Disparate effects of endostatin on tumor vascular perfusion and hypoxia in two murine mammary carcinomas

PURPOSE: Recent results in the literature have demonstrated that the antiangiogenic agent endostatin can enhance antitumor effects when administered before or during radiotherapy. To better understand the underlying pathophysiologic basis for this radiosensitization, the current study investigated whether short-term endostatin administration is linked to alterations in tumor vascular perfusion and oxygen delivery. METHODS AND MATERIALS: Three daily doses of recombinant endostatin (20 mg/kg) were administered to two murine mammary carcinomas, the highly vascularized MCa-35 and the less vascularized MCa-4. Image analysis techniques were used to quantify (1) total and perfused vascular spacing, and (2) changes in tumor hypoxia as a function of distance from the nearest blood vessel. RESULTS: In MCa-35 tumors, endostatin had no effect on vessel spacing, tumor hypoxia, or tumor growth. In MCa-4 tumors, total and perfused vessel spacings were also unchanged, but tumor growth was inhibited, and tumor hypoxia significantly decreased. These tumors demonstrated an increased vascular functionality suggestive of an increase in the number of intermittently perfused vessels, without corresponding alterations in tumor oxygen consumption rate. CONCLUSIONS: Poorly vascularized, hypoxic mammary carcinomas were much more responsive to short-term endostatin treatment than well-vascularized, more homogeneously oxygenated tumors. Oxygen levels in the responsive tumors were transiently improved after treatment, which could have substantial implications with respect to the therapeutic effectiveness of combining antiangiogenic agents with conventional therapies.     

Increased levels of tissue endostatin in human malignant gliomas.

PURPOSE: Malignant gliomas are typically angiogenic and express greater amounts of angiogenic factors. We examined glioma tissues for their expression of an endogenous inhibitor of angiogenesis, endostatin, a COOH-terminal fragment of collagen XVIII. EXPERIMENTAL DESIGN: We examined frozen tissues from 51 patients with astrocytic tumors (grade 2, 13; grade 3, 9; and grade 4, 29). Frozen tissues were subjected to immunoblot analysis and immunohistochemistry for endostatin. Tumor vascular density was determined by calculating the percentage of tumor capillary vessel areas/tissue section area. Tissue concentrations of vascular endothelial growth factor and basic fibroblast growth factor were examined by enzyme immunoassay. RESULTS: The levels of endostatin protein estimated by immunoblotting were significantly higher in grade 4 than lower-grade glioma tissues. The immunoreactive bands for endostatin were identified as the fragment derived from noncollagenous domain 1 of collagen XVIII, a peptide 15 residues longer than endostatin toward the NH(2)-terminal end, by NH(2)-terminal amino acid sequencing. In addition to an intense immunoreactivity for endostatin in tumor blood vessels, sections from malignant gliomas showed widely distributed immunoreactivity around tumor cells near the hyperplastic microvessels. The tumor vascular density and the levels of vascular endothelial growth factor in grade 4 glioma tissues were significantly higher than grade 2 and grade 3 gliomas, whereas the levels of basic fibroblast growth factor were the same. CONCLUSIONS: The results indicate a positive correlation between the levels of tissue endostatin and malignancy grades in gliomas. The endostatin may be released near the tumor blood vessels with hyperplasia to counteract angiogenic stimuli in malignant gliomas.     

重组内皮抑素对肿瘤血管结构和乏氧改善作用的实验观察

目的 观察重组内皮抑素作用下小鼠Lewis肺癌移植瘤在血管退化前是否存在血管正常化时间窗,在此时间窗内肿瘤乏氧是否改善.方法 激光共聚焦显微镜动态观察重组内皮抑素作用下肿瘤血管的形态学变化,免疫组化染色检测不同时间段肿瘤乏氧细胞比例,观测肿瘤生长情况并绘制肿瘤生长曲线.结果 重组内皮抑素处理后肿瘤血管密度逐渐下降,连续治疗9 d较对照组下降最为明显.重组内皮抑素处理后周细胞覆盖内皮细胞比例逐渐增加,第3天开始明显增加,第5天增加最明显,第7天后明显下降.对照组基底膜与内皮细胞连接松散,厚度增加,而重组内皮抑素处理后,基底膜与内皮细胞连接紧密,厚度下降.在重组内皮抑素处理后第5天肿瘤乏氧细胞比例下降最明显,连续5 d用重组内皮抑素,肿瘤生长未见加快.结论 重组内皮抑素能使肿瘤血管正常化,其时间窗为治疗后3～7 d;此时肿瘤氧供明显改善,为临床联合应用放疗和重组内皮抑素提供了实验依据.

