Metabolic reprogramming and validation of hyperpolarized 13C lactate as a prostate cancer biomarker using a human prostate tissue slice culture bioreactor

Abstract

Background: The treatment of prostate cancer has been impeded by the lack of both clinically relevant disease models and metabolic markers that track tumor progression. Hyperpolarized (HP) (13) C MR spectroscopy has emerged as a new technology to investigate the metabolic shifts in prostate cancer. In this study, we investigate the glucose reprogramming using HP (13) C pyruvate MR in a patient-derived prostate tissue slice culture (TSC) model.

Methods: The steady-state metabolite concentrations in freshly excised human prostate TSCs were assessed and compared to those from snap-frozen biopsy samples. The TSCs were then applied to a perfused cell (bioreactor) platform, and the bioenergetics and the dynamic pyruvate flux of the TSCs were investigated by (31) P and HP (13) C MR, respectively.

Results: The prostate TSCs demonstrated steady-state glycolytic and phospholipid metabolism, and bioenergetics that recapitulate features of prostate cancer in vivo. (13) C spectra following injection of HP (13) C pyruvate showed significantly increased pyruvate to lactate flux in malignant as compared to the benign prostate TSCs. This increased flux in the malignant prostate TSCs correlated with both increased expression of monocarboxylate transporters (MCT) and activity of lactate dehydrogenase (LDH).

Conclusions: We provide the first mechanistic evidence for HP (13) C lactate as a prostate cancer biomarker in living human tissues, critical for the interpretation of in vivo studies. More broadly, the clinically relevant metabolic model system in combination with HP MR can facilitate the identification of clinically translatable biomarkers of prostate cancer presence, aggressiveness, and treatment response.

Fig. 1

a: Change in glucose metabolism when comparing normal prostate glandular epithelial and malignant prostate cells. b: Representative in vivo T₂-weighted image and corresponding 3D ¹H MRSI array of a patient with Gleason 3 + 4 cancer in the right base of the prostate gland. Inlaid spectra corresponding to a normal and malignant voxel demonstrate the observed differences in metabolism for these regions. GLUTs, glucose transporters; MCT1,4, monocarboxylate transporters; ZIP1, zinc transporter, ACON, aconitase; OAA, oxaloacetate; αKG, α-ketogluterate; Pyr, pyruvate; Lac, lactate; LDH, lactate dehydrogenase.

Fig. 2

a: Representative ¹H HR-MAS spectra from benign and malignant snap-frozen human biopsies and TSCs post-2 hr of culture. b: Quantified metabolite ratios for benign biopsies and TSCs (N = 16 and 14, respectively) as well as malignant BY and TSC(N = 12 and 9, respectively) compared to PC-3, primary prostate cancer cells and VCaP cells (all N = 4). c: Representative hematoxylin- and eosin- (H&E) staining and (d) Ki-67 immunohistochemistry of TSCs. e: Average percent positive Ki-67 staining of TSCs as compared to the other prostate tissues and cells. Ala, alanine; Lac, lactate; Glut, glutamate; Cre, creatine; Pol, polyamines; PC + GPC, combined phosphocholine and glycero-phosphocholine; totCho, total choline. All ratios reported as mean ± SE.

Fig. 3

a: Representative 2D ¹H–¹³C HSQC HR-MAS NMR spectra of benign (N = 7) and malignant (N = 4) prostate TSCs post-incubation with 4 mM [3-¹³C] pyruvate for 2 hr. b: Fractional Enrichment of each metabolite after 2 hr of labeling in TSCs. All ratios reported as mean ± SE.

Fig. 4

a: ³¹P spectra from living malignant and benign TSCs perfused in the MR-compatible bioreactor. b: Timecourse of βNTP demonstrating preservation of ATP in the bioreactor. c: Ratios of metabolites to βNTP in benign and malignant TSCs (N = 4). d: Representative H&E staining of TSCs post-perfusion in the bioreactor. PCr, phosphocreatine; PC, phosphocholine; PE, phosphoethanolamine; GPC, glycerophosphocholine; α, β, γ-NTPs, nucleotide triphosphates. All ratios reported as mean ± SE. *P < 0.05.

Fig. 5

a: A single spectrum taken at 90 sec post-injection of HP [1-¹³C] pyruvate in the bioreactor with benign (N = 4) or malignant TSCs (N = 4). b: Average HP Lactate dynamics for TSCs. c: Total normalized lactate area under the curve (AUC) and (d) LDH activity for be nign and malignant TSCs. e: mRNA expression of lactate dehydrogenase A (LDHA), monocarboxylate transporters1 (MCT1) and 4 (MCT4). All plots are represented as mean ± SE.