Real‐time measurement of hyperpolarized lactate production and efflux as a biomarker of tumor aggressiveness in an MR compatible 3D cell culture bioreactor

We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) 13C MR and interrogated HP [1‐13C]lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP 13C MR studies were performed on three immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC, and UOK262, an aggressive and metastatic RCC. The intra‐ (Lacin) and extracellular (Lacex) HP lactate signals were robustly resolved in dynamic 13C spectra of the cell lines due to a very small but reproducible chemical shift difference (0.031 ± 0.0005 ppm). Following HP [1‐13C]pyruvate delivery, the ratio of HP Lacin/Lacex was significantly lower for UOK262 cells compared with both UMRC6 and HK2 cells due to a significant (p < 0.05) increase in the Lacex pool size. Lacin/Lacex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lacex pool size. The extension of these studies to living patient‐derived RCC tissue slices using HP [1,2‐13C2]pyruvate demonstrated a similarly split lactate doublet with a high Lacex pool fraction; in contrast, only a single NMR resonance is noted for HP [5‐13C]glutamate, consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export. Copyright © 2015 John Wiley & Sons, Ltd.

[1]  Josef Pfeuffer,et al.  Detection of intracellular lactate with localized diffusion {1H-13C}-spectroscopy in rat glioma in vivo. , 2005, Journal of magnetic resonance.

[2]  W. Bubb,et al.  pH and cell volume effects on H2O and phosphoryl resonance splitting in rapid-spinning NMR of red cells. , 2007, Biophysical journal.

[3]  Franz Schilling,et al.  In vivo measurement of apparent diffusion coefficients of hyperpolarized 13C‐labeled metabolites , 2014, NMR in biomedicine.

[4]  Albert P. Chen,et al.  Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive biomarkers for prostate cancer detection and grading. , 2008, Cancer research.

[5]  J. Kurhanewicz,et al.  Evaluation of the ERETIC method as an improved quantitative reference for 1H HR‐MAS spectroscopy of prostate tissue , 2009, Magnetic resonance in medicine.

[6]  H. Grossman,et al.  Human renal carcinoma: Characterization of five new cell lines , 1985, Journal of surgical oncology.

[7]  A. Schousboe,et al.  Cellular Distribution and Kinetic Properties of High-Affinity Glutamate Transporters , 1998, Brain Research Bulletin.

[8]  David M. Wilson,et al.  Chemistry and biochemistry of 13C hyperpolarized magnetic resonance using dynamic nuclear polarization. , 2014, Chemical Society reviews.

[9]  L. de Geus-Oei,et al.  Differences in metabolism between adeno- and squamous cell non-small cell lung carcinomas: spatial distribution and prognostic value of GLUT1 and MCT4. , 2012, Lung cancer.

[10]  R. Gillies,et al.  Dimethyl methylphosphonate (DMMP): a 31P nuclear magnetic resonance spectroscopic probe of intracellular volume in mammalian cell cultures. , 1993, Biochemistry.

[11]  Wouter Olthuis,et al.  Lactate biosensors: current status and outlook , 2013, Analytical and Bioanalytical Chemistry.

[12]  W. Guder,et al.  Renal substrate metabolism. , 1986, Physiological reviews.

[13]  G. Radda,et al.  In Vivo hyperpolarized carbon‐13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an insulin‐resistant mouse model , 2013, Hepatology.

[14]  R. Zager,et al.  HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. , 1994, Kidney international.

[15]  A. Longatto-Filho,et al.  Monocarboxylate Transporters 1 and 4 Are Associated with CD147 in Cervical Carcinoma , 2009, Disease markers.

[16]  Rinaldo Bellomo,et al.  Bench-to-bedside review: Lactate and the kidney , 2002, Critical care.

[17]  Karl J. Dykema,et al.  Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target , 2012, The Journal of pathology.

[18]  N. Danbolt.  Glutamate uptake , 2001, Progress in Neurobiology.

[19]  E. Repasky,et al.  Characterization of monocarboxylate transport in human kidney HK-2 cells. , 2006, Molecular pharmaceutics.

[20]  C Boesch,et al.  Dipolar coupling and ordering effects observed in magnetic resonance spectra of skeletal muscle , 2001, NMR in biomedicine.

[21]  W. Bubb,et al.  Chemical shift and magnetic susceptibility contributions to the separation of intracellular and supernatant resonances in variable angle spinning NMR spectra of erythrocyte suspensions , 2004, Magnetic resonance in medicine.

[22]  John Kurhanewicz,et al.  Hyperpolarized 13C spectroscopy and an NMR‐compatible bioreactor system for the investigation of real‐time cellular metabolism , 2010, Magnetic resonance in medicine.

[23]  M. Casal,et al.  Role of monocarboxylate transporters in human cancers: state of the art , 2012, Journal of Bioenergetics and Biomembranes.

[24]  J. Kurhanewicz,et al.  Hyperpolarized 13C-pyruvate magnetic resonance reveals rapid lactate export in metastatic renal cell carcinomas. , 2013, Cancer research.

[25]  Kristen Scott,et al.  Hyperpolarized 13C MR spectroscopic imaging can be used to monitor Everolimus treatment in vivo in an orthotopic rodent model of glioblastoma , 2012, NeuroImage.