Abstract：

Objective To investigate whether recombinant human endostatin can create a time window of vascular normalization prior to vascular pruning to alleviate hypoxia in Lewis lung carcinoma in mice. Methods Kinetic changes in morphology of tumor vasculature in response to recombinant human endostatin were detected under a confocal microscope with immunofluorescent staining in Lewis lung carcinomas in mice. The hypoxic cell fraction of different time was assessed with immunohistochemical staining . Effects on tumor growth were monitored as indicated in the growth curve of tumors . Results Compared with the control group vascularity of the tumors was reduced over time by recombinant human endostatin treatment and significantly regressed for 9 days. During the treatment, pericyte coverage increased at day 3, increased markedly at day 5, and fell again at day 7. The vascular basement membrane was thin and closely associated with endothelial cells after recombinant human endostatin treatment, but appeared thickened, loosely associated with endothelial cells in control tumors. The decrease in hypoxic cell fraction at day 5 after treatment was also found. Tumor growth was not accelerated 5 days after recombinant human endostatin treatment. Conclusions Recombinant human endostatin can normalize tumor vasculature within day 3 to 7, leading to improved tumor oxygenation. The results provide important experimental basis for combining recombinant human endostatin with radiation therapy in human tumors.     

Intratumoral administration of endostatin plasmid inhibits vascular growth and perfusion in MCa-4 murine mammary carcinomas

Endostatin, a fragment of the COOH-terminal domain of mouse collagen XVIII is a recently demonstrated endogenous inhibitor of tumor angiogenesis and endothelial cell growth. Antiangiogenic therapy with endostatin in animals requires multiple and prolonged administration of the protein. Gene therapy could provide an alternative approach to continuous local delivery of this antiangiogenic factor in vivo. Established MCa-4 murine mammary carcinomas, grown in immunodeficient mice, were treated with intratumoral injection of endostatin plasmid at 7-day intervals. At the time of sacrifice, 14 days after the first injection, endostatin-treated tumor weights were 51% of controls (P < 0.01). Tumor growth inhibition was accompanied by a marked reduction in total vascular density. Specifically, computerized image analysis showed a 18-21% increase in the median distances between tumor cells and both the nearest anatomical (CD31-stained) vessel [48.1 +/- 3.8 versus 38.3 +/- 1.6 microm (P < 0.05)] and the nearest tumor-specific (CD105-stained) vessel [48.5 +/- 1.5 versus 39.8 +/- 1.5 microm (P < 0.01)]. An increased apoptotic index of tumor cells in endostatin-treated tumors [3.2 +/- 0.5% versus 1.9 +/- 0.3% (P < 0.05)] was observed in conjunction with a significant decrease in tumor perfused vessels (DiOC7 staining), and an increase in tumor cell hypoxia (EF5 staining). Hypoxia resulting from endostatin therapy most likely caused a compensatory increase of in situ vascular endothelial growth factor (VEGF) and VEGF receptor mRNA expression. Increased immunoreactivity of endostatin staining in endostatin-treated tumors was also associated with an increased thrombospondin-1 staining [1.12 +/- 0.16 versus 2.44 +/- 0.35]. Our data suggest that intratumoral delivery of the endostatin gene efficiently suppresses murine mammary carcinoma growth and support the potential utility of the endostatin gene for cancer therapy.     

Endostatin overexpression inhibits lymphangiogenesis and lymph node metastasis in mice

