Erythropoietin Promotes the Growth of Tumors Lacking Its Receptor and Decreases Survival of Tumor-Bearing Mice by Enhancing Angiogenesis

Erythropoietin (Epo), a known hematopoietic growth factor, has been reported to promote tumor growth and angiogenesis in Epo receptor (EpoR)-positive tumors, but its effects on EpoR-negative tumors have not been clearly shown. Here, we show that Epo accelerates the growth of EpoR-negative tumors by promoting tumor angiogenesis. Mice were inoculated with Lewis lung carcinoma cells and treated with Epo. Erythropoietin accelerated tumor growth and increased intratumoral microvessel density, although it did not accelerate Lewis lung carcinoma cell tumor proliferation in vitro. To observe the direct effect of Epo on endothelial cells, we examined human dermal microvascular endothelial cells (HMVECs) that expressed EpoR. Erythropoietin induced the proliferation of HMVECs and protected them from H₂O₂-induced cell death. Erythropoietin activated the extracellular signal-regulated kinase signaling pathway and up-regulated the expression of the downstream antiapoptotic protein Bcl-xL in HMVECs. Moreover, in both the absence and presence of tumors, in vivo treatment of mice with Epo increased circulating endothelial progenitor cells. To investigate the role of Epo in a primary tumor model, we inoculated the chemical carcinogen methylcholanthrene (MCA) subcutaneously into mice at two doses, a high or a low dose, which induced fibrosarcoma, and treated them with Epo. Erythropoietin promoted tumor growth after MCA inoculation at both doses and decreased the overall survival of the mice inoculated with the high-dose MCA. However, Epo did not increase the incidence of fibrosarcoma at either dose. Lewis lung carcinoma cells and MCA-induced fibrosarcomas did not express EpoR. These results suggest that Epo accelerates the growth of tumors that lack EpoR expression by promoting tumor angiogenesis.     

TIMP-3 deficiency in the host, but not in the tumor, enhances tumor growth and angiogenesis

Tumor cells, stromal cell compartment and the extracellular matrix (ECM) together generate a multifaceted tumor microenvironment. Matrix metalloproteinases and their tissue inhibitors (TIMPs) provide a means for tumor-stromal interaction during tumorigenesis. Among TIMPs, TIMP-3 is uniquely localized to the ECM and is frequently silenced in human cancers. Here, we asked whether the absence of TIMP-3 in the tumor cell or the host affects the process of tumorigenesis. Timp-3(-/-) ES-cell clones were generated and used to develop teratomas in nude mice. Timp-3(-/-) teratomas showed similar tumor take, growth, and angiogenesis compared to timp-3(+/+) teratomas. To study the effect of TIMP-3 ablation in the host stroma, we measured the growth kinetics of subcutaneous B16F10 melanomas in timp-3(-/-) and wild-type littermates. Tumors grew significantly faster in timp-3(-/-) than in wild-type mice and their CD31 content was significantly higher indicating increased angiogenesis. Augmented angiogenesis in timp-3(-/-) mice was directly tested using Matrigel plug and Gelfoam assays. In response to FGF-2, timp-3(-/-) endothelial cells invaded more efficiently, leading to enhanced formation of functional blood vessels. Thus, TIMP-3 deficiency in the host, but not in the tumor per se, leads to enhanced tumor growth and angiogenesis. TIMP-3 located within the tumor microenvironment inhibits tumorigenesis.     

