作用机制.[方法]选用高转移特性的小鼠B16-BL6黑色素瘤和U14宫颈癌细胞株制作皮下移植瘤以及实验性肺转移和自然转移模型,探讨了40BU/kg巴曲酶的治疗效果.应用酶联免疫吸附反应(ELISA)方法检测血浆纤维蛋白原浓度,利用免疫组化方法检测肿瘤组织中纤维蛋白原的沉积.[结果]巴曲酶(40BU/kg)可显著抑制B16-BL6和U14皮下移植瘤的生长,其抑制率分别为85.7%和77.4%.巴曲酶可有效地抑制U14的实验性肺转移,对照组的肺表面转移结节数的中位数为133个,而 不同的给药方案(肌肉注射巴曲酶)治疗时肺转移结节数减少到24、24.5、26和52个,并且隔日给药或每4天给药一次的治疗方法均有效地抑制了转移灶的生长.在U14自然转移模型中,巴曲酶可有效地抑制肌肉内肿瘤的生长,并且可有效抑制肺转移和淋巴结转移.巴曲酶在降低血浆纤维蛋白原浓度的同时有效抑制地肿瘤组织基质内纤维蛋白原的沉积.实验显示,巴曲酶对小鼠体重、骨髓及一般健康状况无不良影响.[结论]巴曲酶对肿瘤的生长和转移具有明显的抑制作用,并且无骨髓抑制等毒副作用,在肿瘤治疗中将具有广阔的应用前景.     

Barbigerone Inhibits Tumor Angiogenesis,Growth and Metastasis in Melanoma

Tumor angiogenesis, growth and metastasis are three closely related processes. We therefore investigated theeffects of barbigerone on all three in the B16F10 tumor model established in both zebrafish and mouse models,and explored underlying molecular mechanisms. In vitro, barbigerone inhibited B16F10 cell proliferation,survival, migration and invasion and suppressed human umbilical vascular endothelial cell migration, invasionand tube formation in concentration-dependent manners. In the transgenic zebrafish model, treatment with10μM barbigerone remarkably inhibited angiogenesis and tumor-associated angiogenesis by reducing blood vesseldevelopment more than 90%. In vivo, barbigerone significantly suppressed angiogenesis as measured by H andE staining of matrigel plugs and CD31 staining of B16F10 melanoma tumors in C57BL/6 mice. Furthermore,it exhibited highly potent activity at inhibiting tumor growth and metastasis to the lung of B16F10 melanomacells injected into C57BL/6 mice. Western blotting revealed that barbigerone inhibited phosphorylation of AKT,FAK and MAPK family members, including ERK, JNK, and p38 MAPKs, in B16F10 cells mainly through theMEK3/6/p38 MAPK signaling pathway. These findings suggested for the first time that barbigerone could inhibittumor-angiogenesis, tumor growth and lung metastasis via downregulation of the MEK3/6/p38 MAPK signalingpathway. The findings support further investigation of barbigerone as a potential anti-cancer drug.     

Mechanisms Regulating the Secretion of the Promalignancy Chemokine CCL5 by Breast Tumor Cells: CCL5's 40s Loop and Intracellular Glycosaminoglycans

The chemokine CCL5 (RANTES) plays active promalignancy roles in breast malignancy. The secretion of CCL5 by breast tumor cells is an important step in its tumor-promoting activities; therefore, inhibition of CCL5 secretion may have antitumorigenic effects. We demonstrate that, in breast tumor cells, CCL5 secretion necessitated the trafficking of CCL5-containing vesicles on microtubules from the endoplasmic reticulum (ER) to the post-Golgi stage, and CCL5 release was regulated by the rigidity of the actin cytoskeleton. Focusing on the 40s loop of CCL5, we found that the ⁴³TRKN⁴⁶ sequence of CCL5 was indispensable for its inclusion in motile vesicles, and for its secretion. The TRKN-mutated chemokine reached the Golgi, but trafficked along the ER-to-post-Golgi route differently than the wild-type (WT) chemokine. Based on the studies showing that the 40s loop of CCL5 mediates its binding to glycosaminoglycans (GAG), we analyzed the roles of GAG in regulating CCL5 secretion. TRKN-mutated CCL5 had lower propensity for colocalization with GAG in the Golgi compared to the WT chemokine. Secretion of WT CCL5 was significantly reduced in CHO mutant cells deficient in GAG synthesis, and the WT chemokine acquired an ER-like distribution in these cells, similar to that of TRKN-mutated CCL5 in GAG-expressing cells. The release of WT CCL5 was also reduced after inhibition of GAG presence/synthesis by intracellular expression of heparanase, inhibition of GAG sulfation, and sulfate deprivation. The need for a ⁴³TRKN⁴⁶ motif and for a GAG-mediated process in CCL5 secretion may enable the future design of modalities that prevent CCL5 release by breast tumor cells.     