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. We studied the development of carcinogen-induced skin tumors in transgenic J4 mice overexpressing endostatin in their keratinocytes. Unexpectedly, we did not observe any differences in tumor incidence and multiplicity between these and control mice, nor in the rate of conversion of benign papillomas to malignant squamous cell carcinomas (SCC). We did find, however, that endostatin regulates the terminal differentiation of keratinocytes because the SCCs in the J4 mice were less aggressive and more often well differentiated than those in the control mice. We observed an inhibition of tumor angiogenesis by endostatin at an early stage in skin tumor development, but more strikingly, there was a significant reduction in lymphatic vessels in the papillomas and SCCs in association with elevated endostatin levels and also a significant inhibition of lymph node metastasis in the J4 mice. We showed that tumor-infiltrating mast cells strongly expressed vascular endothelial growth factor-C (VEGF-C), and that the accumulation of these cells was markedly decreased in the tumors of the J4 mice. Moreover, endostatin inhibited the adhesion and migration of murine MC/9 mast cells on fibronectin in vitro. Our data suggest that endostatin can inhibit tumor lymphangiogenesis by decreasing the VEGF-C levels in the tumors, apparently via inhibition of mast cell migration and adhesion, and support the view that the biological effects of endostatin are not restricted to endothelial cells because endostatin also regulates tumor-associated inflammation and differentiation, and the phenotype of epithelial tumors.     

Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin.

PURPOSE: Angiogenesis is a target for the treatment of cancer and other diseases, and its complex biology suggests that establishing the appropriate dose and schedule for antiangiogenic treatment will require extensive study. We present the initial results of a dose-finding clinical trial of recombinant human endostatin (rh-Endo) that examined potential surrogates for response to antiangiogenic therapy. PATIENTS AND METHODS: Twenty-five patients were treated with escalating doses of rh-Endo. Positron emission tomography (PET) was used to assess tumor blood flow (with [15O]H2O) and metabolism (with [18F]fluorodeoxyglucose) before the start of therapy and then every 4 weeks. To directly assess the effects of rh-Endo on endothelial cells within the tumors, biopsy specimens of tumor tissue were obtained before therapy and again at 8 weeks and evaluated for endothelial cell and tumor cell apoptosis. RESULTS: Tumor blood flow and metabolism as measured by PET scans generally decreased with increasing doses of rh-Endo; however, the effects were complex and in some analyses nonlinear. Tumor biopsy analysis revealed a significant increase in tumor cell apoptosis (P =.027) and endothelial cell apoptosis (P =.027) after 8 weeks of therapy. However, there was no statistically significant relationship between rh-Endo dose and induction of tumor cell or endothelial cell apoptosis. CONCLUSION: These initial data suggest that rh-Endo has measurable effects on tumor blood flow and metabolism and induces endothelial and tumor cell apoptosis even in the absence of demonstrable anticancer effects. Further study and validation of these biomarkers in the context of antiangiogenic therapy will be required.     

Significance of blood vessel leakiness in cancer

Despite major advances in the field of tumor angiogenesis, relatively little attention has been paid to the permeability of blood vessels in tumors. The leakiness of tumor vessels is well documented in experimental tumor models and in human cancer, but the mechanism is poorly understood, as are the implications to the rate of cancer growth, predisposition to metastasis, and delivery of macromolecular therapeutics to tumor cells. Sixteen experts in the fields of cancer biology and vascular biology gathered at the William Guy Forbeck "Focus on the Future" Conference to discuss this topic. The meeting was the first of its kind focused on the significance of blood vessel leakiness in tumors. The participants discussed the cellular basis of tumor vessel leakiness, endothelial barrier function of blood vessels, monitoring tumor vessel leakiness, mediators of endothelial leakiness, consequences of tumor vessel leakiness, genomic analysis of vascular targets, targeting drugs to tumor vessels, and therapeutic manipulation of tumor vessels. The group concluded that a more complete understanding of the basic biology of tumor vessels will be necessary to fully appreciate the consequences of vessel leakiness in cancer. New research tools such as intravital measurements of tumor blood flow and vessel leakiness, in vivo phage display, magnetic resonance imaging, and use of selective angiogenesis inhibitors will contribute to this understanding.     

Endostatin expression by MDA-MB-435 breast cancer cells effectively inhibits tumor growth

Tumors must induce the formation of new blood vessels in order to grow and metastasize. Endostatin, a cleaved product of collagen XVIII, inhibits endothelial cell proliferation and suppresses tumor growth and metastases. Several recent reports have questioned the efficacy of endostatin as a tumor suppressor in experimental animals. Our objective was to determine whether endostatin expression in breast cancer cells inhibits neovascularization and tumor growth in nude mice. MDA-MB-435 cells were transfected with an endostatin expression vector while control cells were transfected with an empty vector. Endostatin expression and secretion were confirmed by RT-PCR and a dot blot assay. No differences were observed in the growth rates of the endostatin-expressing and control clones in vitro. When injected into male and female nude mice, tumors from the control clones increased in size 10-15 fold over 8-10 weeks. In contrast, the endostatin clones formed small tumors which did not increase in size after the first 3 weeks. The endostatinderived tumors had a significantly higher apoptotic index (5.6%) compared to controls (2.0%) and showed a marked reduction in vascularization. In conclusion, expression of endostatin in MDA-MB-435 breast cancer cells effectively suppressed breast tumor growth by inhibiting angiogenesis and increasing apoptosis.     