Cellular changes in the tumor microenvironment of human esophageal squamous cell carcinomas

The growth, invasiveness, and metastasis of human cancers are not only determined by the cancer cells but also by their microenvironment. The purpose of this study was to extend our previous studies and to examine the cellular changes in tumor microenvironment (stroma) of esophageal squamous cell carcinomas (ESCCs). The proliferative activity, cellular components, and angiogenesis status in different compartments (non-tumor stroma, tumor stroma, and tumor periphery stroma) of ESCCs were evaluated by immunohistochemistry. The results revealed a hyperproliferative rate labeled by Ki-67 in stromal cells in tumor area as compared with that in stromal cells in non-tumor area, which resulted in the increased densities of myofibroblasts (labeled by smooth muscle actin (SMA)-alpha), lymphocytes (labeled by CD3), macrophages (labeled by CD68), and the activation of angiogenesis characterized by increased microvessel density (MVD) and the increased expression of the proangiogenic factors (vascular endothelial growth factor and interleukin 8) in the tumor stroma. Further analysis showed that the changes of stromal cell density were more significant in the area of periphery tumor stroma than that of stroma between tumor nests. Most cellular changes were significantly associated with lymph node involvement. Double immunohistochemistries with PCNA/CD3, PCNA/CD68, and PCNA/SMA-alpha revealed that these cells present in the ESCC tumor stroma had a proliferative capacity. The cells present in the tumor microenvironment of ESCCs were greatly activated, suggesting that microenvironmental components may be involved in the cancer growth and progression.     

Promotion of angiogenesis by ps20 in the differential reactive stroma prostate cancer xenograft model

Human prostate cancer is associated with a reactive stroma typified by an increase in the proportion of myofibroblast type cells and elevated synthesis of extracellular matrix proteins. Increased vascular density has been identified in the reactive stroma compartment adjacent to both precancerous and cancerous prostate lesions. The differential reactive stroma (DRS) prostate cancer xenograft model has been developed to investigate the role of reactive stroma in prostate cancer progression. Using this model, we have shown that human prostate stromal cells promote angiogenesis and growth of LNCaP human prostate carcinoma cell tumors, and that these increases are transforming growth factor (TGF) beta1 regulated. Our laboratory isolated and identified previously the ps20 protein (WFDC1 gene) as a prostate stromal cell secreted protein. The ps20 protein contains a whey acidic protein-type four-disulfide core domain, which is a functional motif characterized by serine protease inhibition activity in a number of whey acidic protein domain-containing proteins. In the present study, we show ps20 expression by normal human prostate stromal smooth muscle cells and vascular smooth muscle cells indicating a possible role of ps20 in vessel wall biology. Using in vitro assays, we show that ps20 promotes endothelial cell motility but has no effect on endothelial cell proliferation. To address the potential effects of ps20 in a tumor microenvironment, we used the DRS model to evaluate both angiogenesis and tumorigenesis of tumors generated under either ps20 or control conditions. DRS tumors generated with LNCaP and human prostate stromal cells in the presence of ps20 showed a 67% increase in microvessel density compared with control tumors. Elevated DRS tumor growth in the ps20-treated tumors was reflected by a 29% increase in wet weight and a 58% increase in volume compared with controls. Similar tumors composed of GeneSwitch-3T3 cells engineered to express ps20-V5-His under mifepristone regulation showed a 129% increase in microvessel density after induction of ps20-V5-His. GeneSwitch-3T3 cells expressing ps20-V5-His were localized to vessel walls in a mural cell (pericyte) position indicating a possible direct stabilizing interaction with endothelial cells. In addition, we show that ps20 mRNA synthesis is induced by TGF-beta1, a known regulator of endothelial cell-pericyte interactions and of stromal cell-induced angiogenesis in DRS tumors. These findings suggest that ps20 may be a TGF-beta1-induced regulator of angiogenesis that functions by either promoting endothelial cell migration or by contributing to pericyte stabilization of newly formed vascular structures.     

