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PubMed2004

Proteomics to diagnose human tumors and provide prognostic information.

Proteomics is a rapidly emerging scientific discipline that holds great promise in identifying novel diagnostic and prognostic biomarkers for human cancer. Technologic improvements have made it possible to profile and compare the protein composition within defined populations of cells. Laser capture microdissection is a tool for procuring pure populations of cells from human tissue sections to be used for downstream proteomic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used traditionally to separate complex mixtures of proteins. Improvements in this technology have greatly enhanced resolution and sensitivity providing a more reproducible and comprehensive survey. Image analysis software and robotic instrumentation have been developed to facilitate comparisons of complex protein expression patterns and isolation of differentially expressed proteins spots. Differential in-gel electrophoresis (DIGE) facilitates protein expression by labeling different populations of proteins with fluorescent dyes. Isotope-coded affinity tagging (ICAT) uses mass spectroscopy for protein separation and different isotope tags for distinguishing populations of proteins. Although in the past proteomics has been primarily used for discovery, significant efforts are being made to develop proteomic technologies into clinical tools. Reverse-phase protein arrays offer a robust new method of quantitatively assessing expression levels and the activation status of a panel of proteins. Surface-enhanced laser-desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy rapidly assesses complex protein mixtures in tissue or serum. Combined with artificial intelligence-based pattern recognition algorithms, this emerging technology can generate highly accurate diagnostic information. It is likely that mass spectroscopy-based serum proteomics will evolve into useful clinical tools for the detection and treatment of human cancers.

Early Diagnosis↗
PubMed2001

High frequency of mitochondrial DNA mutations in glioblastoma multiforme identified by direct sequence comparison to blood samples.

In an earlier study, we showed that heteroplasmy in the mitochondrial genome of gliomas sometimes occurs in a D-loop polycytosine tract. We extended this study by pairwise comparisons between glioma samples and adjacent brain tissue of 55 patients (50 glioblastomas, 1 astrocytoma WHO grade III, 4 astrocytomas WHO grade II). We used a combination of laser microdissection and PCR to detect and quantify variations in the polycytosine tract. New length variants undetectable in the adjacent brain tissue were observed in 5 glioblastomas (9%). In 2 of these cases, samples from a lower tumor stage (WHO grade II) could be analyzed and revealed the early occurrence of these mutations in both cases. Since the mitochondrial D-loop contains additional repeats and highly polymorphic non-coding sequences, we compared 17 glioblastomas with the corresponding blood samples of the same patients by direct sequencing of the complete D-loop. In 6 of these tumors (35%), instability was detected in 1 or 2 of 3 repeat regions; in 1 of these repeats, the instability was linked to a germline T-to-C transition. Furthermore, of 2 tumors (12%) 1 carried 1 and the other 9 additional transitions. In the latter patient, 6.7 kb of the protein coding mtDNA sequence were analyzed. Six silent transitions and 2 missense mutations (transitions) were found. All base substitutions appeared to be homoplasmic upon sequencing, and 89% occurred at known polymorphic sites in humans. Our data suggest that the same mechanisms that generate inherited mtDNA polymorphisms are strongly enhanced in gliomas and produce somatic mutations.

Adult↗
PubMed2004

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

Aged↗
PubMed2006

Primary cutaneous B-cell lymphoma (marginal zone) with prominent T-cell component and aberrant dual (T and B) genotype; diagnostic usefulness of laser-capture microdissection.

The presence of a dominant B- or T-cell clone is an important diagnostic criterion for distinguishing cutaneous lymphomas from lymphoid reactive infiltrates. Rarely, a combined B- and T-cell rearrangement can be detected from a single sample. In such instances, genotypic analysis does not permit differentiation of the coexistence of a T- and B-cell lymphoma from a single clone harbouring a monoclonal rearrangement for both immunoglobulin heavy chain and T-cell receptor genes. We herein report a case of a skin tumour consistent with a dense cutaneous lymphoid infiltrate showing a double prominent B- and T-cell component. A dual B- and T-cell clonality was detected by polymerase chain reaction from whole-tissue DNA sample. Genotypic analysis with DNA, obtained after laser-assisted microdissection from the B-cell population, again showed both T- and B-cell monoclonal rearrangements. Conversely, the microdissected T-cell population did not reveal a clonal pattern. The diagnosis of cutaneous B-cell lymphoma with a dual B- and T-cell genotype was established. This description illustrates the diagnostic usefulness of laser-capture microdissection in cutaneous lymphomas presenting dual genotype.

