Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS- deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [ l - N ⁶ -(1-iminoethyl) lysine, l -NIL] and an iNOS- deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) ≥ WT with l -NIL or iNOS ^−/− > eNOS ^−/− ≥ eNOS ^−/− with l -NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS- deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS ^−/− mice but not in eNOS ^−/− mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo .

Background: The use of oncolytic adenoviruses as cancer gene therapy is limited by their uneven penetration and distribution in tumors. We investigated whether the expression of the cell matrix–degradative protein relaxin by adenovirus could improve adenovirus distribution and penetration in tumors. Methods: We generated relaxin-expressing, replication-incompetent (dl-lacZ-RLX) and -competent (Ad-ΔE1B-RLX) adenoviruses by inserting a relaxin gene into the E3 adenoviral region. Controls were parental adenoviruses (dl-lacZ and Ad-ΔE1B) and phosphate-buffered saline (PBS) (vehicle). Replication-incompetent viruses, which do not lyse cells, were used to assess transduction efficiency. Viral spread in tumor spheroids, made by dissecting tumor tissue into homogeneous fragments, was assessed by reporter gene (i.e., lacZ) expression. Tumor growth inhibition was assessed by injecting adenoviruses into xenograft tumors in athymic mice (n = 8 or 9). Overall survival was assessed by the Kaplan–Meier method. Extracellular matrix was examined with Masson's trichrome staining. Therapeutic efficacy was evaluated by assessing spontaneous pulmonary metastasis in the B16BL6 melanoma mouse model and growth inhibition of orthotopically implanted hepatoma (n = 4–6). All statistical tests were two-sided. Results: In tumor spheroids and established solid tumors in vivo, transduction with dl-lacZ-RLX, compared with parental virus or vehicle, elicited higher transduction efficiency and viral spread throughout the tumor mass. Infection with Ad-ΔE1B-RLX, compared with parental virus, elicited greater viral persistence and spread, leading to increased survival (e.g., 100%, 95% confidence interval [CI] = 63.1% to 100%, for C33A tumor-bearing mice treated with Ad-ΔE1B-RLX, and 50%, 95% CI = 15.7% to 84.3%, for C33A tumor-bearing mice treated with Ad-ΔE1B). Infection with Ad-ΔE1B-RLX substantially decreased the collagen content of tumor tissue but not of adjacent normal tissue, compared with noninfected tissues. Intratumoral injection of Ad-ΔE1B-RLX inhibited the formation of lung metastases in mice (PBS = 268 mg of metastatic tumor per mouse and Ad-ΔE1B-RLX = 10 mg; difference = 258 mg, 95% CI = 94 to 426; P = .003, Mann–Whitney test). Systemic treatment with Ad-ΔE1B-RLX completely inhibited the growth of Hep1 hepatocellular carcinomas (PBS = 20.2 mg of tumor per mouse and Ad-ΔE1B-RLX = 0 mg; difference = 20.2 mg, 95% CI = 3.7 to 36.7; P = .004, Mann–Whitney test). Conclusion: Extracellular matrix degradation by relaxin expressed by adenoviruses increased viral distribution and tumor penetration, inhibited tumor growth and metastasis, and increased survival of mice.

Despite significant improvement in modalities for treatment of cancer that led to a longer survival period, the death rate of patients with solid tumors has not changed during the last decades. Emerging studies have identified several physical barriers that limit the therapeutic efficacy of cancer therapeutic agents such as monoclonal antibodies, chemotherapeutic agents, anti-tumor immune cells, and gene therapeutics. Most solid tumors are of epithelial origin and, although malignant cells are de-differentiated, they maintain intercellular junctions, a key feature of epithelial cells, both in the primary tumor as well as in metastatic lesions. Furthermore, nests of malignant epithelial tumor cells are shielded by layers of extracellular matrix (ECM) proteins (e.g., collagen, elastin, fibronectin, laminin) whereby tumor vasculature rarely penetrates into the tumor nests. In this chapter, we will review potential strategies to modulate the ECM and epithelial junctions to enhance the intratumoral diffusion and/or to remove physical masking of target receptors on malignant cells. We will focus on peptides that bind to the junction protein desmoglein 2 and trigger intracellular signaling, resulting in the transient opening of intercellular junctions. Intravenous injection of these junction openers increased the efficacy and safety of therapies with monoclonal antibodies, chemotherapeutics, and T cells in mouse tumor models and was safe in non-human primates. Furthermore, we will summarize approaches to transiently degrade ECM proteins or downregulate their expression. Among these approaches is the intratumoral expression of relaxin or decorin after adenovirus- or stem cell-mediated gene transfer. We will provide examples that relaxin-based approaches increase the anti-tumor efficacy of oncolytic viruses, monoclonal antibodies, and T cells.

RNA interference is being developed to treat cancer. Although highly target specific, its use has been limited by its short duration of expression. To overcome this shortcoming, we constructed an oncolytic adenovirus (Ad)-based short hairpin RNA (shRNA) expression system (Ad-B7-shVEGF) against vascular endothelial growth factor (VEGF), a key mediator in angiogenesis. To demonstrate the VEGF-specific nature of this Ad-based shRNA, replication-incompetent Ad expressing VEGF-specific shRNA (Ad-E1-shVEGF) was also generated. Ad-E1-shVEGF was highly effective in reducing VEGF expression, and elicited an antiangiogenic effect in vitro and in vivo. Similarly, Ad-B7-shVEGF exhibited potent antiangiogenic effects in the matrigel plug assay. Moreover, Ad-B7-shVEGF demonstrated a greater antitumor effect and enhanced survival compared to the cognate control oncolytic Ad, Ad-B7. Ad-B7-shVEGF induced significant reduction in tumor vasculature, verifying the antiangiogenic mechanism. Furthermore, both the duration and magnitude of gene silencing by Ad-B7-shVEGF was greater than Ad-E1-shVEGF. These results suggest that the combined effects of oncolytic viral therapy and cancer cell-specific expression of VEGF-targeted shRNA elicits greater antitumor effect than an oncolytic Ad alone.

Approximately 15% of non-small cell lung cancer cases are associated with a mutation in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. With the goal of treating mutated EGFR-mediated lung cancer, we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system to discriminate between the oncogenic mutant and wild-type EGFR alleles and eliminate the carcinogenic mutant EGFR allele with high accuracy. We targeted an EGFR oncogene harboring a single-nucleotide missense mutation (CTG > CGG) that generates a protospacer-adjacent motif sequence recognized by the CRISPR/Cas9 derived from Streptococcus pyogenes. Co-delivery of Cas9 and an EGFR mutation-specific single-guide RNA via adenovirus resulted in precise disruption at the oncogenic mutation site with high specificity. Furthermore, this CRISPR/Cas9-mediated mutant allele disruption led to significantly enhanced cancer cell killing and reduced tumor size in a xenograft mouse model of human lung cancer. Taken together, these results indicate that targeting an oncogenic mutation using CRISPR/Cas9 offers a powerful surgical strategy to disrupt oncogenic mutations to treat cancers; similar strategies could be used to treat other mutation-associated diseases.