Monocyte chemoattractant protein-1 expression correlates with macrophage infiltration and tumor vascularity in human gastric carcinomas

The purpose of this study was to investigate the role of interactions between tumor cells and macrophages during angiogenesis in human gastric carcinomas. Macrophage infiltration into tumors and monocyte chemoattractant protein-1 (MCP-1) expression was assessed in 72 archival specimens of gastric carcinoma for comparison with tumor vascularity. The mRNA expression of MCP-1 was examined by RT-PCR in 6 gastric carcinoma cell lines and in fresh biopsy specimens from 18 patients. Immunolocalization of representative angiogenic factors, vascular endothelial growth factor (VEGF), and platelet-derived endothelial cell growth factor (PD-ECGF) was also done. MCP-1 expression in tumor cells increased with the depth of tumor invasion (Tis 9.5%, T1 19.4%, T2-4 60.0%), as did microvessel density and macrophage infiltration. Macrophage counts correlated with vessel counts, and both were significantly higher in MCP-1-positive than in negative tumors. Of the 6 gastric carcinoma cell lines, 2 constitutively expressed MCP-1 mRNA. In 6 (33.3%) of 18 biopsy samples, MCP-1 mRNA was expressed at higher levels in tumor tissues than in normal mucosa. VEGF protein was expressed by gastric carcinoma cells, whereas PD-ECGF protein was expressed mainly by stromal mononuclear cells. MCP-1 expression correlated significantly with VEGF but not PD-ECGF expression in gastric carcinomas. These results suggest that MCP-1 produced by human gastric carcinoma cells plays a role in angiogenesis via macrophage recruitment and activation.     

The role of stromal myofibroblast and extracellular matrix in tumor angiogenesis

Tumor angiogenesis, the building of blood vessels in an expanding tumor mass, is an elegantly coordinated process that dictates tumor growth and progression. Stromal components of the tumor microenvironment, such as myofibroblasts and the extracellular matrix, collaborate with tumor cells in regulating development. Such myofibroblasts and the extracellular matrix have ever-expanding roles in the angiogenic process as well. This review summarizes how stromal myofibroblasts and the extracellular matrix can modulate tumor angiogenesis, highlighting recent findings.     

Syndecan-1 expression by stromal fibroblasts promotes breast carcinoma growth in vivo and stimulates tumor angiogenesis

The induction of the cell surface heparan sulfate proteoglycan syndecan-1 (Sdc1) in stromal fibroblasts is observed in more than 70% of human breast carcinomas. Using a coculture model, we have recently shown that stromal cell-derived Sdc1 stimulates carcinoma cell proliferation in vitro, and that this activity requires Sdc1 glycanation. In the present study, we investigated the effect of stromal cell Sdc1 on breast carcinoma growth in vivo. MDA-MB-231 human breast carcinoma cells were inoculated into the flanks of athymic nude mice either alone, or as mixed suspensions with Sdc1-transfected or mock-transfected 3T3 mouse fibroblasts. The mixed tumors showed an intimate association between carcinoma cells and stromal fibroblasts and histologically closely resembled poorly differentiated human breast carcinomas. The presence of fibroblasts led to significantly accelerated tumor growth, which was further augmented (88% increase) by forced expression of stromal Sdc1. The hyperemic macroscopic appearance of tumors containing Sdc1-positive stromal cells contrasted with pale tumors developing in the presence of mock-transfected fibroblasts, which prompted us to examine tumor microvessels. Stromal Sdc1 expression was associated with a significantly elevated microvessel density (36% increase) and a larger vessel area (153% increase). To evaluate the relevance of this finding in human breast cancer, the relationship between stromal Sdc1 and tumor vascularity was also examined in a tissue array containing 207 human breast carcinoma samples. Similar to the xenografts, stromal Sdc1 expression correlated with both vessel density (P=0.013) and total vessel area (P=0.0026). In conclusion, stromal fibroblast-derived Sdc1 stimulates breast carcinoma growth and angiogenesis in vivo.     

