Vascular targeting conference
Exploiting the differential production of angiogenic factors within the tumor microenvironment in the design of a novel vascular-targeted gene therapy-based approach to the treatment of cancer

Presented in part at the First International Conference on Vascular Targeting, Cambridge, MA, June 2002.
https://doi.org/10.1016/S0360-3016(02)03921-4Get rights and content

Abstract

Purpose: The aim of this study is to explore a novel strategy through which the differential production of pro-angiogenic cytokines within the tumor microenvironment can be exploited as a means of selectively killing the vascular endothelial cells upon which the survival and growth of a tumor depend.

Methods and Materials: Adenoviral vectors encoding a chimeric cell surface receptor composed of the extracellular domain of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR fused in frame to the membrane spanning and cytoplasmic domain of Fas were constructed and used to transduce primary human endothelial cells in vitro. The apoptotic response of these cells induced upon ligation of the chimeric receptor with VEGF was determined by measuring caspase-3 activation, AnnexinV-FITC binding, and the release of glucose-6-phosphate dehydrogenase.

Results: The chimeric Flk-1/Fas protein is stable and expressed at high levels on the surface of adenovirally transduced cells. Upon the addition of exogenous VEGF, these cells undergo rapid apoptosis.

Conclusions: Receptor/Fas chimeras that recognize and bind pro-angiogenic cytokines represent a novel means by which the signal transduction events normally triggered in vascular endothelial cells upon the binding of angiogenic cytokines may be redirected toward the induction of apoptotic cell death. It is proposed that these constructs will prove of value in the further development of safe and effective vascular-targeted gene therapy-based approaches to the treatment of cancer.

Introduction

As a result of the dramatic progress that has been made in recent years with respect to our understanding of the molecular mechanisms that underlie the malignant process, there is now no shortage of genes that are potentially useful in the context of cancer gene therapy (1). Among the most promising are cytotoxins such as Pseudomonas Exotoxin A, which can directly kill tumor cells; enzymes (most notably cytosine deaminase and thymidine kinase) that convert various prodrugs to their biologically active metabolites; a large and diverse group of genes involved in the regulation of DNA repair, cellular proliferation, differentiation, and apoptosis, which may either directly kill tumor cells or modulate their response to various chemotherapeutic agents and/or ionizing radiation; and finally, genes encoding either cell surface costimulatory proteins or biologically active soluble mediators that are capable of stimulating the generation of locally acting and/or systemic antitumor immune responses (2). Many of these genes have been shown to be effective in vitro and in various animal models, and a number are currently being evaluated in ongoing clinical trials 1, 2, 3, 4. The results obtained to date, although generally encouraging, are perhaps less dramatic than might have been hoped based on in vitro efficacy 1, 3, 4. This finding most likely reflects the difficulties implicit in achieving efficient in vivo gene transfer (5).

Direct intratumoral or locoregional injection remains the most popular means of transducing established solid tumors in vivo3, 5. The approach is, however, limited to easily accessible tumors, such as melanomas, or those in defined body cavities, such as the bladder, peritoneum, or pleural space. Although transduction efficiencies around sites of intratumoral injection can be fairly impressive, because of the physical obstacles that tumor tissue presents to vector diffusion, regions immediately adjacent to a site of injection may be poorly transduced such that the overall impact of treatment on tumor growth is often modest (3).

Systemic delivery of genes to tumor sites, although attractive from a practical perspective, is similarly problematic. In particular, the poor perfusion of tumors that results from intermittent blood flow and the generally chaotic nature of the tumor vasculature, combined with the high interstitial pressures produced as a consequence of inadequate lymphatic drainage, tend to prevent the efficient egress of i.v.-administered vectors into tumor sites (2). The presence within tumors of a dense extracellular matrix and variable numbers of host-derived cells also limits vector diffusion, further reducing transduction efficiencies (2).

It is for these various reasons that the vasculature upon which the survival and growth of tumor depend is such an attractive target for cancer gene therapy 2, 6. In particular, the fact that vascular endothelial cells are in direct contact with the circulation obviously facilitates their transduction with i.v.-administered vectors. Moreover, because each endothelial cell supplies oxygen and nutrients to many thousands of tumor cells, it follows that even the very limited transduction efficiencies that can realistically be obtained using currently available vectors are likely to translate into massive “bystander” killing of tumor cells, as is seen with vascular targeting agents such as combretastatin A4 7, 8 and ZD6126 (9). Rather more speculatively, because vascular endothelial cells are diploid and nontransformed, it is possible that they will respond more predictably to a particular therapeutic approach and that resistance is less likely to emerge. Finally, because the vascular endothelial cells present within tumors are undergoing active angiogenesis and are exposed to various tumor-derived microenvironmental signals, it is likely that they will possess unique phenotypic characteristics that could permit the targeting of therapy.

To succeed, a gene therapy-based approach to the treatment of cancer must be not only effective, but also safe and practical. Because transduction of normal tissues is a legitimate safety concern in the context of i.v.-administered vectors, for a vascular-targeted gene therapy strategy to be viable, it must incorporate efforts to restrict the expression and/or activity of therapeutic genes to tumor-associated vascular endothelial cells in vivo. Various approaches have been explored in this regard, including use of endothelial cell-specific promoter elements and vectors with restricted cellular tropism 10, 11, 12, 13. The present study describes a novel strategy in which the differential expression of angiogenic factors within the tumor microenvironment can be exploited as a means of targeting the neovasculature upon which growth and survival of a tumor depend. In this instance, specificity is achieved not by regulating expression of the transgene, but rather by taking advantage of the differential spatial and/or temporal expression of a naturally occurring ligand that is needed to activate the cytotoxic activity of a chimeric receptor that incorporates the extracellular ligand-binding domain of Flk-1/KDR fused in frame to the membrane spanning and cytoplasmic domains of human Fas.

Section snippets

Cell culture

Normal human microvascular endothelial cells from adult dermal tissue (HMVEC-d) were obtained from Clonetics (San Diego, CA) and were cultured in microvascular endothelial growth media EGM-2 supplemented according to the manufacturer’s instructions. Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics and cultured in EGM-2 media supplemented according to the manufacturer’s instructions. Cells between 3rd and 6th passages were used for all experiments described below.

Transduction of endothelial cells with Ad.Flk-1/Fas

An adenoviral vector was constructed and used to introduce and express within HUVEC and HMVEC-d a chimeric cDNA encoding the extracellular domain of the VEGF receptor Flk-1 fused in frame to the membrane spanning and cytoplasmic “death” domain of the pro-apoptotic protein Fas. As shown in Fig. 1, FACS analysis confirmed that endothelial cells can be readily infected with adenoviral vectors, at least in vitro, and that the encoded chimeric Flk-1/Fas protein is both stable and expressed at high

Discussion

The safety and potential efficacy of vascular-targeted gene therapy-based approaches to the treatment of cancer would be greatly enhanced if practical means could be developed to specifically target the neovasculature of solid tumors in vivo(2). The present study explored a novel approach to achieving this objective, in which chimeric receptor proteins are used to redirect the signal transduction events normally triggered in vascular endothelial cells upon the binding of angiogenic cytokines

References (19)

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