Synergy between angiostatin and endostatin: inhibition of ovarian cancer growth

Ovarian cancer is the leading cause of fatality among gynecological malignancies. Ovarian cancer growth is angiogenesis-dependent, and an increased production of angiogenic growth factors such as vascular endothelial growth factor is prognostically significant even during early stages of the disease. Therefore, we investigated whether antiangiogenic treatment can be used to inhibit the growth of ovarian cancer in an experimental model system. Mouse angiostatin (kringle 1-4) and endostatin were expressed in yeast. Purified angiostatin and endostatin were then used to treat established ovarian cancers in athymic mice. These studies showed that both angiostatin and endostatin inhibited tumor growth. However, angiostatin treatment was more effective in inhibiting ovarian cancer growth when compared with endostatin in parallel experiments. Residual tumors obtained from angiostatin- and endostatin-treated animals showed decreased number of blood vessels and, as a consequence, increased apoptosis of tumor cells. Subsequently, the efficacy of a combined treatment with angiostatin and endostatin was investigated. In the presence of both angiostatic proteins, endothelial cell proliferation was synergistically inhibited. Similarly, a combination regimen using equal amounts of angiostatin and endostatin showed more than additive effect in tumor growth inhibition when compared with treatment with individual angiostatic protein. These studies demonstrate synergism between two angiostatic molecules and that antiangiogenic therapy can be used to inhibit ovarian cancer growth.     

Effect of VEGF receptor-2 antibody on vascular function and oxygenation in spontaneous and transplanted tumors.

BACKGROUND AND PURPOSE: The primary objectives of this study were to address two major questions. (1) Does VEGF receptor-2 antibody (DC101) produce detrimental effects on tumor vascular function and oxygenation that could compromise adjuvant therapies? (2) Is pathophysiological response to such antiangiogenic strategies different in transplanted versus primary spontaneous tumors? MATERIALS AND METHODS: The effects of early and late initiation DC101 treatment were evaluated using spontaneous murine mammary carcinomas and two markedly different transplanted mammary tumors, MCa-35 and MCa-4. Mice were administered DC101 or saline, tumors were frozen, and immunohistochemical staining was quantified using image analysis of multiply-stained frozen sections. Total blood vessels were identified using antibodies to CD31 or panendothelial antigen, perfused vessels via i.v. injection of fluorescent DiOC7, and tumor hypoxia by hypoxia marker (EF5) uptake. RESULTS: Tumor growth was significantly inhibited following DC101 administration in all tumor models. In general, early initiation DC101 treatment reduced perfused vessel counts and increased tumor hypoxia, while late initiation treatment had no significant impact on either. Results indicate that DC101 slows tumor growth through a decrease in vascular function, leading to increased tumor cell apoptosis and necrosis at sites distant from perfused blood vessels, and suggest that DC101 accelerates the rate at which tumor cells outgrow their functional vascular supply. CONCLUSIONS: Although highly variable among individual spontaneous tumors, the overall effects of DC101 on tumor hypoxia were quite similar between spontaneous and transplanted tumors. Since reductions in tumor oxygenation due to antiangiogenic treatment were transient, initial pathophysiological deficiencies that could compromise conventional therapies over the short-term may be of less relevance when administered over more extended treatment schedules.     

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.     

