We found that oHSV-mediated expression of the MMP inhibitor, TIMP-3, increased antitumor efficacy via enhanced direct cellular cytotoxicity, elevated peak i.t. virus titers, and inhibition of neovascularization. Herein, we report the novel finding that a TIMP-3–expressing oHSV reduced systemic numbers of bone marrow–derived endothelial progenitor cells. Although we cannot be certain about a causal relationship of this finding with an antitumor effect, the increased efficacy of oHSV by TIMP-3 transgene expression seems to be mediated via direct effects of TIMP-3 on tumor cells as well as indirect, overlapping effects upon the tumor microenvironment. The relative contribution of each mechanism is unknown.
Previous studies have documented the ability of oncolytic viruses to reduce i.t. vascular density and impede tumor blood flow (28
). Vascular-disrupting oncolytic viruses can infect and kill dividing endothelial cells and have been used to deliver antiangiogenic transgenes such as platelet factor 4, soluble VEGFR, and interleukin-12 (38
). Recent reports have highlighted a role for Tie2-expressing bone marrow–derived endothelial progenitors (including noninflammatory monocytes) in vasculogenesis of ischemic tissues such as infarcts, wounds, and tumors (41
); therefore, we sought to determine if an oncolytic virus could affect these cells. Although rQLuc-treated mice showed a trend toward reduced CEPs, rQT3-treated animals contained a further significant reduction, implicating TIMP-3. Our findings suggest that therapeutic viruses can act systemically by regulating both the mobilization and recruitment of bone marrow–derived progenitors, both CEPs and others, that contribute to the tumor microenvironment and growth.
The importance of SDF-1α, VEGF signaling, and MMP activity has been established in the trafficking of hematopoietic progenitors and vasculogenesis (35
). In our study, peripheral blood from saline-treated, tumor-bearing mice contained human and murine VEGF and SDF-1α as well as an identifiable population of cells consistent with CEPs. Although rQT3-treated mice contained fewer CEPs and elevated plasma SDF-1α levels, they did not contain measurable VEGF. The initially counterintuitive finding of increased SDF-1α and fewer CEPs is consistent with recent reports implicating a role for MMP activity in SDF-1α–directed endothelial cell invasion and formation of neovasculature (14
). Elevation of SDF-1α in rQT3-treated animals in our study may represent a compensatory response to reduced vascular density and increased tumor hypoxia, with mobilization being blocked by MMP inhibition or by a lack of VEGF. The role of MMP activity in regulation of CEP mobilization and recruitment is controversial as these proteases can cleave SDF-1α and CXCR4 rendering them nonfunctional (45
). This receptor turnover may be important for efficient signaling through the CXCR4 receptor. In a recent report, SDF-1α mobilization of bone marrow–derived cells required a costimulatory signal from VEGF (46
). There was a bone marrow contribution to tumor vasculature, although plasma SDF-1α levels were higher than observed in our study. Indeed, the SDF-1α/CXCR4 signaling axis has been implicated in neuroblastoma metastasis to the marrow cavity (44
). Our data are consistent with a model whereby SDF-1α and VEGF signaling may simultaneously regulate tumor vasculogenesis and metastasis, in a MMP-dependent manner, making this axis a highly relevant therapeutic target (see model, Supplementary Fig. S6
TIMP-3 expression may also increase the antitumor effect of oHSV via additional mechanisms. Although rQT3 did not show increased in vitro
virus replication compared with rQLuc, rQT3 reached significantly higher levels in vivo
within tumor tissue. A number of hypotheses might explain this unanticipated finding. Increased levels of i.t. virus may be achieved via more productive virus replication or alternatively, a peak achievable virus load may be achieved via increased virus persistence (prolonged half-life). Our in vitro
data and the fact that we did not observe even higher virus titers at later time points in vivo
argue for the latter possibility. Interestingly, HSV-1, TIMP-3, MMPs, and cytokines including VEGF, βFGF, SDF-1α, etc., are capable of binding heparan sulfate glycosaminoglycans and may be competing for docking sites. As HSV-1 uses heparan sulfate for its initial binding, expression of TIMP-3 may interfere with virus entry into tumor cells, possibly increasing the half-life of extracellular, infectious virus. As MMPs cleave many nonmatrix substrates, including growth factor binding proteins and cellular receptors, we speculate that MMPs (or other proteases), via their intimate arrangement with viruses while docked to heparan sulfates, may cleave viral surface proteins leading to virus inactivation. These interactions would likely be markedly enhanced in a cellular tumor compared with a subconfluent two-dimensional culture. Inhibition of growth factor cleavage and secretion of factors such as VEGF may also explain the systemic effects of TIMP-3 on CEP mobilization. Finally, MMP inhibition within tumors may impede penetration of virus-clearing cells into tumor tissue. Recent reports have elegantly shown that tumor extracellular matrix hinders replication and spread of oncolytic viruses, prompting the emergence of strategies to degrade the tumor microenvironment to enhance virus spread (50
). If such strategies rely upon MMPs to degrade tumor extracellular matrix, care should be taken to avoid tumor-potentiating MMPs.
