Suppression of tumor growth by oncolytic adenovirus-mediated delivery of an antiangiogenic gene, soluble Flt-1.

Armed oncolytic adenoviruses represent an appealing tumor treatment approach, as they can attack tumors at multiple levels. In this study, considering that angiogenesis plays a central role in tumor growth, we inserted an antiangiogenic gene, sflt-1(1-3) (the first three extracellular domains of FLT1, the hVEGF receptor-1), into an E1B-55-kDa-deleted oncolytic adenovirus (ZD55) to construct ZD55-sflt-1. Although soluble (s) Flt-1 did not affect tumor cell growth, ZD55-sflt-1 could specifically induce a cytopathic effect in tumor cells, like ONYX-015. The secretion of sFlt-1 from ZD55-sflt-1 was much higher than that from replication-deficient Ad-sflt-1 upon infection of SW620 human colon tumor cells, leading to a stronger inhibitory effect on VEGF-induced proliferation and tube formation ability of HUVECs. Moreover, marked reduction of tumor growth and long-term survival rates were observed in ZD55-sflt-1-treated nude mice with subcutaneous SW620 tumor. Its efficacy correlated with a decrease in microvessel density and an increase in apoptotic tumor cells. In addition, ZD55-sflt-1 showed a synergic effect with the chemotherapeutic agent 5-FU. These results indicate that ZD55-sflt-1, combining the advantages of oncolytic adenovirus and antiangiogenic gene therapy, is a powerful agent for human tumor treatment.     

Interferon-alpha gene therapy by lentiviral vectors contrasts ovarian cancer growth through angiogenesis inhibition

Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.     

Periocular gene transfer of sFlt-1 suppresses ocular neovascularization and vascular endothelial growth factor-induced breakdown of the blood-retinal barrier

Vascular endothelial growth factor (VEGF) is a critical stimulus for both retinal and choroidal neovascularization, and for diabetic macular edema. We used mouse models for these diseases to explore the potential of gene transfer of soluble VEGF receptor-1 (sFlt-1) as a treatment. Intravitreous or periocular injection of an adenoviral vector encoding sFlt-1 (AdsFlt-1.10) markedly suppressed choroidal neovascularization at rupture sites in Bruch's membrane. Periocular injection of AdsFlt-1.10 also caused significant reduction in VEGF-induced breakdown of the blood-retinal barrier, but failed to significantly inhibit ischemia-induced retinal neovascularization. Periocular delivery of an adenoviral vector encoding pigment epithelium-derived factor (PEDF), another secreted protein, resulted in high levels of PEDF in the retinal pigmented epithelium and choroid, but not in the retina. This may explain why periocular injection of AdsFlt-1.10 inhibited choroidal, but not retinal neovascularization. Periocular delivery offers potential advantages over other routes of delivery and the demonstration that sFlt-1 enters the eye from the periocular space in sufficient levels to achieve efficacy in treating choroidal neovascularization and retinal vascular permeability is a novel finding that has important clinical implications. These data suggest that periocular gene transfer of sFlt-1 should be considered for treatment of choroidal neovascularization and diabetic macular edema.     

Suppression of lymphangiogenesis by soluble vascular endothelial growth factor receptor-2 in a mouse lung cancer model