Suramin Treatment of Human Glioma Xenografts; Effects on Tumor Vasculature and Oxygenation Status

In this study the effect of suramin on tumor growth, vascularity and oxygenation of a human glioma xenografted in the nude mouse was examined. Vascular parameters and oxygenation status of the xenografts were determined immunohistochemically in frozen sections of the tumors, using the hypoxia marker pimonidazole-hydrochloride to detect hypoxic areas. Tumor vessels in these sections were stained by an endothelial cell marker and perfusion of vessels was visualized by administration of the perfusion marker Hoechst 333342 before harvesting the tumors. The vascular parameters were quantified with an image analysis system. The results show that tumor growth was reduced considerably after suramin treatment. This growth suppression was accompanied by marked changes in vascular architecture. Although the total vascular area and perfused fraction of tumor vessels remained unchanged after suramin treatment, vascular density increased, indicating that more but smaller vessel structures had developed during therapy. These vessel structures were also more homogeneously spread over the tumor area. Control tumors showed extensive areas of hypoxia while in treated tumors hypoxic areas had mostly disappeared. This effect was probably due to the higher density of homogeneously distributed perfused vessel structures in the treated tumors, contributing to an increased oxygenation of the tumor. These observations suggest that suramin therapy can result in marked changes not only in tumor vascularity but also in tumor oxygenation status which may have important consequences for sensitivity of these tumors to other therapies such as radiation treatment.     

Addition of an aminopeptidase N-binding sequence to human endostatin improves inhibition of ovarian carcinoma growth

BACKGROUND: Blood vessels in tumors express higher level of aminopeptidase N (APN) compared with normal tissues. It has been reported that peptides that contain asparagine-glycine-arginine (NGR) sequence home to APN in tumor vasculature. Increased expression of APN in tumor vascular endothelium, therefore, offers an opportunity to target NGR peptide-linked therapeutic reagents to tumors. METHODS: To determine whether an additional NGR sequence could improve endothelial homing and biologic activity, human endostatin was modified genetically to introduce an NGR motif (NGR-endostatin) and was expressed in yeast. In vitro biologic activity of NGR-endostatin was compared with the native protein in endothelial cell proliferation and migration. NGR-modified endostatin was used in tumor localization studies. Finally, the effects of endostatin and NGR-endostatin on tumor growth were determined in two model systems. RESULTS: Human endostatin has an internal NGR sequence, which is not accessible to bind APN. However, the addition of an NGR-sequence at the amino terminus resulted in strong binding and inhibition of endothelial cell APN. NGR-endostatin showed increased binding to endothelial cells compared with the native protein. Increased binding of endostatin also coincided with improved antiangiogenic properties of endostatin. NGR modification improved tumor localization and, as a consequence, effectively inhibited ovarian carcinoma growth in athymic nude mice. CONCLUSIONS: These studies demonstrated that human endostatin can be modified genetically to improve its ability to inhibit tumor growth.     

Endostatin improves radioresponse and blocks tumor revascularization after radiation therapy for A431 xenografts in mice

PURPOSE: Clinical trials of antiangiogenic agents used alone for advanced malignancy have been disappointing but preclinical studies suggest that the addition of radiation therapy could improve antitumor efficacy. To test the hypothesis that antiangiogenic therapy combined with radiation therapy can overcome the limitations of antiangiogenic monotherapy, we studied the effects of endostatin combined with radiation on the growth and vascularization of A431 human epidermoid carcinomas growing intramuscularly in the legs of mice. METHODS AND MATERIALS: Mice with established A431 human epidermoid leg tumors were treated with radiation, endostatin, both radiation and endostatin, or vehicle control. The experiment was repeated and mice from each group were killed at 2, 7, and 10 days after irradiation so that tumor tissue could be obtained to further analyze the kinetics of the antitumor, antivascular, and antiangiogenic response to therapy. RESULTS: Endostatin enhanced the antitumor effects of radiation, and prolonged disease-free survival was observed in the combined treatment group. Endothelial cell proliferation was increased in tumors after irradiation but was blocked by the concurrent administration of endostatin, and the combination of endostatin with radiation enhanced endothelial cell apoptosis within 48 h after irradiation. Expression of vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 were increased in tumors after irradiation, and this increase was blocked by concurrent administration of endostatin. CONCLUSION: These data indicate that endostatin can block tumor revascularization after radiation therapy and thereby augment radioresponse.     

