Cancer cells are most clearly characterized by their abnormal and uncontrolled cell growth. One of the most notable theories that explains the vast proliferative capacity of tumorigenic cells is the Warburg effect, a significant shift in metabolism wherein cancer cells preferentially fuel cell division using aerobic glycolysis instead of aerobic respiration. This upregulation of glycolytic fermentation in aerobic environments is highly unusual - glycolysis is typically utilized in anaerobic conditions, but nonetheless dominates cancer metabolic activity in spite of the presence of oxygen. Since the discovery the Warburg effect in the 1920s, researchers have struggled to identify whether aerobic glycolysis is a cause or consequence of carcinogenesis. Interestingly, a new theory recently emerged that challenges this widely-accepted metabolic paradigm for cancer. Known as the reverse Warburg effect, this new mechanism shows that in carcinomas such as breast cancer, the Warburg effect occurs not in cancer cells, but rather in tumor-adjacent stromal fibroblasts. These cancer-associated fibroblasts (CAFs) in the greater tumor microenvironment produce lactate - a high-energy metabolite formed as a byproduct of aerobic glycolysis - to fuel aerobic respiration and rapid tumorigenesis in neighboring cancer cells. This emerging theory emphasizes the pivotal role of the tumor microenvironment in determining whether cancer cells undergo aerobic glycolysis or aerobic respiration. Central to this lactate-linked metabolic intersection are two critical enzymes that regulate a cell's metabolic commitment - lactate dehydrogenase (LDH) and pyruvate dehydrogenase complex (PDHc). In order to clarify the mechanisms through which CAFs induce tumorigenesis in breast cancer, we plan to carry out two specific aims: (1) evaluate the enzymatic activity of LDH and PDHc, and (2) compare LDH and PDHc enzyme content. Using co-culture techniques to study the breast cancer tumor microenvironment in vitro, we will compare the enzymatic activity and enzyme content of both MCF7 breast cancer cells and CAFs to identify whether the reverse Warburg effect occurs due to post-translational enzyme activation or increased enzyme synthesis.