Stromal cells promote angiogenesis and growth of human prostate tumors in a differential reactive stroma (DRS) xenograft model

Reactive stroma has been reported in many cancers, including breast, colon,and prostate. Although changes in stromal cell phenotype and extracellular matrix have been reported, specific mechanisms of how reactive stroma affects tumor progression are not understood. To address the role of stromal cells in differential regulation of tumor incidence, growth rate, and angiogenesis, LNCaP xenograft tumors were constructed in nude mice with five different human prostate stromal cell lines as well as GeneSwitch-3T3 cells engineered to express lacZ under mifepristone regulation. Alone, LNCaP prostate carcinoma cells were essentially nontumorigenic, whereas combinations of LNCaP cells with three different human prostate stromal cell lines (L/S tumors) resulted in a tumor incidence (50-63%) similar to that of control LNCaP plus Matrigel (L/M) tumors over a 9-week period. In contrast, LNCaP combinations with two other human prostate stromal cell lines were nontumorigenic, illustrating that stromal cell effects are differential. L/S tumors exhibited well-developed blood vessels at 2 weeks, whereas control L/M tumors were avascular at 2 weeks and exhibited blood lakes in lieu of extensive vessels at later time points. Xenografts constructed under three-way conditions (LNCaP, Matrigel, and stromal cells; L/M/S tumors) exhibited a 100% tumor incidence and showed rapid blood vessel formation as early as day 7 with mature vessels formed by day 10. L/M/S tumors exhibited a 10.3-fold increase in microvessel density, and the corresponding hemoglobin:tumor weight ratio was increased 2-fold relative to L/M control tumors at day 10. L/M/S tumor wet weight and volume increased by 1.6- and 2.4-fold, respectively, by day 21, compared with control L/M tumors. L/M/S tumors made with LNCaP cells plus GeneSwitch-3T3-pGene/lacZ stromal cells showed similar results. Mifepristone-regulated gene expression was observed in stromal cells immediately adjacent to clusters of carcinoma cells and in vessel walls in a mural cell (pericyte) position. This study shows that regulation of angiogenesis is one mechanism through which stromal cells affect LNCaP tumor incidence and growth rate. This regulation may be mediated through direct recruitment and interactions of stromal cells with endothelial cells. Furthermore, this study describes for the first time a model system with regulated transgene expression in the stromal compartment of an experimental carcinoma. These findings point to the stromal compartment as a potential source of new prognostic markers and therapeutic targets and show the utility of the carcinoma-stromal xenograft model system in dissecting specific mechanisms of reactive stroma.     

Angiopoietin‐1 alters tumor growth by stabilizing blood vessels or by promoting angiogenesis

The maturation of blood vessels requires mural cell adhesion to endothelial cells. Angiopoietin‐1 (Ang1), a ligand for Tie2 receptor expressed on endothelial cells, plays a critical role in cell adhesion between mural cells and endothelial cells and in endothelial cell sprouting from preexisting vessels in the absence of mural cells. Much information has been amassed on the Tie2–Ang1 system in physiological blood vessel formation during embryogenesis; however, the role of Ang1 in the tumor environment and its interaction with mural cells has not been well documented. Here we studied how Ang1 regulates maturation of blood vessels using the human colon cancer cell line HT29 and the human prostate cancer cell line PC3, and studied how Ang1 affects tumor growth. In a xenograft tumor model using female nude mice, we found that Ang1 enhanced angiogenesis and resulted in tumor growth in the case of PC3 tumors but suppressed tumor growth in the case of HT29 tumors. In PC3 tumors, the number of mural cells adhering to endothelial cells was less than that in HT29 tumors. Ang1 induced sprouting angiogenesis in PC3 tumors although there was little maturation of blood vessels. On the other hand, there was abundant mural cell adhesion to endothelial cells in HT29 tumors and Ang1 did not induce angiogenesis. These results suggest that Ang1 alters tumor growth in a manner that is dependent on the adhesion of mural cells and their localization in the tumor environment. (Cancer Sci 2008; 99: 2373–2379)     

Angiogenesis inhibition by vascular endothelial growth factor receptor-2 blockade reduces stromal matrix metalloproteinase expression, normalizes stromal tissue, and reverts epithelial tumor phenotype in surface heterotransplants