Interestingly, there seemed to be an incongruence of in vitro and in vivo results: In S462 cells, the TIMP-3 effect was relatively modest in vitro, but there was a marked effect in vivo. In contrast, the TIMP-3 effect in LA-N-5 was larger in vitro, but there was only a modest effect in vivo. The lack of correlation is likely due to the complex interplay of mitigating factors. For example, the extracellular matrix barrier can vary widely in composition and quantity among different tumor types and can thus both variably impede virus spread and variably affect TIMP-3 function. In addition, because TIMP-3 is bound to the extracellular matrix, it may be more or less accessible to cells in culture than in vivo. Thus, the predominant mechanism by which TIMP-3 exerts an antitumor effect may differ in different models.
Antivascular effects of TIMP-3 have been previously described. In a study using retroviral-mediated stable transduction of TIMP-3 in murine neuroblastoma and melanoma, tumors showed reduced blood vessels by gross appearance (24
). Surprisingly, TIMP-3– overexpressing tumors contained an increased absolute number of endothelial cells; however, these cells had not undergone functional capillary morphogenesis evidenced by a lack of vessel continuity, pericyte recruitment, and vascular endothelial cadherin. These results are consistent with our study and show potent antitumor effects of TIMP-3 via inhibition of tumor neovascularization. Further, our finding of decreased CEPs represents a novel antitumor mechanism in which an oHSV-expressing TIMP-3 interferes with tumor vasculogenesis.
Although many reports highlight antitumor effects of TIMP-3, one study using an oncolytic adenovirus did not find enhancement of antitumor efficacy in a glioma model (17
). The TIMP-3–expressing virus reduced cellular proliferation and increased apoptosis and MMP inhibition, but it did not significantly improve inhibition of tumor growth or survival of tumor-bearing mice. Despite not reaching statistical significance, the authors note that only the cohort of mice treated with the TIMP-3–expressing virus contained long-term survivors. The authors suggest a number of explanations to interpret their results, including a low level of transgene expression, the size of tumors at treatment initiation, or the dominant oncolytic effect of the adenovirus (~33% cured) to explain the lack of improvement in antitumor efficacy. In our studies, we document robust tumor-selective transgene expression, and antitumor efficacy against both small and large tumor models. Whether the choice of virus (HSV versus adenovirus) is important in this context is unclear.
Clinical translation of MMP inhibition strategies have not been as effective as predicted from preclinical studies. Reasons cited for this failure include inadequate drug levels, issues with respect to timing, and simultaneous inhibition of opposing protease activities (51
). The high local concentrations of TIMP-3 possible with gene transfer may be an advantage of virus-mediated delivery. It also may be critical to determine when tumor growth and neovasculature are most dependent on MMP activity to identify an optimal treatment window. Finally, our results suggest that CEP levels in patients after antitumor therapies, cytostatic drugs, MMP inhibition, viral gene therapy, or any vascular-disrupting agent, may serve as a predictor or biomarker of therapeutic efficacy (52
). Overall, MMP inhibition should be broadly applicable to many human malignancies, and thus still holds much promise as part of future anticancer regimens combining cytostatic-targeted agents, gene therapy, and biologics such as oncolytic viruses.