Aged↗
PubMed1993

Cell- and stage-specific expression of vitamin D receptor and calbindin genes in rat incisor: regulation by 1,25-dihydroxyvitamin D3.

To investigate the extent of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action and its relationships to calbindin gene expression in mineralized tissues, we have analyzed rat incisors with different probes, including a vitamin D receptor (VDR) antibody and specific cDNAs to rat calbindin-D9K and calbindin-D28K. Developmental and hormonal controls of calbindin gene expression were investigated by Northern blot analysis of ameloblast and odontoblast mRNA. Distribution and hormone-induced changes of VDR were also studied by light microscopic immunocytochemistry. A differential tissue- and stage-specific expression of the calbindin genes was observed in microdissected portions of the continuously erupting incisor. The two calbindins were expressed in ameloblasts, whereas only calbindin-D28K was expressed in odontoblasts. Moreover, in ameloblasts, expression of calbindin-D28K preceded that of calbindin-D9K. Immunoreactivity for VDR was present in all progenitor cells and progressively decreased during the differentiation process, whereas, in differentiated tissues, a hormonal upregulation was restricted to hard tissue-forming cells, i.e., ameloblasts and odontoblasts. Furthermore, calbindin gene expression appeared to be regulated by 1,25(OH)2D3. Taken together, these data indicate that ameloblasts and odontoblasts are target cells for 1,25(OH)2D3 and provide the first insights into the hormonal control of tooth genes during development.

Animals↗
PubMed2005

Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization.

Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.

Brachyspira↗
PubMed2003

An antigen unmasking protocol that satisfies both immunohistochemistry and subsequent PCR amplification.

Immunohistochemical elucidation of many proteins in formalin-fixed, paraffin-embedded tissues requires a prior antigen unmasking treatment, which often damages both the morphology and genetic materials, making subsequent assessments difficult or impossible. This study attempted to develop a method that satisfies both immunohistochemical and genetic analyses. Consecutive sections were made from formalin-fixed, paraffin-embedded breast and other tissues, and a set of four adjacent sections from each case were treated with (1) routine H & E staining; (2) our unmasking protocol; (3) microwave oven irradiation; (4) pressure cooker incubation. After immunohistochemical staining, the tissue in each section was scraped off, or the same cell clusters in four sections were separately microdissected for DNA extraction and PCR amplification. Compared to microwave and pressure cooker methods, our protocol showed the following advantages: (1) a better preservation of the morphology; (2) a substantial reduction of tissue detachments from slides; (3) effectiveness on all antibodies tested, including those requiring enzyme digestion or no prior unmasking; (4) higher PCR yields; (5) larger (higher molecular weight) amplified PCR products. Compared to the routine method on untreated tissues, our method consistently produced a comparable quality and quantity of PCR products. Our protocol, however, takes a longer time to yield results.

Antigens↗
PubMed2005

Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma.

