血管内皮前体细胞与肿瘤血管发生

实体肿瘤的生长和演进依赖于肿瘤血管生成。肿瘤血管生成包括血管新生(angiogenesis)和血管发生(vasculogenesis),内皮前体细胞(endothelial progenitor cells,EPC)参与的肿瘤血管发生在肿瘤血管生成中起主要作用。EPC来源主要包括骨髓来源的EPC、血管壁定留EPC和成体干细胞经血管内皮分化形成的EPC,不同来源EPC参与的血管发生过程在肿瘤血管生成中起重要作用。其中,肿瘤干细胞作为肿瘤血管内皮前体细胞新来源的认识为研究肿瘤血管发生提供了新的思路。     

Endothelial progenitor cells in breast cancer patients

Purpose Development of new capillary blood vessels is essential for the growth of cancer. Two distinct processes, vasculogenesis and angiogenesis implement the formation of the new vascular network. Recently, it was demonstrated that vasculogenesis creates the primary network of vascular endothelial cells that will become major blood vessels in malignant tumors by the recruitment of CD34⁺/vascular endothelial growth factor receptor 2 (FLK-1)⁺ endothelial progenitor cells (EPCs) to sites of the new vessel formation with subsequent differentiation into mature endothelial cell. Therefore the aim of this study was a) to quantitate EPCs in breast cancer patients and b) to evaluate if the release of EPCs into the circulation is mainly regulated by the tumor himself. Experimental design CD34⁺FLK-1⁺ EPCs were measured in the peripheral circulation of patients with breast cancer (n = 47) before and after therapy. Furthermore the potential of EPCs to differentiate into endothelial cells was investigated by late-outgrowth experiments and the metabolic uptake of dil-acetylated-LDL and immunoreactivity against von Willebrand factor. Results In breast cancer patients the amount of CD34⁺FLK-1⁺ EPCs (percent of peripheral blood mononuclear cells) is significantly increased in women with breast cancer. Tumors larger than 2 cm showed significantly higher values of CD34⁺FLK-1⁺ EPCs. After excision of the tumor the amount of CD34⁺FLK-1⁺ EPCs rapidly declines. Conclusions Our findings lead to the tumor, as source of angiogenic chemokines, is most important for recruiting CD34⁺FLK-1⁺ EPCs during breast cancer development. Therefore circulating endothelial progenitor cells may work as a new diagnostic tool in the screening and diagnosis of breast cancer.     

Endothelial progenitor cells display clonal restriction in multiple myeloma

Background  

In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization.     

Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme

The receptor for the urokinase-type plasminogen activator (uPAR) accounts for many features of cancer progression, and is therefore considered a target for anti-tumoral therapy. Only full length uPAR mediates tumor progression. Matrix-metallo-proteinase-12 (MMP12)-dependent uPAR cleavage results into the loss of invasion properties and angiogenesis. MMP12 can be employed in the field of “targeted therapies” as a biological drug to be delivered directly in patient''s tumor mass. Endothelial Progenitor Cells (EPCs) are selectively recruited within the tumor and could be used as cellular vehicles for delivering anti-cancer molecules. The aim of our study is to inhibit cancer progression by engeneering ECFCs, a subset of EPC, with a lentivirus encoding the anti-tumor uPAR-degrading enzyme MMP12. Ex vivo manipulated ECFCs lost the capacity to perform capillary morphogenesis and acquired the anti-tumor and anti-angiogenetic activity. In vivo MMP12-engineered ECFCs cleaved uPAR within the tumor mass and strongly inhibited tumor growth, tumor angiogenesis and development of lung metastasis.The possibility to exploit tumor homing and activity of autologous MMP12-engineered ECFCs represents a novel way to combat melanoma by a “personalized therapy”, without rejection risk.The i.v. injection of radiolabelled MMP12-ECFCs can thus provide a new theranostic approach to control melanoma progression and metastasis.     

Profiling of high-grade central osteosarcoma and its putative progenitor cells identifies tumourigenic pathways

Background:

Osteosarcoma is the most prevalent primary malignant bone tumour in children and young adults, with poor survival in 40% of patients. To identify the signalling pathways involved in tumourigenesis, we compared gene expression in osteosarcoma with that in its presumed normal counterparts.

Methods:

Genome-wide expression profiles were generated from 25 high-grade central osteosarcoma prechemotherapy biopsies, 5 osteoblastomas, 5 mesenchymal stem cell (MSC) populations and these same MSCs differentiated into osteoblasts. Genes that were differentially expressed were analysed in the context of the pathways in which they function using the GenMAPP programme.

