ABSTRACT
A breast cancer-selective oncolytic adenovirus was engineered to express antagonists of vascular endothelial growth factor (VEGF) and Notch signaling to combine direct anticancer activity with disruption of tumor-associated angiogenesis. Replication of the parental virus, AdEHE2F, is stimulated by estrogen receptor (ER), E2F1 and hypoxia, and it mediates selective lysis of breast cancer cells in vitro and in vivo. Here, we encoded soluble Flt-1 (sFlt1) and soluble Dll4 (sDll4) under control of the E3 promoter. sFlt1 (the extra-cellular domain of VEGF receptor 1) binds VEGF-A and inhibits stimulation of VEGFR2, decreasing angiogenic stimulus. Conversely, sDll4 (the extracellular domain of Delta-like 4) antagonizes Notch signaling to prevent endothelial maturation. We hypothesized that these agents might show additive or synergistic activity. In vitro, sFlt1 inhibited endothelial cell proliferation and sprouting, whereas sDll4 increased the number of vascular branchpoints. In ER-positive ZR75.1 tumors in vivo AdEHE2F showed the potent direct virotherapy with no augmentation owing to sFlt1 or sDll4; however, in ER-negative MDA-231 tumors efficacy was enhanced by encoding sFlt1 or sDll4, with survival time extending to double that of controls. There was also a dramatic decrease in the total number of tumour blood vessels, as well as the number of perfused vessels, suggesting that improved efficacy reflects combined anti-tumour and anti-vascular effects.
Subject(s)
Breast Neoplasms/therapy , Embolization, Therapeutic/methods , Oncolytic Virotherapy/methods , Adaptor Proteins, Signal Transducing , Adenoviridae/genetics , Adenoviridae/metabolism , Adenoviridae/physiology , Animals , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Breast Neoplasms/virology , Calcium-Binding Proteins , Cell Growth Processes/physiology , Combined Modality Therapy , Female , Gene Expression , Human Umbilical Vein Endothelial Cells , Humans , Immunohistochemistry , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/genetics , Mice , Mice, Nude , Neovascularization, Pathologic/therapy , Receptors, Estrogen/metabolism , Signal Transduction , Transgenes , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-1/biosynthesis , Vascular Endothelial Growth Factor Receptor-1/genetics , Xenograft Model Antitumor AssaysABSTRACT
Oncolytic viruses are regulated by the tumor phenotype to replicate and lyse cancer cells selectively. To identify optimal strategies for breast cancer we compared five adenoviruses with distinct regulatory mechanisms: Ad-dl922-947 (targets G1-S checkpoint); Ad-Onyx-015 and Ad-Onyx-017 (target p53/mRNA export); Ad-vKH1 (targets Wnt pathway), and AdEHE2F (targets estrogen receptor/G1-S checkpoint/hypoxic signaling). The quantity of virus required to kill 50% of breast cancer cells after 6 days (EC(50), plaque-forming units per cell) was measured. The most potent virus was Ad-dl922-947 (EC(50), 0.01-5.4 in SkBr3, MDA-231, MDA-468, MCF7, and ZR75.1 cells), followed by wild-type (Ad-WT; EC(50), 0.3-5.5) and AdEHE2F (EC(50), 1.4-3.9). Ad-vKH1 (EC(50), 7.2-72.1), Ad-Onyx-017 (EC(50), 8.4-167), and Ad-Onyx-015 (EC(50), 17.7-377) showed less activity. Most viruses showed limited cytotoxicity in normal human cells, including breast epithelium MCF10A (EC(50), >722) and fibroblasts (EC(50), >192) and only moderate cytotoxicity in normal microvascular endothelial cells (HMVECs; EC(50), 42.8-149), except Ad-dl922-947, which was active in HMVECs (EC(50), 1.6). After injection into MDA-231 xenografts, Ad-WT, AdEHE2F, and Ad-dl922-947 showed replication, assessed by hexon staining and quantitative polymerase chain reaction measurement of viral DNA, and significantly inhibited tumor growth, leading to extended survival. After intravenous injection Ad-dl922-947 showed DNA replication (233% of the injected dose was measured in liver after 3 days) whereas AdEHE2F did not. Overall, AdEHE2F showed the best combination of low toxicity in normal cells and high activity in breast cancer in vitro and in vivo, suggesting that molecular targeting using estrogen response elements, hypoxia response elements, and a dysregulated G1-S checkpoint is a promising strategy for virotherapy of breast cancer.
Subject(s)
Adenoviruses, Human/genetics , Breast Neoplasms/therapy , Oncolytic Virotherapy/methods , Oncolytic Viruses/genetics , Adenoviruses, Human/physiology , Animals , Base Sequence , Breast Neoplasms/pathology , Breast Neoplasms/virology , Cell Cycle , Cell Death , Cell Line , Cell Line, Tumor , DNA Primers/genetics , Female , Humans , Mice , Mice, SCID , Neoplasm Transplantation , Oncolytic Viruses/physiology , Transplantation, Heterologous , Virus ReplicationABSTRACT
Direct intratumoural (IT) administration of adenovirus is widely used, however little is known about the resulting distribution of virus particles. Here we have evaluated the influence of tumour size, volume of injectate and occlusion of injection sites (to prevent retrograde seepage) on particle biodistribution and transgene expression. In subcutaneous MDA-231 xenografts, IT injection of relatively large volumes (4 x 20% (vol/vol) injections) resulted in just 40% of the administered dose being retained in tumour tissue after 30 min, with 15% in the liver thought to reflect systemic 'overflow'. Occlusion of the injection sites using surgical adhesive increased retention of the vector to 80% in the tumour with no increase in liver levels. Spread of expression was enhanced using multiple injection sites, but not by using larger injectate volumes. In ZR75.1 breast carcinoma xenografts virus distribution was different, with no evidence of systemic overflow leading to hepatic transduction following IT injection. Typically, clinical doses employ up to 30% vol/vol IT injections. Depending on the tumour, this may give considerable systemic overflow and might account for the high frequency of fevers observed. Virus performance might be improved by tailoring volumes and frequency of IT injection for tumour biology or histotype.
Subject(s)
Adenoviridae/physiology , Breast Neoplasms/therapy , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Transduction, Genetic/methods , Adenoviridae/genetics , Adenoviridae Infections/pathology , Adenoviridae Infections/virology , Animals , Breast Neoplasms/pathology , Breast Neoplasms/virology , Female , Gene Expression , Humans , Injections, Intralesional , Liver/virology , Mice , Mice, Nude , Transgenes , Xenograft Model Antitumor AssaysABSTRACT
Tumor-associated vasculature is a relatively accessible component of solid cancers that is essential for tumor survival and growth, providing a vulnerable target for cancer gene therapy administered by intravenous injection. Several features of tumor-associated vasculature are different from normal vasculature, including overexpression of receptors for angiogenic growth factors, markers of vasculogenesis, upregulation of coagulation cascades, aberrant expression of adhesion molecules and molecular consequences of hypoxia. Many of these differences provide candidate targets for tumor-selective 'transductional targeting' of genetically- or chemically modified vectors and upregulated gene expression can also enable 'transcriptional targeting', regulating tumor endothelia-selective expression of transgenes following nonspecific gene delivery. Tumor vasculature also represents an important site of therapeutic action by the secreted products of antiangiogenic gene therapies that are expressed in non-endothelial cells. In this review we assess the challenges faced and the vectors that may be suitable for gene delivery to exploit these targets. We also overview some of the strategies that have been developed to date and highlight the most promising areas of research.
