PROJECT SUMMARY AND RELEVANCE Summary: A common feature of aggressively growing tumors is intratumoral hypoxia due to the insufficient blood supply. Tumor cells adapt to hypoxia by reprogramming their metabolism to rely on glucose fermentation as the main energetic pathway and, consequently, drastically increase their glucose intake. This glucose dependence of hypoxic tumor cells can be exploited by limiting total available glucose. The objective of the proposal is to test whether glucose limitation checks the proliferation of hypoxic, glycolytic tumors. A combination of a ketogenic diet with clinically relevant doses of metformin significantly reduced blood glucose levels in BALB/c mice without causing morbidity. This proposal will test the efficacy of the diet/metformin combination on the growth and metastatic outcome of orthotopically implanted, aggressively growing tumors in an established murine model of triple-negative breast cancer (TNBC). Guided by preliminary data, we aim to 1. Establish the efficacy of the diet/metformin combination therapy in inhibiting the progression of mammary tumors to metastasis, and 2. Determine the efficacy of standard chemotherapy with the proposed combination therapy to control the growth dynamics and progression to metastasis of developed mammary tumors. This approach shifts the emphasis of cancer treatment discovery from genetic pathway or protein targets, which tend to evolve and adapt to therapy, to a well-known and hard to escape metabolic vulnerability of cancer by using a combination diet/pharmaceutical therapy approach. While carbohydrate dietary restriction and metformin are well-known interventions, they have yet to be considered in combination for cancer treatment. Significantly, low carbohydrate diets and metformin have been shown to be compatible in humans outside of the cancer field. If effective, this treatment will be expanded to other hypoxic and glycolytic tumors beyond TNBC, particularly those tumors that are refractive to conventional therapies, with the ultimate goal of translating this treatment protocol into a human cancer therapy. The combination therapy may also prove to be a significant adjuvant to existing mainline treatments by debulking the hypoxic, typically chemo- and radio-resistant, part of a tumor. This AREA project will provide research opportunities for undergraduate students to gain hands-on experience in biomedical techniques and data analysis. The project is specifically designed to be straightforward and practical for pre-med undergraduate students. Relevance: Limited treatment options for TNBC represent a critical barrier in attempts to increase post- diagnosis patient survival rates. This project is a rational extension of a well-known paradigm of metabolic reprogramming of cancer cells and, if successful, can be translated into a mainline cancer treatment or used as an adjuvant in combination with existing treatment options.