Inhibition of vascular endothelial growth factor (VEGF) signaling, a key regulator of tumor angiogenesis, through blockade of VEGF receptor (VEGFR)-2 by the monoclonal antibody DC101 inhibits angiogenesis, tumor growth, and invasion. In a surface xenotransplant assay on nude mice using a high-grade malignant squamous cell carcinoma cell line (A-5RT3), we show that DC101 causes vessel regression and normalization as well as stromal maturation resulting in a reversion to a noninvasive tumor phenotype. Vessel regression is followed by down-regulation of expression of both VEGFR-2 and VEGFR-1 on endothelial cells and increased association of alpha-smooth muscle actin-positive cells with small vessels indicating their normalization, which was further supported by a regular ultrastructure. The phenotypic regression of an invasive carcinoma to a well-demarcated dysplastic squamous epithelium is accentuated by the establishment of a clearly structured epithelial basement membrane and the accumulation of collagen bundles in the stabilized connective tissue. This normalization of the tumor-stroma border coincided with down-regulated expression of the stromal matrix metalloproteinases 9 and 13, which supposedly resulted in attenuated turnover of extracellular matrix components permitting their structural organization. Thus, in this mouse model of a human squamous cell carcinoma cell line, blockade of VEGF signaling resulted in the reversion of the epithelial tumor phenotype through stromal normalization, further substantiating the crucial role of stromal microenvironment in regulating the tumor phenotype.     

Inhibition of transforming growth factor-β signals suppresses tumor formation by regulation of tumor microenvironment networks

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-β (TGF-β) promotes tumor progression by inducing epithelial–mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-β receptor containing both TGF-β type I (TβRI) and type II (TβRII) receptors (TβRI-TβRII-Fc), which trapped all TGF-β isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TβRI-TβRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TβRI-TβRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TβRI-TβRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1β (IL-1β) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1β and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-β signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1β/EREG pathways and that TβRI-TβRII-Fc protein is a promising tool for targeting the TME networks.     

靶向肿瘤血管内皮细胞抑制人肝癌移植瘤生长的功能性抗体的研制

背景与目的:肿瘤的生长依赖于新生血管形成,阻断其血管形成可有效治疗肿瘤.本研究旨在研制抗人肝癌血管内皮细胞的功能性单克隆抗体,确定其抑制人肝癌移植瘤生长的作用.方法:用从新鲜人肝癌组织中分离、鉴定、培养的人肝癌血管内皮细胞免疫10只BALB/c小鼠.免疫小鼠脾细胞与SP2/0细胞融合后采用甲基纤维素选择培养.使用活细胞免疫荧光、细胞增殖、内皮成管实验及"人源化血管"动物模型体内抑瘤实验筛选和鉴定有治疗潜力的功能性抗体.结果:细胞融合产生1 442个单个集落的杂交瘤株,获得119株阳性克隆,其中53株是具有抑制人肝癌血管内皮细胞增殖、成管的功能性抗体,2株被证实能抑制人肝癌移植瘤在BALB/c裸鼠体内的生长,抑瘤率为66.7%～76.5%.结论:建立了高通量制备、筛选、鉴定内皮细胞功能性单抗技术,获得了2株具有靶向血管内皮细胞抑制人肝癌移植瘤生长的功能性单抗.     