In order to identify new molecular markers for pancreatic intra-epithelial neoplasias (PanINs), the precursor lesions of pancreatic ductal adenocarcinoma, we established a proteomics approach analysing microdissected PanIN cells. Due to the limited amount of proteins available from microdissection, we developed a procedure including fluorescence dye saturation labelling in combination with high resolution two-dimensional gel electrophoresis. With this procedure we were able to analyse proteins extracted from 1000 microdissected cells with a high resolution of up to 2500 protein spots. Using protein lysates from the pancreatic carcinoma tissue as a reference proteome, we were able to successfully identify the proteins. Thus, we could match approximately 2200 protein spots (92%) of the microdissected sample proteome to the reference proteome for protein identification using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and nanoliquid chromatography-electrospray ionisation tandem mass spectrometry after in-gel digestion. The first proteome analysis of microdissected PanIN-2 grades revealed eight differentially expressed proteins. The differential expression of the three actin filament-associated proteins--transgelin, vimentin and MRCL3 as well as actin itself--indicates a relevant role of the actin cytoskeleton during pancreatic tumour progression. Additionally, two members of the annexin family (annexin A2 and A4) implicate a functional contribution of exocytotic and endocytotic pathways at that stage.

Adenocarcinoma↗
PubMed2005

Laser capture microdissection for analysis of macrophage gene expression from atherosclerotic lesions.

Macrophage foam cells are critical mediators in atherosclerosis plaque development. A better understanding of the in vivo transcript profile of foam cells during the formation and progression of lesions may lead to novel therapeutic interventions. Toward this goal, we demonstrate for the first time that foam cell-specific RNA can be purified from atherosclerotic arteries, a tissue of mixed cellular composition. Foam cells from apolipoprotein (apo) E-/- mice were isolated by laser capture microdissection (LCM); RNA was extracted and used for molecular analysis by real-time quantitative polymerase chain reaction. Compared to whole tissue, a significant enrichment of foam cell-specific RNA transcripts was achieved. Furthermore, to test the ability to quantify differences in gene expression in response to an inflammatory stimulus, apoE-/- mice were injected with lipopolysaccharide, after which the transcriptional induction of the inflammatory mediators, VCAM, ICAM, and MCP-1, was observed in lesional macrophage foam cell RNA. These approaches will facilitate the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.

Animals↗
PubMed1999

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors.

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.

Colorectal Neoplasms↗
PubMed1982

Human retinal vessels in tissue culture. A preliminary report of the effect of acute glucose poisoning on cultured vascular cells.

To delineate the factors involved in the pathogenesis of human retinal vasculopathies, as in vitro model of human retinal vascular cells was developed by using cadaver eyes enucleated four to eight hours after death and stored at +4 C for three to seven days. A pure, viable capillary explant was obtained by microdissection and gentle agitation; the more rapidly occurring postmortem changes in the surrounding nervous tissue of the retina facilitated separation of the vascular explants. Factor VIII indirect immunofluorescent staining revealed that 85% to 90% of the cells harvested from capillaries of 3- to 5-day-old cadaver eyes and all cells cultured from 1-week-old postmortem eyes reacted positively, indicating their endothelial nature. High-glucose medium caused degenerative changes in the cells of the initial explant as well as in the cells of confluent cultures within 24 to 72 hours. The cytotoxic effect of glucose was manifested by accumulation of PAS-positive granules, cytoplasmic vacuolation, and eventual cell degeneration.

Cells, Cultured↗
PubMed2005

Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed-Sternberg (HRS) cells: high expression of interleukin-11 receptor alpha.

We microdissected Hodgkin and Reed-Sternberg (HRS) cells from 14 Hodgkin's lymphoma tissue samples (nodular sclerosis = 5; mixed cellularity = 9), and after isolation and amplification of mRNA, analyzed the expression profile of 140 genes of chemokines, cytokines and their receptors by cDNA microarray methods. We also compared the profile with those of germinal center (GC) cells in reactive lymphadenitis. Unsupervised clustering revealed a relatively homogeneous expression profile in HRS cells. HRS cells tended to express mainly Th2 T cell-associated molecules rather than those of Th1, compared with GC cells. Interleukin-11 receptor alpha (IL-11Ralpha), a previously unknown HRS cell-specific gene, was detected in addition to known genes. Immunohistochemical staining confirmed the expression of IL-11Ralpha at the protein level. In contrast, only few cases were positive for IL-11Ralpha in B cell lymphoma, diffuse large cell lymphoma and follicular lymphoma. This is the first analysis report of tissue HRS cells with cDNA microarray technique.