Results:

MSCs, osteoblasts, osteoblastomas and osteosarcomas clustered separately and thousands of differentially expressed genes were identified. The most significantly altered pathways are involved in cell cycle regulation and DNA replication. Several upstream components of the Wnt signalling pathway are downregulated in osteosarcoma. Two genes involved in degradation of β-catenin protein, the key effectors of Wnt signalling, Axin and GSK3-β, show decreased expression, suggesting that Wnt signalling is no longer under the control of regular signals. Comparing benign osteoblastomas with osteosarcomas identified cell cycle regulation as the most prominently changed pathway.

Conclusion:

These results show that upregulation of the cell cycle and downregulation of Wnt signalling have an important role in osteosarcoma genesis. Gene expression differences between highly malignant osteosarcoma and benign osteoblastoma involve cell cycle regulation.     

携带干扰素基因的内皮祖细胞靶向抑制肿瘤的实验研究

目的:研究携带阿尔法干扰素(hαIFN)基因的内皮祖细胞(EPCs)对肿瘤免疫治疗的效果。方法:用健康捐献者的骨髓体外诱导培养EPCs,携带hαIFN基因的腺病毒Ad5-hαIFN转染EPCs制备EPCs-hαIFN,ELISA法检测EPCs-hαIFN培养上清液中hαIFN浓度。体外EPCs-hαIFN与HepG-2肝癌细胞按照1∶5的浓度混合培养,观察肿瘤细胞增殖情况。制作裸鼠肝癌模型,静脉注射EPCs-hαIFN,观察对肿瘤的抑制作用和对荷瘤动物生存期的影响。结果:使用人骨髓可以培养出CD133、KDR和CD34阳性的EPCs。携带hαIFN基因的EPCs-hαIFN培养液中可以测得hαIFN,最高浓度可以达1400pg/ml以上,在相当长的一段时间内细胞培养液的hαIFN浓度保持稳定。EPCs-hαIFN与HepG-2细胞按照1∶5的浓度混合培养4天,HepG-2细胞的存活率为(70.50±3.33)%(P<0.05)。通过静脉注射EPCs-hαIFN可以抑制肿瘤的生长[肿瘤重量:EPCs-hαIFN组(0.71±0.23)g,空白对照组(3.27±1.28)g,P<0.05],而皮下注射hαIFN和静脉注射EPCs-EGFP对肿瘤没有抑制作用。尾静脉注射EPCs-hαIFN可延长荷瘤裸鼠的生存时间(中位生存时间:EPCs-hαIFN组为39天,空白对照组为33天,P<0.05)。结论:肿瘤靶向性的EPCs-hαIFN可以通过分泌hαIFN抑制肿瘤的进展。     

内皮祖细胞作为干扰素基因载体的体外抑瘤实验

目的 研究用人骨髓诱导培养内皮祖细胞（EPCs）并以其作为免疫性基因载体治疗肿瘤的可行性。方法 采用梯度离心法从人骨髓中分离单个核细胞,加入人血管内皮生长因子(VEGF165）诱导培养,用流式细胞仪测定细胞表面标志CD133、KDR和CD34的表达情况。以增强型绿色荧光蛋白基因（EGFP）作为报告基因,用流式细胞仪检测携带EGFP基因的腺病毒载体（Ad-EGFP）对EPCs的感染效率。携带人干扰素γ（hγIFN）基因的腺病毒载体（Ad-hγIFN）感染EPCs（EPCs-hγIFN）,ELISA法检测细胞EPCs-hγIFN分泌 hγIFN的情况。将EPCs-hγIFN与肺癌细胞株H460、胃癌细胞株SGC7901、大肠癌细胞株LoVo混合培养,观察EPCs-hγIFN对肿瘤细胞的作用。结果 经过诱导培养2周后,贴壁的单核细胞开始表达CD133、KDR和CD34。Ad-EGFP的感染复数（MOI）为150pfu／EPCs时,感染率达90％以上。EPCs-hγIFN培养上清可以测得浓度稳定且高水平的hγIFN,第1天上清液中的hγIFN浓度为1305.28pg／ml,第14天为1419.10pg／ml。EPCs-hγIFN与H460、SGC7901、LoVo细胞株混合培养4天,肿瘤细胞的生长受到明显的抑制,肿瘤细胞存活率分别为（77.87 ± 6.50）％、（79.36 ± 4.35）％和（69.52 ± 3.78）％, 均显著低于对照组（P＜0.05）。结论携带hγIFN目的基因的腺病毒转染EPCs后基因表达情况良好,体外实验中表现为抑制肿瘤细胞的生长,可作为免疫性基因载体开展进一步的研究。     