Macrophage Ablation Reduces M2-Like Populations and Jeopardizes Tumor Growth in a MAFIA-Based Glioma Model

Monocytes/macrophages are an influential component of the glioma microenvironment. However, understanding their diversity and plasticity constitute one of the most challenging areas of research due to the paucity of models to study these cells'' inherent complexity. Herein, we analyzed the role of monocytes/macrophages in glioma growth by using a transgenic model that allows for conditional ablation of this cell population. We modeled glioma using intracranial GL261-bearing CSF-1R–GFP⁺ macrophage Fas-induced apoptosis (MAFIA) transgenic mice. Conditional macrophage ablation was achieved by exposure to the dimerizer AP20187. Double immunofluorescence was used to characterize M1- and M2-like monocytes/macrophages during tumor growth and after conditional ablation. During glioma growth, the monocyte/macrophage population consisted predominantly of M2 macrophages. Conditional temporal depletion of macrophages reduced the number of GFP⁺ cells, targeting mainly the repopulation of M2-polarized cells, and altered the appearance of M1-like monocytes/macrophages, which suggested a shift in the M1/M2 macrophage balance. Of interest, compared with control-treated mice, macrophage-depleted mice had a lower tumor mitotic index, microvascular density, and reduced tumor growth. These results demonstrated the possibility of studying in vivo the role and phenotype of macrophages in gliomas and suggested that transitory depletion of CSF-1R⁺ population influences the reconstitutive phenotypic pool of these cells, ultimately suppressing tumor growth. The MAFIA model provides a much needed advance in defining the role of macrophages in gliomas.     

Extratumoral Macrophages Promote Tumor and Vascular Growth in an Orthotopic Rat Prostate Tumor Model

Tumor-associated macrophages are involved in angiogenesis and tumor progression, but their role and specific site of action in prostate cancer remain unknown. To explore this, Dunning R-3327 AT-1 rat prostate tumor cells were injected into the prostate of syngenic and immunocompetent Copenhagen rats and analyzed at different time points for vascular proliferation and macrophage density. Endothelial proliferation increased with tumor size both in the tumor and importantly also in the extratumoral normal prostate tissue. Macrophages accumulated in the tumor and in the extratumoral normal prostate tissue and were most abundant in the invasive zone. Moreover, only extratumoral macrophages showed strong positive associations with tumor size and extratumoral vascular proliferation. Treatment with clodronate-encapsulated liposomes reduced the monocyte/macrophage infiltration and resulted in a significant inhibition of tumor growth. This was accompanied by a suppressed proliferation in microvessels and in the extratumoral prostate tissue also in arterioles and venules. The AT-1 tumors produced, as examined by RT² Profiler PCR arrays, numerous factors promoting monocyte recruitment, angiogenesis, and tissue remodeling. Several, namely, chemokine (C-C) ligand 2, fibroblast growth factor 2, matrix metalloproteinase 9, interleukin 1β, interferon γ, and transforming growth factor β, were highly upregulated by the tumor in vivo compared with tumor cells in vitro, suggesting macrophages as a plausible source. In conclusion, we here show the importance of extratumoral monocytes/macrophages for prostate tumor growth, angiogenesis, and extratumoral arteriogenesis. Our findings identify tumor-associated macrophages and several chemotactic and angiogenic factors as potential targets for prostate cancer therapy.     

C-Kit and Its Ligand Stem Cell Factor: Potential Contribution to Prostate Cancer Bone Metastasis

The tyrosine kinase receptor c-kit and its ligand stem cell factor (SCF) have not been explored in prostate cancer (PC) bone metastasis. Herein, we found that three human PC cell lines and bone marrow stromal cells express a membrane-bound SCF isoform and release a soluble SCF. Bone marrow stromal cells revealed strong expression of c-kit, whereas PC cells showed very low levels of the receptor or did not express it all. Using an experimental model of PC bone metastasis, we found that intraosseous bone tumors formed by otherwise c-kit-negative PC3 cells strongly expressed c-kit, as demonstrated using immunohistochemical and Western blot analyses. Subcutaneous PC3 tumors were, however, c-kit-negative. Both bone and subcutaneous PC3 tumors were positive for SCF. Immunohistochemical analysis of human specimens revealed that the expression frequency of c-kit in epithelial cells was of 5% in benign prostatic hyperplasia, 14% in primary PC, and 40% in PC bone metastases, suggesting an overall trend of increased c-kit expression in clinical PC progression. Stem cell factor expression frequency was more than 80% in all the cases. Our data suggest that the bone microenvironment up-regulates c-kit expression on PC cells, favoring their intraosseous expansion.