INTRODUCTION: The treatment of prostate cancer has been impeded by both the lack of clinically relevant disease models and metabolic markers that track tumor progression. Hyperpolarized (HP) C MR spectroscopy has emerged as a new technology to investigate the metabolic shifts in prostate cancer [1], and the safety of C pyruvate as a metabolic probe was confirmed in a recent clinical trial [2]. The present study provides the first validation of HP C lactate as a prostate cancer biomarker in human tissues, critical for the interpretation of in vivo studies. A patientderived prostate tissue slice culture (TSC) model that recapitulates the metabolic profile of prostate cancer in vivo was developed, applied to a perfused cell (bioreactor) platform, and investigated by C MR. C spectra following injection of hyperpolarized C pyruvate demonstrated significantly increased pyruvate to lactate flux in malignant as compared to the normal 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), providing mechanistic evidence for HP C lactate as a prostate cancer biomarker.

INTRODUCTION: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common cause of chronic liver disease in North America, with a prevalence approaching 35% in some population groups. The disease is closely associated with obesity and the metabolic syndrome. Although the etiology is multifactorial, current understanding of the metabolic abnormalities of NAFLD, as well as of the factors that lead some patients to progress to hepatic inflammation and end-stage liver cirrhosis while others do not, is limited. Magnetic resonance imaging at high field allows the acquisition of anatomic images with exquisite spatial resolution and detail. Additionally, specialized pulse sequences can be used to determine the degree of adiposity of tissues in vivo, providing a method to visualize the severity of steatosis within the liver. Hyperpolarized C MR continues to be a valuable tool for the investigation of metabolic and biochemical processes in a variety of organs and pathologic conditions. The goal of these studies was to utilize hyperpolarized C MR to identify and quantify metabolic derangements in mice fed a diet deficient in methionine and choline (MCD diet), an animal model of Non-Alcoholic Fatty Liver Disease.

hyperpolarized [C]urea over 30 seconds in a normal rat, overlaid on H T2-weighted FSE images. Top halfKidneys and a portion of liver. Bottom halfHeart, descending aorta, and lungs. Fig. 3Hyperpolarized C-urea (green) and Clactate (red), overlaid on T2-weighted FSE H images (grayscale) of mouse liver, combining results of multiple experiments. Image 1normal mouse. Images 2&3liver tumor mice. Fig. 1bSSFP pulse sequence for dynamic imaging of hyperpolarized media