Endothelial progenitor cells do not contribute to tumor endothelium in primary and metastatic tumors

Despite extensive research, the contribution of bone‐marrow‐derived endothelial progenitor cells (BM‐EPC) to tumor angiogenesis remains controversial. In previous publications, the extent of incorporation of BM‐EPCs into the endothelial cell (EC) layer in different tumor models has been reported as significant in some studies but undetectable in others. Here, we studied the differentiation of BM‐EPCs and its contribution to tumor vessels in experimental and spontaneous lung metastasis (B16 melanoma and prostate carcinoma), in an autochthonous transgenic model of prostate tumorigenesis, in orthotopically implanted lung tumors [Lewis lung carcinoma (LLC)], in heterotopic subcutaneous models (LLC and C1 prostate carcinoma) growing in green fluorescent protein (GFP)‐expressing bone marrow (BM) chimeras. Immunofluorescence was performed with a set of endothelial and hematopoietic markers and confocal microscopy was used to generate 3D reconstruction images. By performing rigorously conducted morphological studies, we found no evidence of BM‐EPCs differentiation into tumor endothelium independently of tumor type, grade and organ site in primary and metastatic tumors. The vast majority of GFP⁺ cells were trafficking leucocytes or periendothelial myeloid cells. To explore the possibility that local overexpression of vascular endothelial growth factor (VEGF) might increase the numbers of incorporated BM‐EPCs, we analyzed tumors genetically manipulated to overexpress VEGF₁₆₄. Local VEGF production induces a massive infiltration of bone‐marrow‐derived cells, but did not lead to vessel wall integration of these cells. Collectively, these findings suggest that during tumor progression vascularization occurs primarily via classical tumor angiogenesis (e.g., sprouting of pre‐existing ECs), whereas BM‐EPCs do not incorporate into the vessel wall to any significant extent. © 2009 UICC     

Endothelial differentiation of hematopoietic stem and progenitor cells from patients with multiple myeloma.

PURPOSE: Vasculogenesis is a physiologic process typical of fetal development in which new blood vessels develop from undifferentiated precursors (or angioblasts). In tumors, near angiogenesis, vasculogenesis contributes to the formation of the microvascular plexus that is important for diffusion. Here, we show that hematopoietic stem and progenitor cells (HSPC) of multiple myeloma (MM) patients are able to differentiate into cells with endothelial phenotype on exposure to angiogenic cytokines. EXPERIMENTAL DESIGN: Circulating HSPCs were purified with an anti-CD133 antibody from patients with newly diagnosed MM before autologous transplantation and exposed to vascular endothelial growth factor (VEGF), fibroblast growth factor-2 and insulin-like growth factor in a 3-week culture. RESULTS: HSPCs gradually lost CD133 expression and acquired VEGF receptor-2, factor VIII-related antigen, and vascular endothelial-cadherin expression. The expression pattern overlapped with paired MM endothelial cells (MMEC). During culture, cells adhered to fibronectin, spread, and acquired an endothelial cell shape. Differentiated HSPCs also became capillarogenic in the Matrigel assay with maximal activity at the third week of culture. Bone marrow biopsies revealed HSPCs inside the neovessel wall in patients with MM but not in those with monoclonal gammopathy of undetermined significance. CONCLUSIONS: In patients with MM, but not in those with monoclonal gammopathy of undetermined significance, HSPCs contribute to the neovessel wall building together with MMECs. Therefore, besides angiogenesis, HSPC-linked vasculogenesis contributes to neovascularization in MM patients. Tentatively, we hypothesize that in HSPC cultures a multipotent cell population expressing low VEGF receptor-2 levels corresponds to the endothelial progenitor cell precursor and seems to be the MMEC precursor.     