Roles of cell adhesion molecules in tumor angiogenesis induced by cotransplantation of cancer and endothelial cells to nude rats

Roles of cell adhesion molecules mediating the interaction of cancer and endothelial cells in tumor angiogenesis were investigated using new in vitro and in vivo model systems with a cultured murine endothelial cell line (F-2) and human cultured epidermoid cancer cells (A431). The A431 cells exhibited typical in vitro cell adhesion to the endothelial F-2 cells. The initial step of adhesion was mediated by sialyl Lewis(x) (Le(x)) and sialyl Le(a), the carbohydrate determinants expressed on the cancer cells, and E-selectin expressed constitutively on F-2 cells. Prolonged culture led to the implantation of cancer cells into the monolayer of the F-2 cells, which was mediated mainly by alpha(3)beta(1)-integrin. F-2 cells cultured on Matrigel showed evident tube formation, and coculture of F-2 cells with A431 cells led to the formation of A431 cell nests constantly surrounded by tube-like networks consisting of F-2 cells. This in vitro morphogenesis was inhibited by the addition of anti-sialyl Le(x)/Le(a) or anti-beta(1)-integrin antibodies, which led to the formation of cancer cell aggregates that were independent from the F-2 cell networks. This in vitro morphological appearance was exactly reproduced in the in vivo tumors, which were formed when the mixture of A431 and F-2 cells at the ratio of 10:1 were cotransplanted s.c. into the back of nude rats. The tumors of A431 supplemented with F-2 cells were profoundly vascularized throughout by the tubular structures formed by F-2 cells, the lumen of which contained the host rat blood cells. The tumor mass thus formed was an average 5.8-fold as large as control A431 tumors that were grown without F-2 cells. The co-injection of anti-Le(x)/Le(a) or anti-beta(1)-integrin antibodies produced a marked reduction in the size of A431 tumors, which were not vascularized and accompanied an independent tiny remnant clump of F-2 cells. The size of these A431 tumors did not differ significantly from those of control A431 tumors raised without F-2 cells. These results indicate that the interaction of tumor cells and endothelial cells in orderly tumor angiomorphogenesis is highly dependent on the action of cell adhesion molecules mediating the adhesion of cancer cells to endothelial cells, inhibition of which remarkably retards tumor growth and angiogenesis.     

Identification of epigenetically silenced genes in tumor endothelial cells

Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated by DNMTs and HDAC are involved in regulation of endothelial cell gene expression during tumor angiogenesis. To understand the mechanisms behind the epigenetic regulation of tumor angiogenesis, we used microarray analysis to perform a comprehensive screen to identify genes down-regulated in tumor-conditioned versus quiescent endothelial cells, and reexpressed by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA). Among the 81 genes identified, 77% harbored a promoter CpG island. Validation of mRNA levels of a subset of genes confirmed significant down-regulation in tumor-conditioned endothelial cells and reactivation by treatment with a combination of DAC and TSA, as well as by both compounds separately. Silencing of these genes in tumor-conditioned endothelial cells correlated with promoter histone H3 deacetylation and loss of H3 lysine 4 methylation, but did not involve DNA methylation of promoter CpG islands. For six genes, down-regulation in microdissected human tumor endothelium was confirmed. Functional validation by RNA interference revealed that clusterin, fibrillin 1, and quiescin Q6 are negative regulators of endothelial cell growth and angiogenesis. In summary, our data identify novel angiogenesis-suppressing genes that become silenced in tumor-conditioned endothelial cells in association with promoter histone modifications and reactivated by DNMT and HDAC inhibitors through reversal of these epigenetic modifications, providing a mechanism for epigenetic regulation of tumor angiogenesis.     