Inhibition of the mammary carcinoma angiogenic switch in C3(1)/SV40 transgenic mice by a mutated form of human endostatin

Cancer therapies based on the inhibition of angiogenesis by endostatin have recently been developed. We demonstrate that a mutated form of human endostatin (P125A) can inhibit the angiogenic switch in the C3(1)/Tag mammary cancer model. P125A has a stronger growth-inhibitory effect on endothelial cell proliferation than wild-type endostatin. We characterize the angiogenic switch, which occurs during the transition from preinvasive lesions to invasive carcinoma in this model, and which is accompanied by a significant increase in total protein levels of vascular endothelial growth factor (VEGF) and an invasion of blood vessels. Expression of the VEGF(188) mRNA isoform, however, is suppressed in invasive carcinomas. The VEGF receptors fetal liver kinase-1 (Flk-1) and Fms-like tyrosine kinase-1 (Flt-1) become highly expressed in epithelial tumor and endothelial cells in the mammary carcinomas, suggesting a potential autocrine effect for VEGF on tumor cell growth. Angiopoietin-2 mRNA levels are also increased during tumor progression. CD-31 (platelet-endothelial cell adhesion molecule [PECAM]) staining revealed that blood vessels developed in tumors larger than 1 mm The administration of P125A human endostatin in C3(1)/Tag females resulted in a significant delay in tumor onset, decreased tumor multiplicity and tumor burden and prolonged survival of the animals. Endostatin treatment did not reduce the number of preinvasive lesions, proliferation rates or apoptotic index, compared with controls. However, mRNA levels of a variety of proangiogenic factors (VEGF, VEGF receptors Flk-1 and Flt-1, angiopoietin-2, Tie-1, cadherin-5 and PECAM) were significantly decreased in the endostatin-treated group compared with controls. These results demonstrate that P125A endostatin inhibits the angiogenic switch during mammary gland adenocarcinoma tumor progression in the C3(1)/Tag transgenic model.     

腹膜后肿瘤术中重大血管的切除及重建

目的提高切除侵及腹部重大血管的腹膜后肿瘤的手术技能。方法总结近年来本院收治的腹膜后肿瘤中２５例侵及重大血管的手术切除及血管重建方面的经验。结果经过术前准备和术前评估,精心设计手术方案,对２５例患者成功实施了腹膜后肿瘤（包括受累血管）完整切除和相应的重大血管重建,无手术死亡病例。结论侵及重大血管的腹膜后肿瘤已不是手术禁忌证,应尽可能将受累血管及肿瘤完整切除,再行必要的血管重建,从而达到减少术后局部复发以及延长存活时间的目的。     

Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin

Endothelial cells in growing tumors express activated Akt, which when modeled by transgenic endothelial expression of myrAkt1 was sufficient to recapitulate the abnormal structural and functional features of tumor blood vessels in nontumor tissues. Sustained endothelial Akt activation caused increased blood vessel size and generalized edema from chronic vascular permeability, while acute permeability in response to VEGF-A was unaffected. These changes were reversible, demonstrating an ongoing requirement for Akt signaling for the maintenance of these phenotypes. Furthermore, rapamycin inhibited endothelial Akt signaling, vascular changes from myrAkt1, tumor growth, and tumor vascular permeability. Akt signaling in the tumor vascular stroma was sensitive to rapamycin, suggesting that rapamycin may affect tumor growth in part by acting as a vascular Akt inhibitor.     

Enhancement of radiation effects by pXLG-mEndo in a lung carcinoma model

PURPOSE: Endostatin is a potent antiangiogenesis protein with little or no toxicity that has potential to enhance radiotherapy. The major goal of this study was to evaluate the combination of radiation and endostatin gene therapy in a preclinical lung cancer model. METHODS: Plasmid pXLG-mEndo, constructed in our laboratory, includes the mouse endostatin gene cloned into the pWS4 vector. The kinetics of endostatin expression and efficacy of the pXLG-mEndo and radiation ((60)Co gamma-rays) combination was evaluated in the C57BL/6 mouse-Lewis lung carcinoma (LLC) model. The LLC cells were implanted s.c. and pXLG-mEndo was injected intratumorally 12-14 days later without any transfection agent; a dose of 10 Gy radiation was applied approximately 16 h thereafter. Some groups received each modality twice. Endostatin, vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1) were quantified in plasma and tumors, and tumor vasculature was examined. RESULTS: Endostatin expression within LLC tumors peaked on Day 7 after pXLG-mEndo injection. Addition of radiation to pXLG-mEndo significantly enhanced the level of tumor endostatin compared with plasmid alone (p < 0.05). Tumor growth was significantly delayed in mice receiving pXLG-mEndo plus radiation compared with no treatment (p < 0.005), radiation (p < 0.05), and control plasmid (p < 0.05). The number of LLC tumor vessels was reduced after combined treatment (p < 0.05), and significant treatment-related changes were observed in both VEGF and TGF-beta1. CONCLUSIONS: The data demonstrate that delivery of endostatin by pXLG-mEndo as an adjuvant to radiation can significantly enhance the antitumor efficacy of radiotherapy in the LLC mouse tumor model and support further investigation of this unique combination therapy.     