Adolescent↗
PubMedDate not supplied

The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis--preliminary findings.

Proteomics-based studies offer a powerful complementary approach to DNA/RNA-based investigations and are now being applied to investigate aspects of many diseases including cancer. However, the heterogeneous nature of tissue samples often makes interpretation difficult. We have undertaken a study into the potential use of a novel laser capture microdissection (LCM) system to isolate cells of interest for subsequent proteomic analysis. Retrieval of selected cells is achieved by activation of a transfer film placed in contact with a tissue section, by a laser beam (30 or 60 microm diameter) which is focused on a selected area of tissue using an inverted microscope. The precise area of film targeted by the laser bonds to the tissue beneath it and these cells are then lifted free of surrounding tissue. Although the technique has been shown to be readily compatible with subsequent analysis of nucleic acids, little information is yet available regarding the application of protein-based analyses to the captured tissue. We report here preliminary data regarding the potential use of the LCM system in combination with two-dimensional electrophoresis to examine protein profiles of selected tissue areas. Electrophoretic profiles of proteins from normal and malignant renal tissue samples showed little change following LCM, nine selected proteins showed identical mass spectrometric sequencing profiles, and two selected proteins retained antigenicity. Dissection of epithelial tissue from a sample of normal human cervix resulted in enrichment of some proteins compared with analysis of the whole tissue. LCM will be a valuable adjunct to proteomic studies although further detailed validation is necessary.

Amino Acid Sequence↗
PubMed2002

Isolating vessels from the mouse brain for gene expression analysis using laser capture microdissection.

Studies of gene expression often examine a pool of RNA extracted from the diverse cell types making up a tissue. We have developed a method for isolating vessels from the brain in order to understand the changes occurring in the vessels during the pathogenesis of cerebral malaria. Vessels were visualised by incubating sections of mouse brain with a substrate for alkaline phosphatase. Vessels were collected by laser capture microdissection and the specificity was monitored by measuring the expression of cell-specific markers. RNA from the captured vessels was highly enriched in mRNA for genes associated with endothelial cells and pericytes. Measurement of indoleamine 2,3-dioxygenase mRNA indicated it was possible to detect changes in gene expression, due to malaria infection, occurring specifically within the vessels. Laser capture microdissection can be used to study changes in gene expression occurring at the blood-brain barrier.

Alkaline Phosphatase↗
PubMed2003

Elevated osteopontin and thrombospondin expression identifies malignant human breast carcinoma but is not indicative of metastatic status.

BACKGROUND: Our previous characterization of a human breast tumor metastasis model identified several candidate metastasis genes. The expression of osteopontin (OPN) correlated with the metastatic phenotype, whereas thrombospondin-1 (TSP-1) and tyrosinase-related protein-1 (TYRP-1) correlated with the nonmetastatic phenotype of independent MDA-MB-435 cell lines implanted orthotopically into athymic mice. The aim of the present study was to examine the cellular distribution of these molecules in human breast tissue and to determine whether the relative expression level of these three genes is associated with human breast tumor metastasis. METHODS: Sixty-eight fresh, frozen specimens including 31 primary infiltrating ductal carcinomas, 22 nodal metastases, 10 fibroadenomas, and five normal breast tissues were evaluated for OPN expression, TSP-1 expression and TYRP-1 expression. Immunohistochemistry was performed to monitor the cellular distribution and to qualitatively assess expression. Quantitative analysis was achieved by enrichment of breast epithelial cells using laser-capture microdissection and subsequent real-time, quantitative PCR. RESULTS: The epithelial components of the breast tissue were the source of OPN and TSP-1 expression, whereas TYRP-1 was present in both the epithelial and stromal components. Both OPN and TSP-1 expression were significantly higher in malignant epithelial sources over normal and benign epithelial sources, but no difference in expression levels was evident between primary tumors with or without metastases, nor between primary and metastatic carcinomas. CONCLUSION: Elevated expression of OPN and TSP-1 may play a role in the pathogenesis of breast cancer. The multiplex analysis of these molecules may enhance our ability to diagnose and/or prognosticate human breast malignancy.