The vascular endothelial growth factor (VEGF) family has a key role in the formation of blood vessels and lymphatics. Among the members of this family, VEGF-C is one of the most important factors involved in lymphangiogenesis via binding with two receptors (vascular endothelial growth factor receptor-2 and -3: VEGFR-2 and VEGFR-3). Soluble VEGFR-2 (sVEGFR-2) has a role in maintaining the alymphatic state of the cornea associated with binding to VEGF-C, and selectively inhibits lymphangiogenesis but not angiogenesis. In this study, we introduced sVEGFR-2 into lung cancer cells and evaluated the influence on tumor progression and on genes regulating lymphatic formation and metastasis in vivo. A retroviral vector was used to introduce the sVEGFR-2 gene into Lewis lung carcinoma cells (LLC), which were designated as LLC-sVEGFR-2 cells. Proteins secreted into the culture supernatant by these cells were detected by western blotting using specific antibodies. To examine lymphangiogenesis by primary lung cancer in vivo, LLC-sVEGFR-2 cells were subcutaneously injected into C57BL/6 mice. At 14 days after injection, immunohistochemistry was performed using an antibody directed against lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a marker of lymphatics. Expression of mRNA for VEGFR-2, VEGFR-3 and matrix metalloproteinases (MMPs) was also determined by real-time PCR. Furthermore, LLC-sVEGFR-2 cells were directly inoculated into the left lung in C57BL/6 mice and the number of micro-metastases in pulmonary lymph nodes was determined. Introduction of sVEGFR-2 into LLC cells resulted in secretion of sVEGFR-2 protein into the culture supernatant. There were fewer LYVE-1 positive lymphatics after inoculation of LLC-sVEGFR-2 into mice compared with the control group. In addition, VEGFR-2, VEGFR-3, and MMPs gene expression was suppressed in the primary tumors of the LLC-sVEGFR-2 group compared with the control group. Furthermore, there were fewer micro-metastases in the pulmonary lymph nodes of the LLC-sVEGFR-2 group compared with the control group after cells were directly inoculated into the lung. These findings indicate that introduction of sVEGFR-2 suppressed lymphangiogenesis in primary lung cancer and also suppressed lymphogenic metastasis by inhibiting VEGF-C, followed by down-regulation of VEGFR-2, VEGFR-3 and MMPs. Accordingly, sVEGFR-2 might be a promising target for treatment of cancer by regulating lymphangiogenesis and lymphogenic metastasis.     

FAK regulates platelet extravasation and tumor growth after antiangiogenic therapy withdrawal

Recent studies in patients with ovarian cancer suggest that tumor growth may be accelerated following cessation of antiangiogenesis therapy; however, the underlying mechanisms are not well understood. In this study, we aimed to compare the effects of therapy withdrawal to those of continuous treatment with various antiangiogenic agents. Cessation of therapy with pazopanib, bevacizumab, and the human and murine anti-VEGF antibody B20 was associated with substantial tumor growth in mouse models of ovarian cancer. Increased tumor growth was accompanied by tumor hypoxia, increased tumor angiogenesis, and vascular leakage. Moreover, we found hypoxia-induced ADP production and platelet infiltration into tumors after withdrawal of antiangiogenic therapy, and lowering platelet counts markedly inhibited tumor rebound after withdrawal of antiangiogenic therapy. Focal adhesion kinase (FAK) in platelets regulated their migration into the tumor microenvironment, and FAK-deficient platelets completely prevented the rebound tumor growth. Additionally, combined therapy with a FAK inhibitor and the antiangiogenic agents pazopanib and bevacizumab reduced tumor growth and inhibited negative effects following withdrawal of antiangiogenic therapy. In summary, these results suggest that FAK may be a unique target in situations in which antiangiogenic agents are withdrawn, and dual targeting of FAK and VEGF could have therapeutic implications for ovarian cancer management.     

Soluble Vascular Endothelial Growth Factor Decoy Receptor FP3 Exerts Potent Antiangiogenic Effects

The binding of vascular endothelial growth factor (VEGF) to its receptors stimulates tumor growth; therefore, modulation of VEGF would be a viable approach for antiangiogenic therapy. We constructed a series of soluble decoy receptors containing different VEGF receptor 1 (FLT1) and VEGF receptor 2 (KDR) extracellular domains fused with the Fc region of human immunoglobulin (Ig) and evaluated their antiangiogenic effects and antitumor effects. Results of in vitro binding and cell proliferation assays revealed that decoy receptor FP3 had the highest affinity to VEGF-A and -B. Compared with bevacizumab, FP3 more effectively inhibited human umbilical vein endothelial cell (HUVEC) migration and vessel sprouting from rat aortic rings. FP3 significantly reduced phosphorylation of AKT and ERK1/2, critical proteins in the VEGF-mediated survival pathway in endothelial cells. Moreover, FP3 inhibited tumor growth in human hepatocellular carcinoma (HepG2), breast cancer (MCF-7), and colorectal cancer (LoVo) tumor models, and reduced microvessel density in tumor tissues. The FP3-mediated inhibition of tumor growth was significantly higher than that of bevacizumab at the same dose. FP3 also demonstrated synergistic antitumor effects when combined with 5-fluorouracil (5-FU). Taken together, FP3 shows a high affinity for VEGF and produced antiangiogenic effects, suggesting its potential for treating angiogenesis-related diseases such as cancer.     