Cancer cells as targets for lentivirus-mediated gene transfer and gene therapy

Lentiviruses have been used as gene transfer vectors for almost 10 years and their utility has been demonstrated in a variety of different applications. However, their value in cancer gene therapy has not been studied thoroughly. Here we show that VSV-G pseudotyped HIV-1-based lentiviruses are efficient vectors for human tumor cells in vitro and in vivo. Lentiviral gene transfer efficiency was demonstrated by transducing 42 different cell lines, representing 10 different human tumor types. It was shown that most of the cell lines were good or excellent targets for lentiviral transduction, allowing 50-95% gene transfer efficiency. These results were comparable to those obtained with an E1/E3 deleted, serotype 5 adenovirus vector. Analysis of lentivirus vector structure revealed that virus particles devoid of HIV-1 accessory proteins appeared to be more efficient, but the presence of enhancing elements cPPT and WPRE did not play a major role in transduction efficiency to four different human tumor cell lines. However, their effect on the gene expression level in these cells was apparent. To examine the impact of lentiviral gene expression level on suicide gene therapy approach, human osteosarcoma cells were transduced with lentivirus- or adenovirus vectors carrying the fusion gene HSV-TK-GFP and exposed to ganciclovir. Cell viability analysis after the treatment revealed that both vector types induced similar level of cytotoxicity, suggesting that lentiviral expression of a suicide gene is adequate for tumor cell destruction. Finally, in vivo transduction studies with subcutaneous tumors showed that lentivirus vectors can yield similar gene transfer efficiency than adenovirus vector, despite three orders of magnitude lower titer of the lentiviral preparation. In conclusion, these data show that lentiviruses are efficient gene transfer vehicles for human tumor cells and justify their use in further preclinical cancer gene therapy studies.     

Alpha-fetoprotein producing cells act as cancer progenitor cells in human cholangiocarcinoma

We aimed to demonstrate that alpha-fetoprotein (AFP)-producing cells in cholangiocarcinomas possessed cancer stem cell (CSC)-like properties. AFP enhancer/promoter-driven EGFP gene was transfected into human cholangiocarcinoma cell lines. One cell line, RBE, expressed both AFP and EGFP. Clonal analyses revealed that one EGFP-positive cell generated both EGFP-positive and EGFP-negative cell fractions. However, one EGFP-negative cell never produced EGFP-positive cells. The EGFP-positive cells had a greater tumorigenic potential. Only the EGFP-positive cells expressed Notch1. AFP and Notch1 expression was observed in clinical intrahepatic cholangiocarcinomas. The AFP-producing cells were suggested to be CSCs. The Notch pathway might play an important role in maintaining the CSC characteristics.     

Engineered embryonic endothelial progenitor cells as therapeutic Trojan horses

While the hematogenic contribution of circulating endothelial cells to tumor angiogenesis is not entirely understood, one can exploit this phenomenon as a therapeutic strategy. In this issue of Cancer Cell, show that murine embryonic endothelial progenitor cells preferentially home to sites of lung metastases, evade immunological rejection, and can exert a bystander antitumor effect when modified to contain a suicide gene construct that activates a prodrug. Treatment with the prodrug led to improved survival in syngeneic and nonsyngeneic tumor-bearing mouse models. The conceptual advance put forward by this study might result in translational applications.     

血管抑素联合顺铂治疗Lewis肺癌

目的探讨血管抑素联合顺铂对内皮细胞增殖和Lewis肺癌生长抑制作用.方法将内皮细胞接种到96孔板中,加入不同试药72 h后,作MTT检测每孔中的细胞数.将Lewis肺癌细胞接种到C57小鼠右腋下,随机分为4组,分别用不同试药处理.每2日测量瘤体积,20天后处死动物,称取瘤重,常规病理和免疫组织化学染色检查,观察微血管密度和血管内皮细胞生长因子表达情况.结果顺铂与血管抑素在抑制血管内皮细胞增殖方面具有协同作用.顺铂组和顺铂 血管抑素组瘤体积、瘤重和血管密度均明显＜对照组.其中以顺铂与血管抑素在同时应用时瘤体积与瘤重又明显低于顺铂组.结论血管抑素与顺铂同时使用对lewis肺癌生长的抑制作用优于先后使用.     

Differentiation-induced ML-1 cells as targets for transformation by a chemical carcinogen

