Metabolic, pathologic, and genetic analysis of prostate tissues: Quantitative evaluation of histopathologic and mRNA integrity after HR-MAS spectroscopy

Department of Radiology, University of California, San Francisco, CA, USA.
NMR in Biomedicine (Impact Factor: 3.04). 01/2009; 23(4):391-8. DOI: 10.1002/nbm.1474
Source: PubMed


The impact of high-resolution magic angle spinning (HR-MAS) spectroscopy on the histopathologic and mRNA integrity of human prostate tissues was evaluated. Forty prostate tissues were harvested at transrectal ultrasound (TRUS) guided biopsy (n = 20) or radical prostatectomy surgery (n = 20), snap-frozen on dry ice, and stored at -80°C until use. Twenty-one samples (n = 11 biopsy, n = 10 surgical) underwent HR-MAS spectroscopy prior to histopathologic and cDNA microarray analysis, while 19 control samples (n = 9 biopsy, n = 10 surgical) underwent only histopathologic and microarray analysis. Frozen tissues were sectioned at 14-µm intervals and placed on individual histopathology slides. Every 8th slide was stained with hematoxylin and eosin (H&E) and used to target areas of predominantly epithelial tissue on the remaining slides for mRNA integrity and cDNA microarray analysis. Histopathologic integrity was graded from 1 (best) to 5 (worst) by two 'blinded' pathologists. Histopathologic integrity scores were not significantly different for post-surgical tissues (HR-MAS vs controls); however, one pathologist's scores were significantly lower for biopsy tissues following HR-MAS while the other pathologist's scores were not. mRNA integrity assays were performed using an Agilent 2100 Bioanalyzer and the electrophoretic traces were scored with an RNA integrity number (RIN) from 1 (degraded) to 10 (intact). RIN scores were not significantly different for surgical tissues, but were significantly lower for biopsy tissues following HR-MAS spectroscopy. The isolated mRNA then underwent two rounds of amplification, conversion to cDNA, coupling to Cy3 and Cy5 dyes, microarray hybridization, imaging, and analysis. Significance analysis of microarrays (SAM) identified no significantly over- or under-expressed genes, including 14 housekeeping genes, between HR-MAS and control samples of surgical and biopsy tissues (5% false discovery rate). This study demonstrates that histopathologic and genetic microarray analysis can be successfully performed on prostate surgical and biopsy tissues following HR-MAS analysis; however, biopsy tissues are more fragile than surgical tissues.

Download full-text


Available from: Mark Swanson, Jul 08, 2015
13 Reads
  • Source
    • "Even though HR MAS MRS leaves the tumor tissue intact, the procedure exposes the tissue to several potential stresses, including hypoxic conditions and lack of nutrients by being embedded in a surrounding buffer at 4°C for approximately an hour, as well as high centrifugal force and magnetic field during the HR MAS MRS acquisition. In our parallel study to address this issue, total RNA integrity was not significantly affected by HR MAS MRS (p-value = 0.86), and findings in a similar evaluation in prostate tissue support this result [31]. The pairs of tumor samples from each patient that had or had not been analyzed by HR MAS MRS cluster together (Additional file 3: Plots illustrating the effect of HR MAS MRS on the transcriptome), indicating that patient to patient variation is larger than the effect of HR MAS MRS. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Combining gene expression microarrays and high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) of the same tissue samples enables comparison of the transcriptional and metabolic profiles of breast cancer. The aim of this study was to explore the potential of combining these two different types of information. Breast cancer tissue from 46 patients was analyzed by HR MAS MRS followed by gene expression microarrays. Two strategies were used to combine the gene expression and metabolic data; first using multivariate analyses to identify different groups based on gene expression and metabolic data; second correlating levels of specific metabolites to transcripts to suggest new hypotheses of connections between metabolite levels and the underlying biological processes. A parallel study was designed to address experimental issues of combining microarrays and HR MAS MRS. In the first strategy, using the microarray data and previously reported molecular classification methods, the majority of samples were classified as luminal A. Three subgroups of luminal A tumors were identified based on hierarchical clustering of the HR MAS MR spectra. The samples in one of the subgroups, designated A2, showed significantly lower glucose and higher alanine levels than the other luminal A samples, suggesting a higher glycolytic activity in these tumors. This group was also enriched for genes annotated with Gene Ontology (GO) terms related to cell cycle and DNA repair. In the second strategy, the correlations between concentrations of myo-inositol, glycine, taurine, glycerophosphocholine, phosphocholine, choline and creatine and all transcripts in the filtered microarray data were investigated. GO-terms related to the extracellular matrix were enriched among the genes that correlated the most to myo-inositol and taurine, while cell cycle related GO-terms were enriched for the genes that correlated the most to choline. Additionally, a subset of transcripts was identified to have slightly altered expression after HR MAS MRS and was therefore removed from all other analyses. Combining transcriptional and metabolic data from the same breast carcinoma sample is feasible and may contribute to a more refined subclassification of breast cancers as well as reveal relations between metabolic and transcriptional levels. See Commentary:
    BMC Cancer 11/2010; 10(1):628. DOI:10.1186/1471-2407-10-628 · 3.36 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The diffusion mechanism of arsenic (As) in SiO2 was studied by the diffusion of oxygen in SiO2 using isotope 18O as a component element of the matrix oxide. The result suggests that As loosens SiO2 network bonds and drives a subsequent rearrangement of the network which enhances diffusion of As and oxygen in N2 annealing. However, when 10% H2 was added to N2, As atoms were released from the network in a molecular form and were completely independent of the network rearrangement
    Electron Devices Meeting, 1988. IEDM '88. Technical Digest., International 01/1997; DOI:10.1109/IEDM.1997.650479
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Prostate cancer has been shown to undergo unique metabolic changes associated with neoplastic transformation, with associated changes in citrate, alanine, and lactate concentrations. (13)C high resolution-magic angle spinning (HR-MAS) spectroscopy provides an opportunity to simultaneously investigate the metabolic pathways implicated in these changes by using (13)C-labeled substrates as metabolic probes. In this work, a method to reproducibly interrogate metabolism in prostate cancer cells in primary culture was developed using HR-MAS spectroscopy. Optimization of cell culture protocols, labeling parameters, harvesting, storage, and transfer was performed. Using [3-(13)C] pyruvate as a metabolic probe, (1)H and (13)C HR-MAS spectroscopy was used to quantify the net amount and fractional enrichment of several labeled metabolites that evolved in multiple cell samples from each of five different prostate cancers. Average enrichment across all cancers was 32.4 +/- 5.4% for [3-(13)C] alanine, 24.5 +/- 5.4% for [4-(13)C] glutamate, 9.1 +/- 2.5% for [3-(13)C] glutamate, 25.2 +/- 5.7% for [3-(13)C] aspartate, and 4.2 +/- 1.0% for [3-(13)C] lactate. Cell samples from the same parent population demonstrated reproducible fractional enrichments of alanine, glutamate, and aspartate to within 12%, 10%, and 10%, respectively. Furthermore, the cells produced a significant amount of [4-(13)C] glutamate, which supports the bioenergetic theory for prostate cancer. These methods will allow further characterization of metabolic properties of prostate cancer cells in the future. Magn Reson Med, 2009. (c) 2009 Wiley-Liss, Inc.
    Magnetic Resonance in Medicine 11/2009; 62(5):1091-8. DOI:10.1002/mrm.22120 · 3.57 Impact Factor
Show more