Regional Cell Proliferation in Microdissected Human Prostate Specimens after Heavy Water Labeling In Vivo: Correlation with Prostate Epithelial Cells Isolated from Seminal Fluid

Abstract

Purpose: Prostate cancer is detected with increasing frequency but has a highly variable natural history and prognosis and active surveillance of men with low-risk prostate cancer would benefit greatly from minimally invasive methods to identify progression. We describe here two novel in vivo metrics of cell proliferation in men with prostate neoplasia.Experimental Design: Three groups of men drank heavy water, a nonradioactive, stable isotopic tracer for 14 to 28 days: (i) healthy men, (ii) men scheduled for transrectal core needle biopsy, and (iii) men scheduled for radical prostatectomy. Prostate epithelial cells (PEC) were isolated from ejaculated seminal fluid in all subjects. Histologically graded lesions were microdissected from tissue slides obtained from subjects undergoing surgery and proliferation rates were measured from isolated cells via mass spectrometry.Results: Proliferation rates of seminal PEC in healthy men (0.10%-0.27%/d) were stable on repeat sampling. Rates above 0.34%/d were seen only in patients with cancer where rates increased progressively from normal tissue through benign prostate hyperplasia, prostate intraepithelial neoplasia, and tumor grades III and IV in all subjects. Seminal PEC kinetics correlated highly with the most proliferative microdissected region in each subject (r 2 ¼ 0.94).Conclusions: Prostate cell proliferation can be measured in vivo from microdissected histopathology sections or noninvasively from seminal fluid where the latter reflects the most proliferative region of the gland. This approach may allow monitoring of progression in men with low-risk prostate cancer.

Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry

“…Conversely, in our predominantly non-dividing C2C12 myotube cultures, the reliance on the DNL pathway was relatively high (~77% palmitate was ‘self-made’), thus indicating the reliance on the DNL pathway is unlikely related to cell proliferation. The 2 H 2 O labelling technique and extraction method described here could easily be applied to 3D culture systems and is also ideally suited for studying in vivo tumor growth and metabolism in pre-clinical animal models [59,60] and human clinical settings [36,61]. …”

“…Following the administration of 2 H 2 O into the body water pool in vivo or into cell culture media in vitro, 2 H atoms become stably incorporated via enzyme catalyzed reactions into C-H bonds of nonessential amino acids, glycerol-3-phosphate, fatty acids, cholesterol, hexoses and pentoses (ribose and deoxyribose). Subsequent analysis of 2 H labelling of these constituents in macromolecules provides a highly sensitive measure of the rates of synthesis/turnover of lipids [15,16,17,18,19,20,21,22,23,24,25,26,27], proteins [15,28,29,30,31,32,33,34], DNA [15,35,36], RNA [15] and glucose/glycogen [25,26,37,38,39,40,41,42,43] and an objective assessment of fundamental cellular processes that include, but are not limited to, rates of cell and organelle proliferation, cell and tissue biomass turnover, de novo lipogenesis and gluconeogenesis. Such measurements have tremendous application to researchers in the field of cancer, immunology, cardiovascular disease, metabolism, diabetes, obesity, developmental biology, as well as the biotechnology and agricultural sectors.…”

Strategies for Extending Metabolomics Studies with Stable Isotope Labelling and Fluxomics

“…and observing their metabolic incorporation and/or dilution in biological systems, it is also possible to use other labelling approaches combined with mass spectrometric and/or NMR analysis to understand cellular metabolism and growth characteristics. For example, the use of deuterated “heavy” water ( 2 H 2 O) has proven to be a particularly powerful approach for measuring the synthesis rates of proteins [71,72,73,74,75], lipids ( de novo lipogenesis, esterification, and chain elongation) [67,76,77,78], DNA [79,80], and RNA [81], thus providing information on biomass synthesis and cell division rates, both in vivo and in vitro . Furthermore, 2 H 2 O labelling can be used to quantify the pathways used to produce glucose and glycogen, thus providing a way to measure gluconeogenic and glycogenolytic/glycogenic pathway activity [82,83,84,85,86,87].…”

In vivo kinetics and nonradioactive imaging of rapidly proliferating cells in graft-versus-host disease

“…Pioneering in vivo kinetics studies were conducted in patients with HIV (18,19), which measured CD4 + and CD8 + T cell kinetics, and then in many other cell types and conditions (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). To date, studies involving stable water isotopes measured T cell kinetics in circulation, a dynamic cellular compartment, while systemic cell half-lives were mathematically estimated.…”