人源化肿瘤血管移植瘤模型的建立及应用

背景与目的:目前在肿瘤内皮细胞基因功能和靶向血管治疗肿瘤的研究中仍缺乏适宜的动物模型。本研究拟建立一种新的小鼠人源化肿瘤血管移植瘤模型。方法:将人肝窦微血管内皮细胞系(humanliversinusoidendothelialcells,HLSEC)分别与人肝癌细胞系BEL7402、人结肠癌细胞系LS174T、人食管癌细胞系NEC按不同比例混合共接种NOD/SCID小鼠或BALB/c裸小鼠,以单独接种肿瘤细胞的小鼠作为对照组,观察小鼠人移植瘤生长的情况。采用绿色荧光蛋白基因(greenfluorescentprotein,GFP)转染HLSEC,结合荧光显微镜检方法观察HLSEC在共接种移植瘤中的存活及血管形成的情况。免疫组化法检测肿瘤内微血管密度(microvesseldensity,MVD)。用抗人肝癌内皮细胞单抗2B6处理人肝癌移植瘤共接种模型,观察2B6对肿瘤生长的影响。结果:HLSEC与BEL7402细胞共接种NOD/SCID小鼠时,共接种组肿瘤生长速度显著加快,平均瘤重可达肿瘤单独接种组的5.1倍。在GFP表达阳性的HLSEC与BEL7402共接种的移植瘤冰冻切片中可见HLSEC的存在,并已形成瘤内新生血管。免疫组化检测发现共接种组移植瘤中的总MVD较肿瘤细胞单独接种组增加85.7%,采用抗人vWF多抗检测结果显示共接种组人源微血管平均MVD可达10.28～29.28,约占总血管数量的41%～65%。进一步将HLSEC分别与NEC、BEL7402及LS174T细胞共接种于BALB/c裸小鼠时,共接种组的瘤重是单独接种组的3.3～6.0倍。2B6单抗能使共接种人肝癌移植瘤中人源肿瘤血管密度减少65.1%,抑制肿瘤瘤重达71.8%。结论:HLSEC与人肿瘤细胞共接种小鼠后,能在移植瘤中存活、增殖,形成大量人源化的肿瘤新生血管,并在促进肿瘤快速生长中起重要作用。该小鼠人源化肿瘤血管移植瘤模型可为肿瘤内皮细胞相关基因及靶向治疗剂的研究提供一个新的有价值的工具。     

An in vitro tumor model: analysis of angiogenic factor expression after chemotherapy

Tumor tissues include malignant cells and a stroma made up of mainly inflammatory cells, endothelial cells, and fibroblasts. To differentiate the effects of treatment on angiogenic cytokine secretion in tumor tissue, exponential and stationary phase human CaKi-1 renal cell carcinoma cells, human SW2 small cell lung carcinoma cells, human umbilical vein endothelial cells (HUVECs), murine NIH-3T3 fibroblasts, and murine RAW264.7 macrophages were exposed to gemcitabine, paclitaxel, carboplatin, and the protein kinase Cbeta inhibitor LY317615, and secretion (24 h) of tumor necrosis factor-alpha, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-beta was determined by a Luminex FlowMetrix assay. After 72 h of exposure, exponential RAW, 3T3, and SW2 cells were sensitive to gemcitabine; exponential and stationary SW2 and HUVECs were sensitive to paclitaxel; and exponential and stationary HUVECs were most sensitive to LY317615. None of the cells secreted detectable tumor necrosis factor-alpha. Generally, exponential cells secreted higher levels of cytokines than stationary cells (stationary cells secreted approximately 10 times less TGF-beta). Only malignant cells secreted VEGF (80-300 pg/10(6) cells). VEGF secretion by exponential SW2 cells decreased in an anticancer agent concentration-dependent manner. Every cell type secreted TGF-beta (40-700 pg/10(6) cells). Exponential 3T3, RAW, CaKi-1, and SW2 cells secreted the most TGF-beta, and levels did not decrease with treatment. Only CaKi-1, SW2, and HUVECs secreted bFGF (0.5-50 pg/10(6) cells). CaKi-1 cells increased secretion of bFGF with therapy. Although malignant cells alone secreted VEGF, stromal cells secreted TGF-beta and bFGF at levels comparable with or greater than malignant cells and thus may be important contributors to tumor growth and progression.     