INTRODUCTION: Philadelphia-positive chronic myelogenous leukemia (CML) is identified by the fusion of BCR with the Abl tyrosine kinase resulting in constitutive activity and uncontrolled myeloid cell proliferation [1]. Imatinib (Gleevec or STI-571) is a small molecule inhibitor of BCR-Abl and a therapeutic agent for the treatment of CML. Resistance to Imatinib has been observed both in patients and experimental cell models [2] although the mechanisms underlying resistance are not fully understood. Recently, an Imatinib-resistant CML cell model, MyLR was generated from normal CML cells, MyL. These Imatinib-resistant cells are independent of BCR-Abl overexpression or mutations [3] and display a multi-drug resistant phenotype [4,5]. NMR spectroscopy is a unique method that identifies and quantitates multiple metabolites in crude cell extracts and can be used for non-invasive metabolite assessment in whole, live cell preparations. Here we examined the global metabolic differences between MyL and MyLR cells. Several metabolites were decreased in the MyLR cells, however the most dramatic change was a ~7-fold increase in the total creatine (Cr). Real time in vivo P NMR experiments were conducted to determine the significance and role the elevated Cr plays in drug resistance. METHODS: MyL and MyLR cells were cultured in T-175 flasks and grown to high density. 10 cells were collected, added to fresh culture media and incubated 2 hours at 37 ̊C. Cells were then collected and metabolites extracted with icecold methanol. Lyophilized extracts were dissolved in D2O containing 1.5 mM TSP as a concentration and chemical shift reference. H NMR spectra were obtained on a narrowbore 16.5T Varian INOVA (125 MHz C, Varian Instruments) equipped with a 5 mm inverse detect probe. For the in vivo studies a custom designed 10 mm NMR compatible bioreactor system, which allows for continuous media flow and temperature regulation was used [6]. Approximately 2.5 x 10 MyLR cells were electrostatically encapsulated into ~500 μm alginate beads and loaded into the bioreactor system. The flow rate during the experiment was 4 mL/ min and the O2 concentration was maintained in the media using a Gas Exchange Module (GEM), which was filled with 95% Air/ 5% CO2. Bioreactor P NMR spectra were acquired on a 14.1T Varian INOVA. Data from both H and P experiments were processed using ACD/Labs 1D and 2D NMR processing software, version 7.0 (Advanced Chemistry Development, Inc. Toronto). In some cases spectra were binned and analyzed by Principal Component Analysis and Mutual Information Analysis to determine what metabolites were significantly different between cell types. Additionally, individual metabolite concentrations were determined for H spectra using Chenomx software (Chenomx, Inc. Alberta). RESULTS: Figure 1 shows representative H NMR spectra from MyL and MyLR cell extracts highlighting the significant increase in Cr. 2D H-C HSQC of the MyLR extract demonstrated that the resonances in the 1D H spectrum are representative of Cr (Fig. 1 Insert). Several other metabolites were found to be decreased in MyLR cells compared to MyL cells, including, choline, phosphocholine, myo-inositol, taurine, and the glycolytic related, alanine and lactate (Fig. 2). We also observed that glucose concentrations in MyLR media were higher, suggesting an alteration in glucose uptake and/ or utilization (glycolysis). As it is difficult to distinguish Cr from phosphocreatine (PCr) using H NMR and due to the rapid degradation of PCr to Cr by creatine kinase, MyLR cells were encapsulated in alginate beads and a NMR-compatible bioreactor was used to examine PCr levels in vivo. Figure 3A shows P NMR spectra collected over an 8 hr period clearly showing PCr in MyLR cells. Moreover, following the addition of 2,4-dinitrophenol (DNP), an uncoupler of mitochondrial oxidative phosphorylation, PCr levels were completely diminished, while βNTP levels remained relatively unaffected (presumably through glycolysis)(Fig. 3B). These data suggest that maintenance of the PCr pool is highly coupled to mitochondrial ATP production in MyLR cells. DISCUSSION AND CONCLUSIONS: We examined the metabolic profile of MyL and MyLR cells and found that drug resistant cells display an altered metabolic phenotype compared to their non-resistant counterparts. These data suggest that drug-resistant MyLR cells have decreased glycolytic flux and display a near 7-fold increase in total Cr. While others have observed an increase in PCr in adriamycinresistant breast cancer, this is the first such example in drug-resistant leukemia cells. We propose that enhanced Cr synthesis provides an additional energy reserve in the form of PCr thereby giving a selective advantage to MyLR cells and possibly facilitating drug resistance. REFERENCES: [1] Sherbenou, D.W. and B.J. Druker, J Clin Invest, 2007. 117(8): p. 2067-74. [2] Shah, N.P. and C.L. Sawyers. Oncogene, 2003. 22(47): p. 7389-95. [3] Ito, T., H. Tanaka, and A. Kimura. Eur J Haematol, 2007. 78(5): p. 417-31. [4] Donato, N.J., et al. Cancer Res, 2004. 64(2): p. 672-7. [5] Raguz, S., et al. Int J Cancer, 2008. 122(5): p. 1058-67. [6] Jeffries, RE, et. al. Intern. Soc. Magn. Reson. Med. 2008. ACKNOWLEDGEMENTS: National Institute of Health, GM075941-01. MyLR MyL