卵巢肿瘤的三维彩色血管能量成像与病理微血管密度对比研究

目的：探讨上皮性卵巢肿瘤的三维彩色血管能量成像（three-dimensional color power angio,3D－CPA)特点及其血管病理学基础,评价3D－CPA鉴别卵巢肿瘤的临床应用价值。方法：对67例上皮性卵巢肿瘤患者（40例卵巢癌,27例良性上皮性卵巢肿瘤）术前应用3D－CPA检测肿瘤的血管分布类型并计算肿瘤血管指数（vascularity index,VI),术后分别应用抗血管内皮生长因子（VEGF）、抗CD34单克隆抗体对组织进行免疫组织化学染色,计数肿瘤的微血管密度（microvessel density,MVD)及VEGF染色强度。结果：卵巢癌的MVD及VEGF表达强度均显著高于良性上皮性卵巢肿瘤（P＜0．05）。以3D－CPA血管指数＞0．02条／cm^3诊断卵巢癌敏感性77．5％,特异性81．5％。卵巢癌的血管指数与MVD呈等级相关（rs= 0.503,P＜0．01）。结论：3D－CPA可用于术前间接评价肿瘤的血管生成,为卵巢肿瘤的诊断及鉴别提供有价值的信息。     

Soluble Flt-1 gene delivery using PEI-g-PEG-RGD conjugate for anti-angiogenesis.

Vascular endothelial growth factor (VEGF), a potent angiogenic molecule specific for vascular endothelial cells, is overexpressed in most tumors and closely associated with tumor growth and metastasis. It has been shown that a soluble fragment of VEGF receptor Flt-1 (sFlt-1) has anti-angiogenic properties by way of its antagonist activity against VEGF. In the present study, we demonstrated that the stable expression of sFlt-1 by endothelial cell targeted non-viral gene delivery inhibited the angiogenesis of endothelial cells. A targeted polymeric gene delivery system, PEI-g-PEG-RGD, was developed by incorporating the alphanubeta3/alphanubeta5 integrin-binding RGD peptide, ACDCRGDCFC (single-letter amino acid code), into the cationic polymer, polyethylenimine (PEI) via a hydrophilic polyethylene glycol (PEG) spacer. The functional analysis of therapeutic gene encoding sFlt-1/carrier complex was performed with an endothelial cell proliferation assay. The complex of sFlt-1 gene with PEI-g-PEG-RGD conjugate efficiently inhibited the proliferation of cultured endothelial cells, representing that expressed sFlt-1 predominantly bound to exogenous VEGF and blocked the binding of VEGF to the full-length Flt-1 receptor. These findings suggest that the combination of targeted gene carrier and sFlt-1 possesses the potential to be an efficient tool for the anti-angiogenic gene therapy to treat cancer.     

Efficient inhibition of ovarian cancer by truncation mutant of FILIP1L gene delivered by novel biodegradable cationic heparin-polyethyleneimine nanogels

Filamin A interacting protein 1-like (FILIP1L), which was reported to be consistently absent in ovarian cancer cell lines, has been identified to hold therapeutic potential for inhibiting tumor growth, and its COOH-terminal truncation mutant (FILIP1LΔC103) was found to be more potent than the wild-type. The use of polymeric nanoparticles to deliver functional gene intraperitoneally holds much promise as an effective therapy for ovarian cancer. In this study, a recombinant plasmid expressing FILIP1LΔC103 (FILIP1LΔC103-p) was constructed, and biodegradable cationic heparin-polyethyleneimine (HPEI) nanogels were prepared to deliver FILIP1LΔC103-p into human ovarian cancer SKOV3 cells. The expression of FILIP1LΔC103 in vitro and in vivo was determined using RT-PCR and western blot analysis. Moreover, a SKOV3 intraperitoneal ovarian carcinomatosis model was established to investigate the antitumor activity of HPEI+FILIP1LΔC103-p complexes in nude mice. Tumor weights were evaluated during the treatment course. Cell proliferation and apoptosis were evaluated by Ki-67 immunochemical staining and TUNEL assay, respectively, and the antiangiogenic effect of FILIP1LΔC103-p was assessed by CD31 immunochemical staining and alginate-encapsulated tumor cell assay. FILIP1LΔC103-p could be efficiently transfected into SKOV3 cells by HPEI nanogels. Intraperitoneal administration of HPEI+FILIP1LΔC103-p complexes led to effective growth inhibition of ovarian cancer, in which tumor weight decreased by almost 72% in the treatment group compared with that in the empty-vector control group. Meanwhile, decreased cell proliferation, increased tumor cell apoptosis, and reduction in angiogenesis were observed in the HPEI+FILIP1LΔC103-p group compared with those in the control groups. These results indicated that HPEI nanogels delivering FILIP1LΔC103-p might be of value in the treatment against human ovarian cancer.     

Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma.

Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin-endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model.     

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p < 0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.     

Preclinical evaluation of a class of infectivity-enhanced adenoviral vectors in ovarian cancer gene therapy

Ovarian carcinoma cells are often infected inefficiently by adenoviruses (Ad) due to low expression of coxsackie-adenovirus receptors (CAR), hindering the application of adenovirus-mediated gene therapy in ovarian cancer. In this study, we explored a class of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs RGD (Ad5.RGD), polylysine (Ad5.pK7), or both (Ad5.RGD.pK7), for their utility in ovarian cancer gene therapy using in vitro and in vivo model systems. We found that these vectors infected established ovarian carcinoma cell lines and primary ovarian cancer cells with significantly enhanced infectivity. Among them, Ad5.RGD.pK7 appeared to be most efficient. Further, we evaluated their gene delivery efficiency using two different ovarian cancer mouse models--subcutaneous and intraperitoneal human ovarian cancer xenografts. All of the modified vectors appeared to be more efficient than the unmodified Ad5 vector in both models, although some of the differences are not statistically significant. Of these, Ad5.RGD.pK7 exhibited the highest efficacy in the subcutaneous tumor model, while Ad5.pK7 worked most efficiently in the intraperitoneal tumor model. These preclinical results suggest that Ad5.RGD.pK7 and Ad5.pK7 may be very useful in ovarian cancer gene therapy.     

Vascular endothelial growth factor and its soluble receptor in benign and malignant ovarian tumors

An imbalance between pro-angiogenic and anti-angiogenic factors is hypothesized in the pathogenesis of ovarian cystic disease. The aim of the following study was to explore the possible role of free vascular endothelial growth factor receptor 1 (sVEGFR-1), a soluble regulator of vascular endothelial growth factor (VEGF) action, in ovarian cystoadenoma, endometriomata and cystoadenocarcinoma. Forty-eight women, of whom fourteen had ovarian serous cysts, twenty-eight had stage III-IV ovarian endometriomata, and six had stage IIIB-IIIC ovarian carcinoma, were included. Sampling of serum, peritoneal and ovarian cystic fluids and of tumor tissue was performed before, during and following surgery, respectively. Levels of VEGF and sVEGFR-1 were measured in serum, peritoneal and cystic fluid. VEGF and sVEGFR-1 expression was evaluated in tumor tissue. There were no differences in serum VEGF and sVEGFR-1 levels nor in VEGF/VEGFR-1 ratio between study groups. Peritoneal fluid VEGF levels were higher in cystoadenocarcinoma patients than in endometriosis and in cystoadenoma patients, while sVEGFR-1 peritoneal fluid concentrations were significantly higher only in endometriosis-affected women. VEGF/VEGFR-1 ratio was highest in the peritoneal fluid of cancer patients with respect to the other two groups of women. Cystic fluid VEGF and VEGFR-1 concentrations were higher in endometriomata and in cystoadenocarcinomas than in cystadenomas but the VEGF/VEGFR-1 ratio was highest in cancer patients. Western blot evidenced a marked expression of VEGF and soluble VEGFR-1 in endometriosis tissue with respect to benign cyst tissue but a lower expression of both molecules, contrary to that expected, in cancer tissue. In conclusion, all in all, our data indicate that an excess of local VEGF with respect to its soluble receptor VEGFR-1 may be a key factor in the onset and maintenance of pathological neo-angiogenesis in ovarian cyst formation.     

