Very little is known of the mechanisms by which the levels of VEGFR3 are regulated on either normal or tumor vessels. In this study, we have shown that IL-12 can suppress the aberrant expression of VEGFR3 on tumor vessels via an IFN-γ–dependent mechanism (). IFN-γ is produced by the TILs and NK cells (); however, it is the T cells that are required for the IL-12–mediated VEGFR3 suppression (). Using IFN-γR−/−
bone marrow chimeras, we have found that in the absence of immune cell responsiveness to IFN-γ, VEGFR3 can still be suppressed via a direct mechanism of IFN-γ acting on vessel endothelial cells (). Interestingly, IFN-γ can also act in an indirect mechanism in animals that have endothelial cells unable to respond to IFN-γ. There exists the possibility that a small population of mesenchymal stem cells may have been transferred with the donor marrow that could result in the presence of fibroblasts and endothelial cells of donor origin that could have a role in the antiangiogenesis in these tumors (41
). To examine this, we grew tumors in chimeric mice in which WT recipients received GFP+
bone marrow. Flow cytometry revealed a subset of tumor-associated GFP+
cells; however, the vast majority (>95%) of these cells were CD45+
and therefore not fibroblasts. In addition, we failed to observe any GFP+
tumor vessels (data not shown). Therefore, we have no evidence to suggest that donor bone marrow can develop into significant numbers of either stromal or endothelial cells within our model. Thus, there appears to be at least two distinct mechanisms by which IFN-γ can suppress VEGFR3.
The indirect mechanism by which IFN-γ can downregulate VEGFR3 requires an immune cell-produced mediator. The candidate for this mediator could be any gene product that is downstream of IFN-γ signaling that can act on endothelial cells. The chemokine CXCL10 is one of the few mediators for which there is published data for a role in the antiangiogenic axis initiated by IL-12. For example, it has been reported that blocking CXCL10 abrogates the antiangiogenic effects of IL-12 within IL-12–treated tumors (42
). Thus, CXCL10 is a likely potential mediator of the indirect mechanism of IFN-γ on tumor vessel endothelial cells. To test the role of CXCL10 and other ligands of CXCR3, we injected B16 and B16/IL-12 into CXCR3−/−
mice. In these experiments, even in the absence of CXCR3 signaling, IL-12 was still able to downregulate VEGFR3 on the tumor vessels (data not shown), suggesting that the ligands of CXCR3, including CXCL10, are not essential for the suppression of VEGFR3. This is in line with our findings that, even when the indirect mechanism is blocked, IFN-γ can act directly on endothelial cells to down-regulate VEGFR3.
Regarding the direct mechanism of IFN-γ–mediated VEGFR3 suppression, it is possible that IFN-γ acts alone on tumor endothelial cells to suppress VEGFR3 by upregulating receptors known to suppress VEGFR3. Recent studies into VEGFR3 regulation have revealed a role for the Notch ligand Delta-like 4 (DLL4) and Notch1 in antiangiogenesis and VEGFR3 downregulation. Interestingly, although VEGF is a strongly proangiogenic factor, it has recently been shown to induce DLL4 in tip cells, the single specialized endothelial cell at the leading edge of a vessel sprout that can define the direction in which the newly formed vessel grows or if it grows at all. The upregulation of DLL4 in tip cells in turn suppresses sprouting in the adjacent endothelial cells through its interaction with Notch1 (44
). Interestingly, Notch signaling can suppress VEGFR3 on the surface of tumor vessels (34
). Therefore, it is possible that IFN-γ acts via endothelial Notch1 regulation; indeed, preliminary experiments in vitro, using an immortalized mouse endothelial cell line (SVEC), have indicated that IFN-γ can upregulate Notch1 mRNA in a CXCL10-independent fashion (data not shown). If IFN-γ has a role in the downregulation of endothelial Notch1, it could be responsible for the direct mechanism of IFN-γ–mediated VEGFR3 downregulation.
Vessels within tumors are known to be chaotic and poorly formed; however, the presence of IL-12 seems to have a normalizing effect on tumor vasculature morphology, as shown by a decrease in the number and the diameter of the tumor vessels, as well as a suppression of VEGFR3 (20
). The suppression of VEGFR3 on tumor vessels can improve tumor control in several ways. It may increase the efficacy of current angiogenic treatments such as Avastin (Genentech, San Francisco, CA). Because this treatment only blocks VEGF-A, and many types of tumors contain vessels that express VEGFR3, VEGF-C and VEGF-D within the tumor can continue to act in a proangiogenic manner. Indeed, even in the absence of VEGF-A, VEGFR2 can enhance VEGFR3 phosphorylation and signaling by heterodimerizing with it (48
). In addition, the outright blockade of VEGFR3 alone within the tumor results in decreased vessel formation and increased tumor control (28
). Aside from the positive effects yielded from VEGFR3 suppression alone, it is thought that overall vascular normalization could possibly lead to more potent tumor control. For example, blockade of VEGF leads to less leaky and dilated vessels, and increases pericyte coverage (18
). Therefore, although both anti-angiogenic therapies and local treatment of tumors with IL-12 cause a decrease in the number of vessels and may lead to more areas of hypoxia, the vessels that remain become more functional as they normalize. Although fewer in number, these normalized vessels could increase drug delivery to the tumor and may enhance lymphocyte function through relieving hypoxia, a condition that can suppress immune function in multiple ways. For example, hypoxia can decrease production of T cell prosurvival and procytotoxic cytokines (IL-2 and IFN-γ) and an induction of cAMP, which can interfere with the effector signaling pathways (50
). In addition, elevated tumor oxygenation can increase efficacy of radiation therapy because of the increase of oxygenation of the tumor cells themselves.
The concept of using the immune system to alter the vasculature is a novel one. Thus, promoting immunotherapy may have 2-fold effects of: 1) enhancing the immune response to recognize and eliminate tumor cells, and 2) suppressing angiogenesis, which not only slows tumor growth, but may also normalize vessels resulting in increased influx of immune cells, which will continue to have antiangiogenic effects on the vasculature. This cyclic process may result in potent antitumor effects.