Tumor pH is physiologically important since it influences a number of processes relevant to tumorigenesis and therapy. Hence, knowledge of localized pH within tumors would contribute to understanding these processes. The destructiveness, poor spatial resolution, and poor signal-to-noise ratio (SNR) of current technologies (e.g., microelectrodes, 31 P magnetic resonance spectroscopy) have limited such studies. An extrinsic chemical extracellular pH (pH e ) probe is described that is used in combination with 1 H magnetic resonance spectroscopic imaging to yield pH e maps with a spatial resolution of 1 ؋ 1 ؋ 4 mm 3 . Since the discovery of lactic acid production in tumors more than 50 years ago (1), it has generally been assumed that the pH of tumors is acidic. Indeed, numerous microelectrode measurements have shown that extracellular tumor pH (pH e ) is acidic (2). This acidic pH e of tumors has been confirmed with less invasive 31 P magnetic resonance spectroscopy (MRS) measurements (3). Although the intracellular pH (pH i ) of tumors remains neutral to alkaline (4,5), it is somewhat influenced by the pH e (6).An acidic pH e of tumors is physiologically important since it influences a number of processes relevant to carcinogenesis and therapy. Knowledge of localized pH within tumors, both intra-and extracellular, would allow more detailed study of these processes and relate them to intratumoral pH heterogeneity. For example, it has been found that low pH e in vitro causes tumorigenic transformation of primary Syrian hamster embryo cells (7) and can lead to chromosomal rearrangements in Chinese hamster embryo cells (8,9). Furthermore, culturing cells at low pH causes them to be more invasive in vitro (10) and metastatic in vivo (11). Finally, the orientation of the pH gradient across the cell membrane may influence cell drug resistance (6,12) Previously reported measurements of extracellular pH using either microelectrodes or 31 P MRS of 3-aminopropylphosphonate (3-APP) (3) have drawbacks. Microelectrodes are invasive and can destroy the membrane integrity, thereby disrupting the mechanism for maintaining the pH e . 31 P MRS does not suffer this drawback and has the additional advantage of permitting simultaneous measurements of intracellular pH. However, the limited sensitivity of 31 P MRS allows measurements of pH e only from relatively large tissue volumes. Hence, 31 P MRS provides measurements of pH ranges rather than different pH values for discrete spatial locations (13).The use of 1 H MRS, inherently more sensitive than 31 P MRS, would allow measurements of pH over smaller tissue volumes. For example, the imidazole protons of histidine have long been useful as intracellular pH indicators in NMR (14,15).Rabenstein and Isab (16) first proposed using imidazoles as extrinsic pH e indicators. Gil et al (17) suggested several modifications of the basic structure of the imidazole molecule to improve its performance as an extrinsic pH probe. To date, the most promising candidate for a 1 H nuclear magnetic resonance ...
MR spectroscopy of cultured cells allows non-invasive analyses of the metabolism of cells with specific phenotypes under defined conditions. This technique can be used to investigate the intracellular metabolism of cells or extended to critically evaluate phenomena observed by in vivo MRS. In this paper, a cell maintenance system is described which allows MR analyses with unparalleled spectral resolution, S/N and stability. This system consists of a 25 mm diameter hollow fiber bioreactor and a supporting circuit. The hollow fiber reactor was chosen because it yields a large filling factor which can be perfused through defined volumes. The fibers were 300 microns diameter microporous (0.2 micron) cellulose acetate/cellulose nitrate membranes with high porosity, which allow bulk convective flow throughout the extracapillary space. This flow (Starling flow) is necessary to disrupt steady-state gradients in substrates and waste products. In many respects, the design of the supporting circuit is more important than the bioreactor itself, since it provides the reactor with the proper chemical and physical environment. Hence, this circuit can be applied to a variety of bioreactor configurations. The circuit consists of a hollow fiber oxygenator and a bleed-and-feed system housed in a temperature-controlled cabinet. Culture of mammalian cells in this reactor yields 31P spectra which have excellent spectral and temporal resolution. At confluence, endogenous 31P line widths were typically < 10 Hz (at 162 MHz) and well resolved spectra were obtained in < 30 s.
BackgroundTwo-thirds of U.S. adult women are overweight or obese. High body mass index (BMI) and adult weight gain are risk factors for a number of chronic diseases, including postmenopausal breast cancer. The higher postmenopausal breast cancer risk in women with elevated BMI is likely to be attributable to related metabolic disturbances including altered circulating sex steroid hormones and adipokines, elevated pro-inflammatory cytokines, and insulin resistance. Metformin is a widely used antidiabetic drug that has demonstrated favorable effects on metabolic disturbances and as such may lead to lower breast cancer risk in obese women. Further, the anti-proliferative effects of metformin suggest it may decrease breast density, an accepted biomarker of breast cancer risk.Methods/designThis is a Phase II randomized, double-blind, placebo-controlled trial of metformin in overweight/obese premenopausal women who have elements of metabolic syndrome. Eligible participants will be randomized to receive metformin 850 mg BID (n = 75) or placebo (n = 75) for 12 months. The primary endpoint is change in breast density, based on magnetic resonance imaging (MRI) acquired fat-water features. Secondary outcomes include changes in serum insulin levels, serum insulin-like growth factor (IGF)-1 to insulin-like growth factor binding protein (IGFBP)-3 ratio, serum IGF-2 levels, serum testosterone levels, serum leptin to adiponectin ratio, body weight, and waist circumference. Exploratory outcomes include changes in metabolomic profiles in plasma and nipple aspirate fluid. Changes in tissue architecture as well as cellular and molecular targets in breast tissue collected in a subgroup of participants will also be explored.DiscussionThe study will evaluate whether metformin can result in favorable changes in breast density, select proteins and hormones, products of body metabolism, and body weight and composition. The study should help determine the potential breast cancer preventive activity of metformin in a growing population at risk for multiple diseases.Trial registrationClinicalTrials.gov Identifier: NCT02028221. Registered on January 2, 2014. Grant #: 1R01CA172444-01A1 awarded on Sept 11, 2013.
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