[26]  M. Weiner,et al.  31P-NMR in vivo measurement of renal intracellular pH: effects of acidosis and K+ depletion in rats. , 1986, The American journal of physiology.

[27]  Franziska Hirschhaeuser,et al.  Lactate: a metabolic key player in cancer. , 2011, Cancer research.

[28]  John Kurhanewicz,et al.  Analysis of cancer metabolism by imaging hyperpolarized nuclei: prospects for translation to clinical research. , 2011, Neoplasia.

[29]  Albert P. Chen,et al.  Probing Early Tumor Response to Radiation Therapy Using Hyperpolarized [1-13C]pyruvate in MDA-MB-231 Xenografts , 2013, PloS one.

[30]  Kevin Brindle,et al.  New approaches for imaging tumour responses to treatment , 2008, Nature Reviews Cancer.

[31]  Ilwoo Park,et al.  Kinetic modeling of hyperpolarized 13C1-pyruvate metabolism in normal rats and TRAMP mice. , 2010, Journal of magnetic resonance.

[32]  John Kurhanewicz,et al.  Metabolic Reprogramming and Validation of Hyperpolarized 13C Lactate as a Prostate Cancer Biomarker Using a Human Prostate Tissue Slice Culture Bioreactor , 2013, The Prostate.

[33]  P. Larson,et al.  Metabolic Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-13C]Pyruvate , 2013, Science Translational Medicine.

[34]  K. Brindle,et al.  Detection of transgene expression using hyperpolarized 13C urea and diffusion‐weighted magnetic resonance spectroscopy , 2015, Magnetic resonance in medicine.

[35]  L. Carpenter,et al.  The kinetics, substrate and inhibitor specificity of the lactate transporter of Ehrlich-Lettre tumour cells studied with the intracellular pH indicator BCECF. , 1994, The Biochemical journal.

[36]  J. Pouysségur,et al.  Disrupting proton dynamics and energy metabolism for cancer therapy , 2013, Nature Reviews Cancer.

[37]  O. Feron,et al.  Pyruvate into lactate and back: from the Warburg effect to symbiotic energy fuel exchange in cancer cells. , 2009, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[38]  C. James,et al.  Detection of early response to temozolomide treatment in brain tumors using hyperpolarized 13C MR metabolic imaging , 2011, Journal of magnetic resonance imaging : JMRI.

[39]  S. Bröer,et al.  The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells. , 2000, The Biochemical journal.

[40]  P. Porporato,et al.  Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis. , 2012, Current pharmaceutical design.

[41]  J. Kurhanewicz,et al.  Metabolic response of prostate cancer to nicotinamide phophoribosyltransferase inhibition in a hyperpolarized MR/PET compatible bioreactor , 2015, The Prostate.

[42]  P. Kuchel,et al.  CHAPTER 12 - Rates of anion transfer across erythrocyte membranes measured with NMR spectroscopy , 1992 .

[43]  Robert J Gillies,et al.  Acidity generated by the tumor microenvironment drives local invasion. , 2013, Cancer research.

[44]  M. Dewhirst,et al.  Tumor metabolism of lactate: the influence and therapeutic potential for MCT and CD147 regulation. , 2010, Future oncology.

[45]  E. T. Gawlinski,et al.  Acid-mediated tumor invasion: a multidisciplinary study. , 2006, Cancer research.

[46]  T. Seyfried,et al.  Cancer as a metabolic disease , 2010, Nutrition & metabolism.

[47]  R K Jain,et al.  Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo. , 2001, Cancer research.

[48]  W. Hull,et al.  Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays , 2005, Magnetic Resonance Materials in Physics, Biology and Medicine.

[49]  A. Lardner.  The effects of extracellular pH on immune function , 2001, Journal of leukocyte biology.

[50]  R. Nolley,et al.  Tissue slice grafts of human renal cell carcinoma: an authentic preclinical model with high engraftment rate and metastatic potential. , 2014, Urologic oncology.

[51]  P. Kuchel,et al.  Exchange of fluorinated glucose across the red-cell membrane measured by 19F-n.m.r. magnetization transfer. , 1990, The Biochemical journal.

[52]  H. Mackay,et al.  Targeting the protein kinase C family: are we there yet? , 2008, Nature Reviews Cancer.

[53]  M. Mccarty,et al.  Manipulating tumor acidification as a cancer treatment strategy. , 2010, Alternative medicine review : a journal of clinical therapeutic.

[54]  A. Haase,et al.  Diffusion of hyperpolarized 13C‐metabolites in tumor cell spheroids using real‐time NMR spectroscopy , 2013, NMR in biomedicine.

[55]  John M Pauly,et al.  Hyperpolarized C‐13 spectroscopic imaging of the TRAMP mouse at 3T—Initial experience , 2007, Magnetic resonance in medicine.

[56]  P W Kuchel,et al.  Spin‐exchange NMR spectroscopy in studies of the kinetics of enzymes and membrane transport , 1990, NMR in biomedicine.

[57]  R Gruetter,et al.  Extracellular–Intracellular Distribution of Glucose and Lactate in the Rat Brain Assessed Noninvasively by Diffusion-Weighted 1H Nuclear Magnetic Resonance Spectroscopy In Vivo , 2000, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[58]  P. Larson,et al.  Rapid in vivo apparent diffusion coefficient mapping of hyperpolarized 13C metabolites , 2015, Magnetic resonance in medicine.