Introduction: The amount of nuclear antigen Ki-67 staining has been associated with cancer grade [1] and metastases [2]. Elevated levels of phospholipid metabolites have also been correlated with the presence of prostate cancer [3]. However, a direct correlation between the levels of individual phospholipid metabolites with cancer proliferation and Gleason grade has not been determined. This is of great clinical significance since most prostate cancers are indolent and will never metastasize, but there is currently no accurate way to identify these patients at diagnosis. HR-MAS twodimensional total correlation spectroscopy (TOCSY) can be used to quantify choline and ethanolamine phospholipid metabolites in intact human prostate tissues prior to pathology and immunohistochemistry of the same tissues [3]. The purpose of this study is to establish the relationship between phospholipid metabolite levels, Ki-67, and Gleason Grade. Methods: TOCSY’s were acquired from snap frozen tissue samples obtained at radical prostatectomy in order to resolve the choline (choline – Cho, phosphocholine – PC, glycerophosphocholine GPC) and ethanolamine-containing compounds (ethanolamine Eth, phosphoethanolamine PE, glycerolphosphethanolamine GPE), that overlap in 1-D HR-MAS tissue spectra. NMR spectra were acquired using a 11.7T (500MHz for H), 1°C, and a 2250 Hx spin rate using a Varian INOVA spectrometer equipped with a 4-mm gHX nanoprobe (Varian, Palo Alto, CA) and processed as previously described to generate absolute concentrations [3]. After HR-MAS, tissues were imbedded in OCT and 5 mm sections were obtained using a Leica CM1850 cryostat. For each sample, adjacent sections were stained with hematoxylin and eosin (H&E) and Ki-67 [1] and the % of the sample that was prostate cancer as well as the % of the cancer that stained positive for Ki-67 was determined by two experienced pathologists who were blinded to the clinical and metabolic findings. For the purpose of the abstract we divided cancers into low grade (≤ 3+4) and high grade (≥ 4+3).

INTRODUCTION: The molecular probes used in hyperpolarized C spectroscopy have important advantages over contrast agents currently in clinical use, expected to have little or no toxicity in humans, even at relatively high concentrations. This feature is particularly appealing given recent concerns about contrast nephropathy associated with iodinated CT contrast, as well as nephrogenic systemic fibrosis (NSF) seen in patients receiving gadolinium chelates for MR[1,2]. Many new C agents may be appropriate for metabolic imaging in humans. In addition to C pyruvate, C lactate itself is a promising primary agent for cancer imaging[3]. Other recent work has demonstrated in vivo pH mapping using C bicarbonate, by hyperpolarization of C cesium bicarbonate followed by an ion exchange method to exchange most of the Cs for sodium[4]. In this abstract, a method for direct polarization of C sodium bicarbonate is reported, that is suitable for use in humans. This method has been combined with a copolarization approach that allows simultaneous polarization of C bicarbonate and C pyruvate, to perform both pH and metabolic mapping in vivo using a single contrast bolus.

Fourier Transform Infrared Imaging (FTIRI) is a new method for quantitatively assessing the spatial-chemical composition of complex materials. This technique has been applied to examine the feasibility of measuring changes in the composition and distribution of collagen and proteoglycan macromolecules in human osteoarthritic cartilage. Human cartilage was acquired post-operatively from total joint replacement patients. Samples were taken at the site of a focal lesion, adjacent to the lesion, and from relatively healthy cartilage away from the lesion. Sections were prepared for FTIRI and histochemical grading. FTIRI spectral images were acquired for the superficial, intermediate, and deep layers for each sample. Euclidean distance mapping and quantitative partial least squares analysis (PLS) were performed using reference spectra for type-II collagen and chondroitin 6-sulphate (CS6). FTIRI results were correlated to the histology-based Mankin scoring system. PLS analysis found relatively low relative concentrations of collagen (38 +/- 10%) and proteoglycan (22 +/- 9%) in osteoarthritic cartilage. Focal lesions were generally found to contain less CS6 compared to cartilage tissue adjacent to the lesion. Loss of proteoglycan content was well correlated to histological Mankin scores (r=0.69, p<0.0008). The evaluation of biological tissues with FTIRI can provide unique quantitative information on how disease can affect biochemical distribution and composition. This study has demonstrated that FTIRI is useful in quantitatively assessing pathology-related changes in the composition and distribution of primary macromolecular components of human osteoarthritic cartilage.