IL-12 has been shown to be effective in enhancing antitumor responses. However, how IL-12 exerts its antiangiogenic effect is largely unknown. In this study, we elucidate this mechanism using B16 transfected to express IL-12 (B16/IL-12), a system that provides constant, local production of IL-12 within the tumor microenvironment. Intratumoral IL-12 resulted in a significant delay in tumor growth and phenotypic changes in the vasculature. Vessels found within B16 tumors are chaotic and poorly formed and express vascular endothelial growth factor receptor 3 (VEGFR3), a growth factor receptor not expressed on normal adult vessels. However, the vessels within B16/IL-12 tumors have a more normal morphology and do not express VEGFR3. We have shown that IFN-γ is required for IL-12 to suppress the aberrant expression of VEGFR3. Indeed, the presence of intratumoral IL-12 stimulates the immune system resulting in more IFN-γ–producing tumor-infiltrating lymphocytes per tumor when compared with parental B16 tumors, which may have a marked effect on control of tumor growth. Interestingly, within B16/IL-12 tumors, T cells are necessary to suppress VEGFR3 expression on tumor vessels. Finally, using IFN-γ receptor knockout mice in a bone marrow chimera system, we show that the IFN-γ produced within the tumor suppresses VEGFR3 expression in two ways: 1) acting directly on tumor vessel endothelial cells, and 2) acting on the tumor-infiltrating lymphocytes to indirectly alter endothelial cells’ VEGFR3 expression. Our data indicate a mechanism in which tumor-infiltrating immune cells regulate tumor vessel phenotype.

Tumor hypoxia occurs because of an increased demand for oxygen by the rapidly growing tumor cells, together with reduction in the oxygen supply due to malformed and nonfunctional tumor vasculature. The effects of tumor hypoxia on radiotherapy (RT) are well known; however, recent findings suggest it may also suppress immunotherapy, although the mechanisms governing this observation remain undetermined. Our laboratory and others have shown that IFN-γ conditions the tumor milieu and is important for the efficacy of RT. Thus, we hypothesized that hypoxia could inhibit IFN-γ–mediated antitumor responses, resulting in decreased RT efficacy. This inhibition could involve the production and/or the cellular response to IFN-γ. To test this, we used murine tumor cell lines B16F0 and Colon38. We observed that hypoxia inhibited upregulation of IFN-γ–dependent MHC class I expression by tumor cells along with the gene expression of IFN-γ–dependent chemokines CXCL9 and CXCL10, essential for immune cell infiltration. Furthermore, CD8⁺ T cells, an important source of IFN-γ, which mediate effector antitumor responses, had reduced ability to proliferate and generate IFN-γ under hypoxic conditions in vitro. Interestingly, reoxygenation restored the cytokine-producing capability of these cells. Studies performed in vivo using a mouse tumor model and the hypoxia marker EF5 demonstrated that RT could reverse the hypoxia within treated tumors. This study has identified a unique mechanism of hypoxia-induced immune suppression involving the downregulation of IFN-γ production and cellular responsiveness to this essential cytokine. These results suggest that therapies that target and reduce tumor hypoxia can potentially boost antitumor immune responses.

New blood vessel formation within tumours is a critical feature for tumour growth. A major limitation in understanding this complex process has been the inability to visualise and analyse vessel formation. Here, we report on the development of a whole-tissue mount technique that allows visualisation of vessel structure. Mice expressing green fluorescent protein (GFP) made it possible to easily see GFP⁺ vessels within non-GFP-expressing B16 melanoma tumours. The small fragments of tumour used in this technique were also effectively stained with fluorescent probe-conjugated antibodies, allowing characterisation of the vessels based on surface marker phenotype. The vessels within tumour tissue were much more irregular and tortuous compared to those within surrounding normal muscle. B16 tumours stably transfected with the genes for IL-12 were used to assess the effects of this cytokine on tumour growth and vessel formation. The IL-12-expressing tumours grew more slowly and had much smaller blood vessels than the large, webbed vessels characteristic of the parental tumours, effects that were dependent on interferon gamma (IFN-γ). Vessels in the parental tumours were found to express VEGFR-3, the receptor for VEGF-C and VEGF-D. Expression of this receptor by the endothelial cells of the blood vessels was lost in the cytokine expressing tumours, thus suggesting a mechanism for the antiangiogenic effects of IL-12. The combination of the whole mount technique and the GFP transgenic mice provides a powerful method for visualising tumour vasculature and characterising the effects of agents such as cytokines.