NMR quantification of lactate production and efflux and glutamate fractional enrichment in living human prostate biopsies cultured with [1,6‐¹³C₂]glucose

Abstract

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 [1,6‐13C2]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 [1,6‐13C2]glucose. Following 24 h of culture, tissue lactate and glutamate pool sizes and fractional enrichments were quantified using quantitative 1H high resolution magic angle spinning Carr‐Purcell‐Meiboom‐Gill (CPMG) spectroscopy at 1°C with and without 13C 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 [3‐13C]lactate (≈70%) and [4‐13C]glutamate (≈24%) in both low‐grade cancer and benign biopsies. Although a significant difference in tissue [3‐13C]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.

Metabonomic Investigation of Biological Effects of a New Vessel Target Protein tTF-pHLIP in a Mouse Model

“…The re-balancing of the levels of TCA cycle intermediates and enzymes in the post-treatment mice is therefore a metabolic reflection of the reduced tumor size observed in mice treated with tTF-pHLIP (Figures A and E). Energy disruption associated with tumor growth also presents with rewired glycolysis, also known as Warburg effect, and is the most important metabolic alteration which rapidly consumes glucose and secret abundant lactate . Our current study observed an elevated level of lactate in plasma, urine, and liver in tumor-bearing mice, which were consistent with the Warburg effect of tumors.…”

Developments in proton MR spectroscopic imaging of prostate cancer

“…Increased signals of lactate, commonly observed for tumors because of high glycolysis (i.e., Warburg effect), are seen in 1 H MR spectra of PCa tissue in vitro [ 49 , 76 ], but these signals appear to be under the detection limit (~1.5 mM) of in vivo 3T MRSI of tumor lesions in the prostate recorded at TE = 144 ms [ 77 ]. Furthermore, the methyl lactate signal, if present, is easily obscured by signals of lipids resonating at a similar chemical shift or suppressed together with these signals, a common procedure embedded in most acquisition sequences.…”

High‐resolution magic angle spinning NMR for intact biological specimen analysis: Initial discovery, recent developments, and future directions

“…These improvements range from early, slow spinning schemes aimed at better preserving tissue architecture during HRMAS analysis to further subsequent pathology evaluations, 20 to recently reported schemes for more comprehensively defining disease metabolomic profiles by diffusion‐filtered 93 and heteronuclear single quantum correlation (HSQC) 94 spectroscopy. HRMAS NMR has also been developed to validate patient‐derived tumor models in a hypoxic microenvironment by evaluating functional, genomic, and metabolomics data, 43 as well as to quantify lactate production from living human prostate biopsies cultured with [1,6‐ 13 C]glucose 95 …”