Systemic cancer gene therapy using adeno-associated virus type 1 vector expressing MDA-7/IL24.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL24), selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and antiangiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for a new anticancer gene therapy. Here, we examined the feasibility of adeno-associated virus type 1 (AAV1) vector-mediated systemic gene therapy using mda-7/IL24. In vitro studies showed that medium conditioned by AAV1-mda7-transducedC2C12 cells induces tumor cell-specific apoptosis and inhibits angiogenesis in a human umbilical vein endothelial cell tube formation assay. To assess the in vivo effects of AAV1-mediated systemic delivery of MDA-7/IL24, we generated a subcutaneous tumor model by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice. A single intravenous injection of AAV1-mda7 (2.0 x 10(11) viral genomes) significantly inhibited tumor growth. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analyses showed significant induction of tumor-cell-specific apoptosis and reduction of microvessel formation within the tumors, and there was a significant increase in survival among the AAV1-mda7-treated mice. These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treatment for cancer. Thus, AAV1 vector-mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to anticancer therapy.     

Tumor-penetrating microparticles for intraperitoneal therapy of ovarian cancer

Intraperitoneal chemotherapy prolongs survival of ovarian cancer patients, but its utility is limited by treatment-related complications and inadequate drug penetration in larger tumors. Previous intraperitoneal therapy used the paclitaxel/Cremophor EL (polyethoxylated castor oil) formulation designed for intravenous use. The present report describes the development of paclitaxel-loaded microparticles designed for intraperitoneal treatment (referred to as tumor-penetrating microparticles or TPM). Evaluation of TPM was performed using intraperitoneal metastatic, human ovarian SKOV3 xenograft tumor models in mice. TPM were retained in the peritoneal cavity and adhered to tumor surface. TPM consisted of two biocompatible and biodegradable polymeric components with different drug release rates; one component released the drug load rapidly to induce tumor priming, whereas the second component provided sustained drug release. Tumor priming, by expanding interstitial space, promoted transport and penetration of particulates in tumors. These combined features resulted in the following advantages over paclitaxel/Cremophor EL: greater tumor targeting (16-times higher and more sustained concentration in omental tumors), lower toxicity to intestinal crypts and less body weight loss, greater therapeutic efficacy (longer survival and higher cure rate), and greater convenience (less frequent dosing). TPM may overcome the toxicities and compliance-related problems that have limited the utility of intraperitoneal therapy.     

Enhanced Antitumor Efficacy of Vasculostatin (Vstat120) Expressing Oncolytic HSV-1

Oncolytic viral (OV) therapy is a promising therapeutic modality for brain tumors. Vasculostatin (Vstat120) is the cleaved and secreted extracellular fragment of brain-specific angiogenesis inhibitor 1 (BAI1), a brain-specific receptor. To date, the therapeutic efficacy of Vstat120 delivery into established tumors has not been investigated. Here we tested the therapeutic efficacy of combining Vstat120 gene delivery in conjunction with OV therapy. We constructed RAMBO (Rapid Antiangiogenesis Mediated By Oncolytic virus), which expresses Vstat120 under the control of the herpes simplex virus (HSV) IE4/5 promoter. Secreted Vstat120 was detected as soon as 4 hours postinfection in vitro and was retained for up to 13 days after OV therapy in subcutaneous tumors. RAMBO-produced Vstat120 efficiently inhibited endothelial cell migration and tube formation in vitro (P = 0.0005 and P = 0.0184, respectively) and inhibited angiogenesis (P = 0.007) in vivo. There was a significant suppression of intracranial and subcutaneous glioma growth in mice treated with RAMBO compared to the control virus, HSVQ (P = 0.0021 and P < 0.05, respectively). Statistically significant reduction in tumor vascular volume fraction (VVF) and microvessel density (MVD) was observed in tumors treated with RAMBO. This is the first study to report the antitumor effects of Vstat120 delivery into established tumors and supports the further development of RAMBO as a possible cancer therapy.     

