Incorporation of d-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since d-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of d-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. d-[3-¹¹C]Alanine and the dipeptide d-[3-¹¹C]alanyl-d-alanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [¹¹C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Gram-positive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, d-[3-¹¹C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation. When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[¹⁸F]fluoro-d-glucose and a gallium citrate radiotracer, d-alanine showed more bacteria-specific uptake. Decreased d-[3-¹¹C]alanine uptake was also observed in antibiotic-sensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of d-[3-¹¹C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of d-[3-¹¹C]alanine to address a number of important human infections.

This study applied a dual-agent, ¹³C-pyruvate and ¹³C-urea, hyperpolarized ¹³C magnetic resonance spectroscopic imaging (MRSI) and multi-parametric (mp) ¹H magnetic resonance imaging (MRI) approach in the transgenic adenocarcinoma of mouse prostate (TRAMP) model to investigate changes in tumor perfusion and lactate metabolism during prostate cancer development, progression and metastases, and after lactate dehydrogenase-A (LDHA) knock-out. An increased Warburg effect, as measured by an elevated hyperpolarized (HP) Lactate/Pyruvate (Lac/Pyr) ratio, and associated Ldha expression and LDH activity were significantly higher in high- versus low-grade TRAMP tumors and normal prostates. The hypoxic tumor microenvironment in high-grade tumors, as measured by significantly decreased HP ¹³C-urea perfusion and increased PIM staining, played a key role in increasing lactate production through increased Hif1α and then Ldha expression. Increased lactate induced Mct4 expression and an acidic tumor microenvironment that provided a potential mechanism for the observed high rate of lymph node (86%) and liver (33%) metastases. The Ldha knockdown in the triple-transgenic mouse model of prostate cancer resulted in a significant reduction in HP Lac/Pyr, which preceded a reduction in tumor volume or apparent water diffusion coefficient (ADC). The Ldha gene knockdown significantly reduced primary tumor growth and reduced lymph node and visceral metastases. These data suggested a metabolic transformation from low- to high-grade prostate cancer including an increased Warburg effect, decreased perfusion, and increased metastatic potential. Moreover, these data suggested that LDH activity and lactate are required for tumor progression. The lactate metabolism changes during prostate cancer provided the motivation for applying hyperpolarized ¹³C MRSI to detect aggressive disease at diagnosis and predict early therapeutic response.

Purpose: The combined hyperpolarized (HP)¹³C pyruvate and urea MRI has provided a simultaneous assessment of glycolytic metabolism and tissue perfusion for improved cancer diagnosis and therapeutic evaluation in preclinical studies. This work aims to translate this dual‐probe HP imaging technique to clinical research.Methods: A co‐polarization system was developed where [1‐¹³C]pyruvic acid (PA) and [¹³C,¹⁵N₂]urea in water solution were homogeneously mixed and polarized on a 5T SPINlab system. Physical and chemical characterizations and toxicology studies of the combined probe were performed. Simultaneous metabolic and perfusion imaging was performed on a 3T clinical MR scanner by alternatively applying a multi‐slice 2D spiral sequence for [1‐¹³C]pyruvate and its downstream metabolites and a 3D balanced steady‐state free precession (bSSFP) sequence for [¹³C,¹⁵N₂]urea.Results: The combined PA/urea probe has a glass‐formation ability similar to neat PA and can generate nearly 40% liquid‐state¹³C polarization for both pyruvate and urea in 3‐4 h. A standard operating procedure for routine on‐site production was developed and validated to produce 40 mL injection product of approximately 150 mM pyruvate and 35 mM urea. The toxicology study demonstrated the safety profile of the combined probe. Dynamic metabolite‐specific imaging of [1‐¹³C]pyruvate, [1‐¹³C]lactate, [1‐¹³C]alanine, and [¹³C,¹⁵N₂]urea was achieved with adequate spatial (2.6 mm × 2.6 mm) and temporal resolution (4.2 s), and urea images showed reduced off‐resonance artifacts due to theJ_CNcoupling.Conclusion: The reported technical development and translational studies will lead to the first‐in‐human dual‐agent HP MRI study and mark the clinical translation of the first HP¹³C MRI probe after pyruvate.

Advanced prostate cancer represents the fifth leading cause of cancer death in men worldwide. Although androgen-receptor signaling is the major driver of the disease, evidence is accumulating that disease progression is supported by substantial metabolic changes. Alterations in de novo lipogenesis and fatty acid catabolism are consistently reported during prostate cancer development and progression in association with androgen-receptor signaling. Therefore, the term “lipogenic phenotype” is frequently used to describe the complex metabolic rewiring that occurs in prostate cancer. However, a new scenario has emerged in which lactate may play a major role. Alterations in oncogenes/tumor suppressors, androgen signaling, hypoxic conditions, and cells in the tumor microenvironment can promote aerobic glycolysis in prostate cancer cells and the release of lactate in the tumor microenvironment, favoring immune evasion and metastasis. As prostate cancer is composed of metabolically heterogenous cells, glycolytic prostate cancer cells or cancer-associated fibroblasts can also secrete lactate and create “symbiotic” interactions with oxidative prostate cancer cells via lactate shuttling to sustain disease progression. Here, we discuss the multifaceted role of lactate in prostate cancer progression, taking into account the influence of the systemic metabolic and gut microbiota. We call special attention to the clinical opportunities of imaging lactate accumulation for patient stratification and targeting lactate metabolism.