The detection and treatment monitoring of inflammatory states remain challenging in part due to the multifactorial mechanisms of immune activation and spectrum of clinical manifestations. Currently, diagnostic strategies tend to be subjective and limited quantitative tools exist to monitor optimal treatment strategies. Pro-inflammatory M1 polarized macrophages exhibit a distinct metabolic glycolytic phenotype compared to the continuum of M2 polarization states. In the present study, the distinct metabolic phenotypes of resting and activated macrophages were successfully characterized and quantified using hyperpolarized carbon-13 (¹³C) labeled pyruvate and its metabolic products, i.e. lactate, as a biomarker of resting, disease and treated states. Methods: Mouse macrophage J774A.1 cells were used as a model system in an NMR compatible bioreactor to facilitate dynamic hyperpolarized ¹³C measurements. The glycolytic metabolism of the cells in the quiescent or resting state were compared with macrophages stimulated by lipopolysaccharide, a classical M1 activator using hyperpolarized ¹³C labeled pyruvate. Additionally, the activated macrophages were also treated with a non-steroidal anti-inflammatory drug to assess the changes in hyperpolarized lactate signal. The hyperpolarized lactate signals were then correlated using biochemical and molecular assays. Results: We first validated our model system of inflammatory cells by the hallmarks of M1 polarization using steady state metabolic profiling with high resolution NMR in conjunction with nitric oxide Greiss assay, enzyme activity, and mRNA expression. Subsequently, we clearly showed that the cutting edge technology of hyperpolarized ¹³C NMR can be used to detect elevated lactate levels in M1 polarized macrophages in comparison to control and non-steroidal anti-inflammatory drug treated M2 states. Conclusion: Hyperpolarized ¹³C lactate has the potential to serve as a biomarker to non-invasively detect and quantify pro-inflammatory state of immune regulatory cells and its response to therapy.

Purpose

Image-guided prostate biopsies are routinely acquired in the diagnosis and treatment monitoring of prostate cancer, yielding useful tissue for identifying metabolic biomarkers and therapeutic targets. We developed an optimized biopsy tissue culture protocol in combination with [1,6-¹³ C₂ ]glucose labeling and quantitative high-resolution NMR to measure glycolysis and tricarboxcylic acid (TCA) cycle activity in freshly acquired living human prostate biopsies.

Methods

We acquired 34 MRI-ultrasound fusion-guided prostate biopsies in vials on ice from 22 previously untreated patients. Within 15 min, biopsies were transferred to rotary tissue culture in 37°C prostate medium containing [1,6-¹³ C₂ ]glucose. Following 24 h of culture, tissue lactate and glutamate pool sizes and fractional enrichments were quantified using quantitative ¹ H high resolution magic angle spinning Carr-Purcell-Meiboom-Gill (CPMG) spectroscopy at 1°C with and without ¹³ C decoupling. Lactate effluxed from the biopsy tissue was quantified in the culture medium using quantitative solution-state high-resolution NMR.

Results

Lactate concentration in low-grade cancer (1.15 ± 0.78 nmol/mg) and benign (0.74 ± 0.15 nmol/mg) biopsies agreed with prior published measurements of snap-frozen biopsies. There was substantial fractional enrichment of [3-¹³ C]lactate (≈70%) and [4-¹³ C]glutamate (≈24%) in both low-grade cancer and benign biopsies. Although a significant difference in tissue [3-¹³ C]lactate fractional enrichment was not observed, lactate efflux was significantly higher (P < 0.05) in low-grade cancer biopsies (0.55 ± 0.14 nmol/min/mg) versus benign biopsies (0.31 ± 0.04 nmol/min/mg).

Conclusion

A protocol was developed for quantification of lactate production-efflux and TCA cycle activity in single living human prostate biopsies, allowing metabolic labeling on a wide spectrum of human tissues (e.g., metastatic, post-non-surgical therapy) from patients not receiving surgery.

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) ¹³ C dehydroascorbate (DHA) and ¹³ C pyruvate magnetic resonance spectroscopy (MRS) to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline and 7 days after unilateral ischemia reperfusion injury (IRI). Compared with the contralateral sham-operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP ¹³ C vitamin C/(vitamin C + DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP ¹³ C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP ¹³ C lactate/pyruvate ratio at day 7 compared with baseline, although the ¹³ C lactate/pyruvate ratio was not significantly different between the IRI and contralateral sham-operated kidneys at day 7. Arterial spin labeling magnetic resonance imaging (MRI) demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS) and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP ¹³ C MRS for the non-invasive assessment of oxidative stress and mitochondrial PDH activity following renal IRI.