Development of the hybrid Sleeping Beauty: baculovirus vector for sustained gene expression and cancer therapy

Antiangiogenesis is an appealing anticancer approach but requires continued presence of the antiangiogenic agents, which can be remedied by gene therapy. Baculovirus is an emerging gene delivery vector but only mediates transient expression (<7 days); thus, this study primarily aimed to develop a hybrid baculovirus for sustained antiangiogenic gene expression and cancer therapy. We first constructed plasmids featuring adeno-associated virus inverted terminal repeats (AAV ITRs), oriP/Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1) or Sleeping Beauty (SB) transposon and compared their efficacies in terms of persistent expression. In human embryonic kidney (HEK293) cells, AAV ITR failed to prolong the expression while oriP/EBNA1 moderately extended the expression to 35 days. In contrast, the SB system led to stable expression beyond 77 days even without antibiotic selection. Given this finding, we constructed a hybrid SB baculovirus expressing the SB transposase and harboring the transgene cassette flanked by inverted repeat/direct-repeat (IR/DR) elements recognizable by SB. The hybrid SB baculovirus efficiently transduced mammalian cells and mediated an expression duration longer than that by conventional baculoviruses, thanks to the transgene persistence and integration. The SB baculovirus (Bac-SB-T2hEA/w) expressing the antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) also enabled prolonged hEA expression. With sustained hEA expression, Bac-SB-T2hEA/w repressed the angiogenesis in vivo, hindered the growth of two different tumors (prostate tumor allografts and human ovarian tumor xenografts) in mice and extended the life span of animals. These data altogether implicated the potential of the hybrid SB-baculovirus vector for prolonged hEA expression and for the treatment of multiple types of angiogenesis-dependent tumors.     

Oncolytic HSV armed with platelet factor 4, an antiangiogenic agent, shows enhanced efficacy.

Oncolytic herpes simplex viruses (HSV) have emerged as a promising platform for cancer therapy. However, efficacy as single agents has thus far been unsatisfactory. Tumor vasculature is critical in supporting tumor growth, but successful antiangiogenic approaches often require maintaining constant levels of antiangiogenic products. We hypothesized that oncolytic HSV has the potential to destroy tumor vasculature and that this effect can be enhanced by combination with antiangiogenic gene transfer. We examined the strategy of arming oncolytic HSV with an antiangiogenic transgene, platelet factor 4 (PF4). The PF4 transgene was inserted into oncolytic HSV G47Delta utilizing a bacterial artificial chromosome construction system. Whereas bG47Delta-empty showed robust cell killing and migration inhibition of proliferating endothelial cells (HUVEC and Py-4-1), the effect was further enhanced by PF4 expression. Importantly, enhanced potency did not impede viral replication. In vivo, bG47Delta-PF4 was more efficacious than its nonexpressing parent bG47Delta-empty at inhibiting tumor growth and angiogenesis in both human U87 glioma and mouse 37-3-18-4 malignant peripheral nerve sheath tumor models. Enhancing the antiangiogenic properties of oncolytic HSV through the expression of antiangiogenic factors such as PF4 is a powerful new strategy that targets both the tumor cells and tumor vasculature.     

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72?h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.     

卵巢癌超声造影与血管生成拟态和MVD的相关性分析

目的 分析不同时期裸鼠卵巢癌移植瘤血管生成拟态(VM)密度、微血管密度(MVD)及其CEUS参数的相关性。方法 分别建立生长21天组、28天组裸鼠移植瘤模型,每组10只,对两组移植瘤模型行CEUS检查,分析各组时间强度曲线(TIC)及相关参数。应用CD31及PAS双重染色检测卵巢癌中VM表达及MVD。对两个时期移植瘤造影的峰值强度(PI)与相应的VM密度及MVD进行相关性分析。结果 成功制备移植瘤模型。CEUS显示,21天组、28天组的峰值强度(PI)、达峰时间(TTP)、持续时间(TTD)差异有统计学意义(P<0.05)。染色检测显示,21天组、28天组VM密度、MVD差异均有统计学意义(P均<0.05)。21天组PI与VM密度、MVD均呈正相关(r=0.657、0.652,P均<0.05);28天组PI与VM密度无相关性(P>0.05),PI与MVD呈正相关(r=0.687,P=0.03)。结论 不同时期卵巢癌CEUS与VM密度或MVD密切相关,对卵巢癌早期诊断有一定价值。