The capacity for continuous proliferation (immortalization) of ML-1 human myeloblastic leukemia cells derives from their sensitivity to growth factors and their insensitivity to differentiation factors (DF) at the limiting concentrations at which these are present in the culture medium. Upon increasing the concentration of DF, or after treatment with DNA-specific anticancer agents, the cells exit the proliferation program and differentiate to monocyte/macrophage-like cells (Y. Honma, C. Honma, and A. Bloch, Cancer Res., 46: 6311-6315, 1986). The study reported here shows that when ML-1 cells, induced to differentiate with DF contained in mitogen-stimulated human leukocyte-conditioned medium (CM) are treated with the carcinogen N-nitroso-N-methylurea, their differentiation program is interrupted and proliferation is resumed at a stably increased rate of growth (doubling time, 25.1 versus 31.3 h). This "step-up" transformation is brought about by only a narrow concentration range of carcinogen, acting at a restricted time interval following differentiation induction. The step-up transformed cells are more sensitive to growth factor signals and less sensitive to DF signals than are untreated ML-1 cells. When retreated with a higher concentration of DF and the same concentration of N-nitroso-N-methylurea, the transformed cells undergo a further decrease in doubling time to 21 h. Differentiation-uninduced ML-1 cells do not respond to treatment with N-nitroso-N-methylurea, indicating that differentiation-induced cells, at an early stage of the maturation process, may be the targets for the carcinogen-mediated transformation.     

Brain tumor stem cells as research and treatment targets

Glioblastoma multiforme (GBM) is one of the most malignant forms of human cancer. Despite intensive treatment, the mean survival of GBM patients remains about 1 year. Recent cancer studies revealed that cancer tissues are pathologically heterogeneous and only a small population of cells has the specific ability to reinitiate cancer. This small cell population is called cancer stem cells (CSCs); in brain tumors these are known as brain tumor stem cells (BTSCs). The identification of BTSCs yielded new insights into chemo-and radioresistance, by which BTSCs can survive selectively and initiate recurrence. Research focused on BTSCs as treatment targets may contribute to the discovery of new therapeutic strategies.     

Differential production of angiostatin by concomitant antitumoral resistance-inducing cancer cells

HCC is a common cancer and HBV and AFB(1) are well-documented, major risk factors. Epidemiologic studies have documented that cigarette smoking also contributes to the development of HCC. PAHs are ubiquitous environmental pollutants and products of incomplete combustion. They are present in both mainstream and sidestream cigarette smoke. PAHs are metabolically activated by phase I enzymes, including CYP1A1, into electrophilic reactants (diol epoxides), which covalently bind to DNA to form adducts. Diol epoxides are also substrates for phase II detoxifying enzymes, including GSTM and GSTP. To examine the association between PAH-DNA adducts and HCC, adduct levels were determined in liver tissue by relative staining intensity with an immunoperoxidase method using a polyclonal antiserum against BPDE-modified DNA. Subjects were also genotyped for polymorphism in several genes involved in the metabolism of PAH, including GSTM1 and GSTP1. Liver tissue was collected from patients with histologically confirmed HCC (n = 105) and from non-HCC controls (n = 37). There was a significant positive correlation (r = 0.3, p < 0.01) between adducts in tumor and adjacent nontumor tissues among HCC cases. The risk of HCC was higher after adjustment for age, sex and HBsAg in the group with the highest tertile tissue levels of PAH-DNA adducts (mean relative nuclear staining intensity of tumor and nontumor tissue > 344) than in the group with the lowest tertile (staining < 241, OR = 3.9, 95% CI = 1.0-14.9). Among non-HCC controls, there were no significant associations between adduct levels and cigarette smoking, GSTM1 null genotype and HBsAg positivity. A strikingly increased HCC risk was observed (OR = 20.3, 95% CI = 5.0-81.8) among HBsAg-positive subjects whose PAH-DNA adduct levels were high (mean relative nuclear staining intensity of tumor and nontumor tissue > 301, median of control tissues) compared to HBsAg-negative subjects who had low PAH-DNA adduct levels. 4-ABP- and AFB(1)-DNA adducts had been measured previously in these same tissues. Subjects with elevated DNA adduct levels of PAH, 4-ABP and AFB(1) had a significantly higher HCC risk with an OR of 36.7 (95% CI 7.2-187.2) compared to those who had low DNA adduct levels. These results suggest that PAHs may play a role in human hepatocarcinogenesis in conjunction with HBsAg carrier status, GSTM1 and GSTP1 genotypes and exposure to 4-ABP and AFB(1).     

Lysosomes as targets for cancer therapy

Tumor invasion and metastasis are associated with altered lysosomal trafficking and increased expression of the lysosomal proteases termed cathepsins. Emerging experimental evidence suggests that such alterations in lysosomes may form an "Achilles heel" for cancer cells by sensitizing them to death pathways involving lysosomal membrane permeabilization and the release of cathepsins into the cytosol. Here, we highlight recent results on cancer-related changes in the composition and function of lysosomes, focusing on possible implications for the development of novel cancer therapeutics that target tumor cell lysosomes.