Biological implications of macrophage infiltration in human tumor angiogenesis

Tumor angiogenesis is believed to be induced by increased production of angiogenic factors and decreased production of angiogenic inhibitors by cancer cells, vascular endothelial cells, and other stromal cell types. Most solid tumor cells are surrounded by stroma comprising interstitial connective tissue, blood vessels, fibroblastic cells, etc. Interaction between the stroma and malignant cells appears to have a critical role in the development of tumor neovasculature. We focused on macrophages, which demonstrate wide heterogeneity in biological function and have an essential role in tumor angiogenesis. Macrophages are terminally differentiated cells which produce a number of potent angiogenic cytokines and growth factors such as vascular endo-thelial growth factor, tumor necrosis factor-alpha, interleukin-8, and basic fibroblast growth factor. They also modulate events in the extracellular matrix through the secretion of extracellular matrix-degrading enzymes and -modulating enzymes. Thus macrophages could influence various stages of angiogenesis either positively or negatively. We found a close correlation between increased macrophage index, malignancy, and high vascular grade in malignant melanoma, and present a model for the possible involvement of activated macrophages in neovascularization in human malignant melanoma.     

Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo

The Eph family of receptor tyrosine kinases and their ligands, known as ephrins, play a crucial role in vascular development during embryogenesis. The function of these molecules in adult angiogenesis has not been well characterized. Here, we report that blocking Eph A class receptor activation inhibits angiogenesis in two independent tumor types, the RIP-Tag transgenic model of angiogenesis-dependent pancreatic islet cell carcinoma and the 4T1 model of metastatic mammary adenocarcinoma. Ephrin-A1 ligand was expressed in both tumor and endothelial cells, and EphA2 receptor was localized primarily in tumor-associated vascular endothelial cells. Soluble EphA2-Fc or EphA3-Fc receptors inhibited tumor angiogenesis in cutaneous window assays, and tumor growth in vivo. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor vascular density, tumor volume, and cell proliferation, but increased cell apoptosis. However, EphA2-Fc had no direct effect on tumor cell growth or apoptosis in culture, yet inhibited migration of endothelial cells in response to tumor cells, suggesting that the soluble receptor inhibited blood vessel recruitment by the tumor. These data provide the first functional evidence for Eph A class receptor regulation of pathogenic angiogenesis induced by tumors and support the function of A class Eph receptors in tumor progression.     

Effects of human bone marrow stroma on the growth of human tumor cells

As some tumors metastasize frequently to marrow we modified the clonogenic assay for human tumor cell growth by culturing tumor cells in the presence of human bone marrow stromal cells. In a bilayer soft agar assay, human tumor cells which had been passaged in nude mice were plated in the agar overlayer on an underlayer containing a suspension of trypsinized human bone marrow stromal cells. These marrow stromal cells stimulated the growth of tumor cells in a dose-dependent fashion, with a growth peak at a stromal cell density of 5-10 x 10(5)/ml. The maximal stimulation of tumour cell growth was 13-fold. We evaluated clonal growth of six separate tumors of five different histological types (small and large cell bronchogenic carcinoma; mammary carcinoma; malignant melanoma; pleural mesothelioma) and demonstrated that in 9 of 11 experiments tumor cell colonies formed in the absence of stromal cells, but colony growth was markedly stimulated by stromal cells in every case. Stromal stimulation persisted after irradiation of the stromal cells with 10 Gy. Growth of five fresh human tumor samples was similarly stimulated by the presence of human bone marrow stromal cells. Tumor cell colonies were characterized morphologically by Pappenheim stain and immunologically for surface antigens by peroxidase-antiperoxidase immunostaining utilizing monoclonal antibodies (carcinoembryonic antigen 26/3/13 and 26/5/1, EMA, HEA125, Sam 2 and Sam 10) which detected epithelial cell antigens. Colonies consisted of cytologically malignant cells which expressed epithelial cell antigens. Thus, the tumor cell origin of colonies from mammary carcinoma and bronchogenic small cell, large cell, and adenocarcinoma was proven. This tumor stem cell assay permits further analyses of human tumor cell biology and may be useful for testing drug sensitivity.