Biomarkers, Tumor↗
PubMed2006

Microchemical analysis of laser-microdissected stone cells of Norway spruce by cryogenic nuclear magnetic resonance spectroscopy.

Stone cells (sclereids) in Norway spruce (Picea abies) bark have been reported to be highly lignified tissues that are important in physical defence against bark beetle invasion. Microchemical analyses of the low-molecular weight compounds in the stone cells of Norway spruce were carried out using laser microdissection in combination with cryogenic nuclear magnetic resonance and mass spectrometry (LMD/NMR/MS). Two phenolic compounds, the stilbene astringin and the dihydroflavonol dihydroxyquercetin 3'-O-beta-D-glucopyranoside, were identified indicating that stone cells are more than just repositories for lignin. Both of these compounds were also found to be present in other phloem tissue at a higher level than in the stone cells based on quantification by cryogenic 1H NMR. Our results suggest that stone cells may be involved in chemical as well as physical defense against bark beetles and their associated microorganisms. This paper reports on the identification of secondary plant metabolites from a single laser-microdissected population of plant cells offering a sensitive new way to determine the chemical profile of specific plant cell types with a high degree of precision.

Lasers↗
PubMed2003

Glycosyltransferase expression in human colonic tissue examined by oligonucleotide arrays.

For an understanding of tumor-related alterations of the complex carbohydrate pattern of carcinomas, it is indispensable to monitor the expression profile of the various glycosyltransferases. The objective of this contribution was to perform an evaluation of the usefulness and the limits of the microarray approach for the identification of enzymes responsible for carbohydrate synthesis with differential expression in carcinomas. Expression profiles of colonic carcinomas were studied by oligonucleotide arrays using a novel strategy: colonic tissue of healthy individuals was compared with early staged colonic carcinomas; 'pure' cell populations were obtained by laser microdissection; RNA samples for hybridization with the oligonucleotide arrays were prepared by in vitro transcription without additional amplification. Expression of 39 glycosyltransferases and of 10 sulfotransferases in colonic tissues was analyzed by Affymetrix GeneChip technology. GeneChip analysis proved the high expression level of ST6Gal-I, beta4Gal-TI, II and III, GalNacT-1, FT-III and showed that ST3Gal-IV was the most abundantly expressed enzyme in healthy tissue. The strong overexpression of FT-VI in healthy tissue has not been described so far, as well as the upregulation of FT-VIII and downregulation of GnT-I in carcinoma tissue. Quantitative RT-PCR confirmed that FT-VI expression was significantly enhanced in healthy tissue. On the other hand, GeneChip analysis failed to detect any expression of GnT-III and GnT-V as well as of ST3Gal-I and ST3Gal-II, although these sequences could be amplified from the samples used for microarray analysis. According to our restricted analysis of only those 39 glycosyltransferases present on the GeneChip U95A, alterations of sialyltransferases ST6Gal-I, ST3Gal-IV, of fucosyltransferases FT-VI, FT-III, and probably FT-VIII, of GalNacT-I, and of beta4GalT-II seem to be of relevance for the aberrant biosynthesis of membrane-bound carbohydrates during colonic carcinogenesis and metastasis.

Aged↗
PubMed2005

Intracellular localization of oxidized low-density lipoproteins in atherosclerotic plaque cells revealed by electron microscopy combined with laser capture microdissection.

Recently, laser capture microdissection (LCM) has become a powerful tool for investigating the genome and protein contents of cells populating normal and pathologically altered tissues. The present work reports a technique for the preparation of tissue specimens for further LCM combined with electron microscopy. In this work, atherosclerotic plaque cells containing oxidized low-density lipoproteins (LDL) were microdissected from human carotid arteries and the intracellular distribution of oxidized LDL was examined, providing novel information about the association of microvesicles containing oxidized LDL with "lipid droplets" in macrophage foam cells.

Aged↗