<p> Prostate cancer is among the most prevalent and deadly of malignancies in both the United States and worldwide. Ongoing diagnostic challenges in prostate cancer include differentiating low-risk and high-risk tumors, and monitoring responses to therapy in patients with aggressive disease. Prostate cancer metabolism is characterized by a shift to aerobic glycolysis with lactate production and efflux, as well as increased tricarboxylic acid cycle activity, which has led to the investigation and development of metabolic imaging strategies such as hyperpolarized 13C MRI. However, it is nontrivial to study human prostate cancer metabolism in vivo, and the capability to better characterize tumor metabolism from a variety of disease states would be valuable for metabolic imaging biomarker development. This dissertation focuses on developing ex vivo strategies to measure metabolism in benign and malignant living human prostate tissue. First, because prostate tissue heterogeneity can impact metabolic measurements, we present the engineering of a 600 MHz radiofrequency (RF) microcoil to assess the heterogeneity of freshly acquired human prostate biopsies using microscale diffusion-weighted imaging (DWI). Next, we demonstrate the capability of micro-DWI to determine the biopsy percentage of glandular tissue, setting the stage for establishing the percentage and grade of cancer using this approach. After this, we develop a protocol for nuclear magnetic resonance (NMR) quantification of lactate production and efflux and glutamate fractional enrichment in freshly acquired living human prostate biopsies cultured with [1,6-13C2]glucose. In this study we demonstrate a significantly higher lactate efflux rate coming from low-grade prostate cancer versus benign biopsies in an early-stage patient population. This sets the stage for studies of metabolic fluxes and steady-state metabolite levels in biopsies from patients with aggressive disease before and after non-surgical therapy. Finally, due to recent interest in the potential role of Myc amplification and glutaminolysis upregulation in treatment insensitive castrate-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC), we present metabolic labeling results from a study of primary human prostate tissue slice cultures (TSCs) obtained at surgery and cultured with either [1,6-13C2]glucose or [3-13C]glutamine. Our results are consistent with prior thinking on the role of glucose and glutamine metabolism in treatment-na&iuml;ve prostate cancer.</p><p>