Obesity is associated with a poorer prognosis following diagnosis of breast cancer, including greater tumor burden and higher-grade tumors. The mechanism by which obesity can promote metastasis of breast cancer remains to be determined. Obesity is also associated with aberrant cytokine production and a chronic inflammatory state, characterized by the recruitment of macrophages into the adipose tissue, which have been shown to promote breast cancer progression. Therefore, we hypothesize that adipocytes and macrophages cooperate to promote breast cancer metastasis.

To start testing this hypothesis, we developed a set of specific aims. We began by assessing and validating THP-1 as a model human macrophage system. First, we optimized conditions for differentiating and treating the cells and then attempted to replicate the activation states described in literature as M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes. We found it difficult to reproduce results from the literature regarding this paradigm and chose to focus on well characterized regulators of tumor metastasis shown to be produced by macrophages, matrix metalloproteinase 9 (MMP9), transforming growth factor beta (TGF-?), and vascular endothelial growth factor (VEGF).

We then set up a system to successfully test the effects of adipocyte exposure on macrophage phenotype. First, we optimized the differentiation of the primary human adipocytes and then developed a protocol for co-culturing them with the macrophages. To determine whether adipocytes could promote a pro-tumor phenotype in macrophages we chose a Transwell system to co-culture the cells, then analyzed macrophage mRNA and protein for changes in metastatic markers. Our preliminary data showed trends toward increases in macrophage production of MMP9, TGF-beta, and VEGF. We then switched from visceral adipocytes to breast adipocytes in order to more closely model the breast tumor microenvironment. With the breast adipocyte co-culture, we again observed trends toward increases in macrophage production of MMP9 and TGF-beta.

Next, we tried to determine if macrophages are critical mediators of the impact of obesity and adipocytes in breast cancer metastasis. We found that macrophages exposed to adipocytes produce significantly increased amounts of the pro-angiogenic protein VEGF. Although increased VEGF expression is important for primary breast tumor growth, it is also a mechanism through which obesity could promote breast cancer metastasis. We then confirmed this macrophage phenotypic change in a murine cell culture system and found that VEGF production significantly increased in adipocyte-exposed macrophages using the same indirect Transwell co-culture system. We also attempted to replicate the dysfunctional state of adipocytes normally found in obesity by pretreating our adipocytes with cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), which have been found at elevated levels in obese adipose tissue, and observed greater increases in VEGF production in the human system, but not significantly more than without the cytokine pretreatment.

Then we attempted to identify potential mediators produced by the adipocytes that could induce VEGF expression in macrophages. Exploring the adipocyte-derived signals that promote VEGF expression in co-cultured macrophages, potential signaling molecules insulin, leptin, IL-6, and TNF-alpha were evaluated. Macrophages were treated with potential secreted mediators and analyzed for changes in VEGF transcription. We found that insulin, leptin, TNF-alpha, and IL-6 significantly increased macrophage VEGF mRNA expression. However, the experiments did not include proper positive controls or independent verification of potential mediator effectiveness and follow up experiments examining secretion of VEGF protein by macrophages treated with these potential mediators remain to be conducted.

We then wished to evaluate and characterize our adipocyte-exposed macrophages in terms of their changes in gene expression patterns as well as phenotype in relation to the common paradigm. We examined the phenotype of our adipocyte-exposed macrophages in relation to the M1/M2 definitions found in the literature and believe that they may have characteristics of both activation states. But no conclusions can be drawn until the corresponding protein levels of the mRNA transcripts are evaluated for changes in expression to prove that adipocyte-exposed macrophages are producing potential biologically significant molecules. To examine the functional effects of adipocyte-exposed macrophage-derived VEGF on an in vitro model of angiogenesis, we employed a human umbilical vein endothelial cell (HUVEC) tube formation assay. Endothelial cells in conditioned media from adipocyte-exposed macrophages showed significant increases in angiogenesis-related parameters when compared to those in conditioned media from macrophages not exposed to adipocytes. (Abstract shortened by ProQuest.).