Mechanisms of Vascular and Immune Alteration via Local Cytokine Treatment of Melanoma.

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2010.One of the earliest immunotherapies for the treatment of cancer involved the systemic administration of cytokines, and interestingly, both metastatic melanoma and renal cell carcinoma are particularly responsive to cytokine-based immunotherapies. However, although many cytokine treatments have been shown to be efficacious in animal models and in human clinical trials, the toxicity caused by high doses given systemically has been a barrier to their use in common clinical practice. The work presented in this thesis utilizes B16, a spontaneous mouse melanoma cell line, transfected to express the cytokines of interest (IL-12 or IL-2) to provide a system of constant, local production of cytokine within the tumor; a treatment modality that has been associated with increased anti-tumor response and decreased toxicity in both mice and humans. In this way, we have been able to elucidate the mechanisms by which these cytokines suppress tumor growth. IL-12 has been shown to be effective in enhancing anti-tumor responses by enhancing immunity and by limiting angiogenesis. However, how IL-12 exerts its anti-angiogenic effect is largely unknown. The majority of solid tumor vessels are chaotic and poorly formed, frequently expressing vascular endothelial growth factor receptor 3 (VEGFR3), a growth factor receptor not expressed on normal adult vessels. However, the vessels within tumors established using B16 cells transfected to express IL-12 (B16/IL-12) have a more normal morphology and do not express VEGFR3. We have shown that IFN- , is required for IL-12 to suppress the aberrant expression of VEGFR3. Further, within B16/IL-12 tumors, T cells are necessary to suppress VEGFR3 expression on tumor vessels. Finally, using IFN-, receptor knockout mice in a bone marrow chimera system, we have shown that the IFN- 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 (TILs) to indirectly alter endothelial cells’ VEGFR3 expression. The indirect mechanism was shown to require CXCR3 expression on the immune cells themselves, indicating that a quaternary mediator, downstream of CXCR3 signaling, is required to suppress VEGFR3 on tumor vessels in the absence of direct IFN- signaling. In addition to the alteration of VEGFR3 on the tumor vessel, we found that B16/IL-12 displayed a significant delay in tumor growth and an upregulation of vascular cell adhesion molecule-1 (VCAM-1). This upregulation required IFN- produced primarily by T cells, which then acted directly on the tumor vessel endothelial cells. Indeed, the presence of intratumoral IL-12 stimulated the immune system, resulting in an increased number of IFN--producing TILs per tumor when compared to parental B16 tumors, which may have a marked effect on control of tumor growth. Our data indicate a mechanism in which tumor infiltrating immune cells regulate tumor vessel phenotype. IL-2, the other cytokine focused on in this study, is a T cell derived growth factor, which has a major role in activating many immune cell subsets, including cytotoxic T cells, natural killer cells, and lymphokine-activated killer cells. Although in clinical trials, it has led to some patients having long-lasting remissions in both melanoma and renal cell carcinoma, it may have a role in stimulating immunosuppressive T regulatory cell development, confounding its role as an anti-tumor agent. Here, we show the significant difference that treatment dose has on the anti-tumor potential of IL-2. Transfecting B16 cells to express a low dose of IL-2 led to a small delay in tumor growth and an increase in tumor infiltrating immune cells, but no long-lasting tumor control. Conversely, using a cell line transfected to produce high doses of IL-2 (B16/IL-2.4) led to lasting tumor control and no palpable tumor growth. Because the high dose IL-2 led to a more significant therapeutic benefit, we focused solely on the mechanism by which IL-2 suppresses tumor growth within B16/IL-2.4 and its effect on the tumor-infiltrating immune cells. There was a significant influx of both NK and T cells as early as day 4 post-injection, and although T cells seem to be required for tumor control, IFN- was not, indicating that the tumor cell suppression was achieved via direct cytotoxic effects or a tumor-suppressing mediator produced by tumor-infiltrating T cells. Cell proliferation experiments indicate that high doses of IL-2 suppress tumor cell division at very early time points after injection. In addition, although no tumor growth was seen unless the tumor cells stopped producing IL-2, small tumor cell foci persisted in the area of injection indicating tumor cell dormancy. These data represent a novel mechanism of dose-dependent IL-2-mediated tumor control, which may lead to insights regarding the treatment of human metastatic melanoma. This work has studied multiple mechanisms by which local cytokine treatment alters the tumor microenvironment and controls tumor growth, with the ultimate goal of understanding how immunotherapies can be better utilized to more effectively treat cancer patients