Non-invasive assessment of the biological aggressiveness of prostate cancer (PCa) is needed for men with localized disease. Hyperpolarized (HP) ¹³C magnetic resonance (MR) spectroscopy is a powerful approach to image metabolism, specifically the conversion of HP [1-¹³C]pyruvate to [1-¹³C]lactate, catalyzed by lactate dehydrogenase (LDH). Significant increase in tumor lactate was measured in high-grade PCa relative to benign and low-grade cancer, suggesting that HP ¹³C MR could distinguish low-risk (Gleason score ≤3 + 4) from high-risk (Gleason score ≥4 + 3) PCa. To test this and the ability of HP ¹³C MR to detect these metabolic changes, we cultured prostate tissues in an MR-compatible bioreactor under continuous perfusion. ³¹P spectra demonstrated good viability and dynamic HP ¹³C-pyruvate MR demonstrated that high-grade PCa had significantly increased lactate efflux compared to low-grade PCa and benign prostate tissue. These metabolic differences are attributed to significantly increased LDHA expression and LDH activity, as well as significantly increased monocarboxylate transporter 4 (MCT4) expression in high- versus low- grade PCa. Moreover, lactate efflux, LDH activity, and MCT4 expression were not different between low-grade PCa and benign prostate tissues, indicating that these metabolic alterations are specific for high-grade disease. These distinctive metabolic alterations can be used to differentiate high-grade PCa from low-grade PCa and benign prostate tissues using clinically translatable HP [1-¹³C]pyruvate MR.

New magnetic resonance (MR) molecular imaging techniques offer the potential for noninvasive, simultaneous quantification of metabolic and perfusion parameters in tumors. This study applied a three-dimensional dynamic dual-agent hyperpolarized ¹³C magnetic resonance spectroscopic imaging approach with ¹³C-pyruvate and ¹³C-urea to investigate differences in perfusion and metabolism between low- and high-grade tumors in the transgenic adenocarcinoma of mouse prostate (TRAMP) transgenic mouse model of prostate cancer. Dynamic MR data were corrected for T1 relaxation and RF excitation and modeled to provide quantitative measures of pyruvate to lactate flux (k_PL ) and urea perfusion (urea AUC) that correlated with TRAMP tumor histologic grade. k_PL values were relatively higher for high-grade TRAMP tumors. The increase in k_PL flux correlated significantly with higher lactate dehydrogenase activity and mRNA expression of Ldha, Mct1, and Mct4 as well as with more proliferative disease. There was a significant reduction in perfusion in high-grade tumors that associated with increased hypoxia and mRNA expression of Hif1α and Vegf and increased k_trans , attributed to increased blood vessel permeability. In 90% of the high-grade TRAMP tumors, a mismatch in perfusion and metabolism measurements was observed, with low perfusion being associated with increased k_PL This perfusion-metabolism mismatch was also associated with metastasis. The molecular imaging approach we developed could be translated to investigate these imaging biomarkers for their diagnostic and prognostic power in future prostate cancer clinical trials. Cancer Res; 77(12); 3207-16. ©2017 AACR.

Currently, no clinical methods reliably predict the development of castration-resistant prostate cancer (CRPC) that occurs almost universally in men undergoing androgen deprivation therapy. Hyperpolarized (HP) ¹³C magnetic resonance imaging (MRI) could potentially detect the incipient emergence of CRPC based on early metabolic changes. To characterize metabolic shifts occurring upon the transition from androgen-dependent to castration-resistant prostate cancer (PCa), the metabolism of [U-¹³C]glucose and [U-¹³C]glutamine was analyzed by nuclear magnetic resonance spectroscopy. Comparison of steady-state metabolite concentrations and fractional enrichment in androgen-dependent LNCaP cells and transgenic adenocarcinoma of the murine prostate (TRAMP) murine tumors versus castration-resistant PC-3 cells and treatment-driven CRPC TRAMP tumors demonstrated that CRPC was associated with upregulation of glycolysis, tricarboxylic acid metabolism of pyruvate; and glutamine, glutaminolysis, and glutathione synthesis. These findings were supported by ¹³C isotopomer modeling showing increased flux through pyruvate dehydrogenase (PDH) and anaplerosis; enzymatic assays showing increased lactate dehydrogenase, PDH and glutaminase activity; and oxygen consumption measurements demonstrating increased dependence on anaplerotic fuel sources for mitochondrial respiration in CRPC. Consistent with ex vivo metabolomic studies, HP [1-¹³C]pyruvate distinguished androgen-dependent PCa from CRPC in cell and tumor models based on significantly increased HP [1-¹³C]lactate.