Tumor neoangiogenesis detection by confocal laser endomicroscopy and anti-CD105 antibody: Pilot study

AIM: To evaluate neoangiogenesis in patients with colon cancer by two fluorescently labeled antibodies on fresh biopsy samples imaged with confocal laser endomicroscopy (CLE).METHODS: CLE is an imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. An important question in validating tumor angiogenesis is what proportion of the tumor vascular network is represented by pre-existing parent tissue vessels and newly formed vessels. CD105 (endoglin) represents a proliferation-associated endothelial cell adhesion molecule. In contrast to pan-endothelial markers, such as CD31, CD105 is preferentially expressed in activated endothelial cells that participate in neovascularization. Thus, we evaluated CD105 and CD31 expression from samples of ten patients with primary rectal adenocarcinoma, using a dedicated endomicroscopy system. A imaging software was used to obtain the Z projection of the confocal serial images from each biopsy sample previously combined into stacks. Vascular density and vessel diameters were measured within two 50 μm x 475 μm rectangular regions of interest centered in the middle of each image in the horizontal and vertical direction. The results were averaged over all the patients and were expressed as the mean ± SE.RESULTS: The use of an anti-CD105 antibody was found to be suitable for the detection of blood vessels in colon cancer. Whereas anti-CD31 antibodies stained blood vessels in both normal and pathologic colon equally, CD105 expression was observed primarily in malignant lesions, with little or no expression in the vessels of the normal mucosa (244.21 ± 130.7 vessels/mm³ in only four patients). The average diameter of anti-CD105 stained vessels was 10.97 ± 0.6 μm in tumor tissue, and the vessel density was 2787.40 ± 134.8 vessels/mm³. When using the anti-CD31 antibody, the average diameter of vessels in the normal colon tissue was 7.67 ± 0.5 μm and the vessel density was 3191.60 ± 387.8 vessels/mm³, while in the tumors we obtained an average diameter of 10.88 ± 0.8 μm and a vessel density of 4707.30 ± 448.85 vessels/mm³. Thus, there were more vessels stained with CD31 than CD105 (P < 0.05). The average vessel diameter was similar for both CD31 and CD105 staining. A qualitative comparison between CLE vs immunohistochemistry lead to similar results.CONCLUSION: Specific imaging and quantification of tumor microvessels are feasible in human rectal cancer using CLE examination and CD105 immunostaining of fresh tissue samples.     

Combretastatin family member OXI4503 induces tumor vascular collapse through the induction of endothelial apoptosis

The mechanism of tumor cell killing by OXI4503 was investigated by studying vascular functional and morphological changes post drug administration. SCID mice bearing MHEC5-T hemangioendothelioma were given a single dose of OXI4503 at 100 mg/kg. Tumor blood flow, measured by microsphere fluorescence, was reduced by 50% at 1 hr, and reached a maximum level 6-24 hr post drug treatment. Tumor vascular permeability, measured by Evan's blue and hemoglobin, increased significantly from 3 hr and peaked at 18 hr. The elevated tumor vessel permeability was accompanied by an increase in vascular endothelial growth factor (VEGF) from 1 hr post drug treatment. Immunohistochemical staining for CD31 and laminin showed that tumor blood vessels were affected as early as 3 hr but more prominent from 6 hr. From 12 hr, the vessel structure was completely destroyed. Histopathological and double immunohistochemical staining showed morphological change and induction of apoptosis in endothelial cells at 1-3 hr, followed by tumor cell necrosis from 6-72 hr. There were no statistically significant changes of Evan's blue and hemoglobin contents in liver tissue over the time course. These results suggest that OXI4503 selectively targets tumor blood vessels, and induces blood flow shutdown while it enhances tumor blood vessel permeability. The early induction of endothelial cell apoptosis leads to functional changes of tumor blood vessels and finally to the collapse of tumor vasculature, resulting in massive tumor cell necrosis. The time course of the tumor vascular response observed with OXI4503 treatment supports this drug for development as a stand alone therapy, and also lends support for the use of the drug in combination with other cancer therapies.