A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage–mediated, vascular-targeted suicide gene transfer

Abstract

Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as the herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells via a novel targeted AAVP vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic bystander effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of co-cultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18α-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexin (Cx)43 and Cx26, which are expressed in both endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative non-mutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted AAVP, and further support the promising role of non-malignant tumor stromal cells as therapeutic targets.

AAVP displaying octreotide for ligand-directed therapeutic transgene delivery in neuroendocrine tumors of the pancreas

“…Of note, because many of the challenges for patients with pancreatic NETs (particularly those with functioning tumors) arise from hormonal hypersecretion and tumor-associated metabolic burden (35), meaningful results that show an unequivocal reduction in both metabolic activity and/or secreted insulin after treatment are clinically relevant. This experimental design mimics the experimental preclinical dosing schedule reported by our group in large animals (dogs) with native malignant tumors (5), and confirms the efficacy of repetitive AAVP administration serially over time, likely owing to additional cellular transduction after each administration and a bystander effect observed with AAVP activity related to the exposure of neighboring cells to cytotoxic TNF generated by transduced cells (36).…”

Clathrin-mediated Endocytosis and Subsequent Endo-Lysosomal Trafficking of Adeno-associated Virus/Phage

“…However, high titers of AAVP are still required for transduction of large numbers of cells in culture. In vivo tumor therapy is therefore likely to be influenced by other factors, such as the heterotypic "bystander effect" which facilitates transport of cytotoxic metabolites between the transduced and neighboring nontransduced tumor cells, thus enhancing therapeutic effect (31).…”

Adeno-associated virus type 2 infection of nude mouse human breast cancer xenograft induces necrotic death and inhibits tumor growth

“…Therefore, the initial mechanism of cell killing in all these different breast cancer cell lines is potentially mediated by a "bystander" effect, in which dying cells release cytotoxic metabolites, cellular and/or AAV2 encoded proteins, which are able to affect the neighboring non-infected cells via movement through cellular gap junctions to mediate cell killing, a phenomenon which has been previously described elsewhere. 25,26 Subsequent mass cytotoxicity of AAV2-infected cells is potentially due to a downstream amplification step, since we also observed that the AAV2-mediated cell killing was positively associated with viral genome amplification (discussed below). Therefore, low levels of AAV2 infectious particles potentially synthesized in the infected breast cancer cell lines and released into the medium could infect neighboring cells, thereby amplifying the bystander effect.…”