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PubMed1998

[PyNPase expression and cancer progression in the colorectum].

We analyzed PyNPase expression discriminating between cancer and tumor stroma of the colorectum by Western blotting using a newly developed extraction method from microdissected tissue sections fixed with buffered formalin. Analysis of 98 colorectal cancers revealed that PyNPase was as high as 70.2 +/- 18.5 unit/mg protein in the stroma fraction (SF), whereas it was 45.1 +/- 10.5 in the cancer fraction (CF) (p < 0.0001). Vessel density was correlated with PyNPase in the SF but not in the CF. In stage IIIb, 11 cases expressing a high level of PyNPase in the CF showed poorer prognosis than 10 cases with low-level PyNPase expression (p < 0.05), although the level of PyNPase expression in the SF did not affected the patients prognosis. Immunohistochemical examination indicated that PyNPase in the SF was mainly produced by macrophages (M phi), and therefore we investigated the profile of PyNPase production by M phi. In in vitro experiments PyNPase production by M phi was greatly enhanced by stimulation with OK-432, and the culture supernatant had the ability to convert 5'DFUR to 5-FU.

Antineoplastic Agents↗
PubMed1999

Perivascular cells harboring multiple endocrine neoplasia type 1 alterations are neoplastic cells in angiofibromas.

Although neoplasia is caused by clonal proliferation of cells, the resulting tumors are frequently heterogeneous, being composed of both neoplastic and reactive cells. Therefore, identification of tumors as neoplastic processes is frequently obscured. We studied cutaneous angiofibroma, which is a tumor of unknown etiology. Combined analysis using immunohistochemistry, selective tissue microdissection, fluorescence in situ hybridization, sequencing analysis, and deletion analysis of the multiple endocrine neoplasia type 1 locus succeeded in the identification of a population of genetically altered, neoplastic cells in these tumors. This approach may be valuable in the future in identifying the etiology of other tumors of unknown etiology.

Angiofibroma↗
PubMed2003

Laser capture microdissection of cells from plant tissues.

Laser capture microdissection (LCM) is a technique by which individual cells can be harvested from tissue sections while they are viewed under the microscope, by tacking selected cells to an adhesive film with a laser beam. Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein spectra from individual cell types. We have optimized LCM for a variety of plant tissues and species, permitting the harvesting of cells from paraffin sections that maintain histological detail. We show that RNA can be extracted from LCM-harvested plant cells in amount and quality that are sufficient for the comparison of RNAs among individual cell types. The linear amplification of LCM-captured RNA should permit the expression profiling of plant cell types.

DNA, Plant↗
PubMed2005

Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix-assisted laser desorption/ionisation-time of flight/time of flight-mass spectrometry.

Appropriate methods for the analysis of microdissected solid tumour tissues by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing approximately 200 000 cells, which were solubilised either in urea containing buffer, trifluoroethanol/NH4HCO3, 0.1% sodium dodecyl sulphate (SDS) or in 0.1% RapiGest solution, then trypsin digested and analysed by MALDI-TOF MS. Solubilisation in 0.1% SDS resulted in detection of the highest number of sample specific peak signals. Interestingly, there was little overlap in detectable peaks using the different buffers, implying that they can be used complementarily to each other. Additionally, we fractionated tryptic digests on a monolithic high-performance liquid chromatography column. Fractionation of tryptic digest from whole tissue sections resulted in a four-fold increase in the total number of peaks detected. To prove this principle, we used 0.1% SDS to generate peptide patterns from 2000 microdissected tumour and stromal cells from five different breast carcinoma tumours. The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI-TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues.

Breast Neoplasms↗
PubMed2001

Molecular analysis of complex tissues is facilitated by laser capture microdissection: critical role of upstream tissue processing.

Every tissue contains heterogeneous cell populations. Laser capture microdissection (LCM) facilitates cell isolation from complex tissues followed by molecular analysis. LCM entails placing a transparent film over a tissue section or a cytological sample, visualizing the cells microscopically, and selectively adhering the cells of interest to the film with a focused pulse from an infrared laser. The film with the procured cells is then removed from the original sample and placed directly into DNA, RNA, or protein-extraction buffer for processing. LCM has revolutionized molecular analysis of complex tissues because it combines the topographic precision of microscopy with the power of molecular genetics, genomics, and proteomics. However, the success of molecular analysis still depends on the experimental design and requires the understanding of each technical step involved in specimen preparation. This review attempts to rationalize and demystify the choice of various technical options in upstream tissue processing supporting global analytical strategies.

Animals↗
PubMed1993

Adenylate cyclase activity in microdissected rat liver tissue: periportal to pericentral activity gradient.

Adenylate cyclase activity was measured in microdissected samples from lyophilized cryostat sections of rat liver by means of an improved assay. Livers were obtained from adult Sprague-Dawley rats fasted for 22 hr. Adenylate cyclase activities, basal and those elicited by various agents, were determined in dissected samples from periportal and pericentral regions of the classic liver lobule. In all samples, enzyme activity was strongly stimulated by glucagon, cholera toxin, guanosine-5'-O-(3-thiotriphosphate), sodium fluoride and forskolin. The beta-adrenergic agonist isoproterenol produced very weak, if any, enzyme stimulation. Angiotensin II did not inhibit the activity elicited by lithium chloride and GTP at high concentrations, and pertussis toxin did not enhance the GTP-stimulated activity. We observed a periportal-to-pericentral gradient for basal and agent-stimulated activities.

Adenylyl Cyclases↗
PubMed2003

Differential gene expression analysis using paraffin-embedded tissues after laser microdissection.

Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.

Acetic Acid↗
PubMed1996

Detection of frequent allelic loss of 6q23-q25.2 in microdissected human breast cancer tissues.

Detection of allelic loss in human breast cancer is hindered by the fact that breast cancer tissues are frequently infiltrated by stromal and inflammatory cells. For this study, we carefully microdissected infiltrating breast cancer tumor cells from contaminating normal cells and analyzed the DNA from these samples for allelic loss on the long arm of chromosome 6 by using a panel of 15 dinucleotide repeat markers. We found 53 of the 66 cases studied (80%) to have allelic loss of either the entire chromosomal arm (37 cases) or a portion of the chromosomal arm (16 cases). One common region that was identified for all tumors with deletions of 6q was the area between markers D6S310/314 and D6S473/255, consistent with a tumor suppressor gene locus at 6q23-6q25.2. The use of tissue microdissection allowed the detection of allelic loss in this chromosomal region in human breast cancer at a much higher frequency than was previously recognized.

Adult↗
PubMed1999

Laser capture microdissection of single cells from complex tissues.

Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 microns. A computer-controlled arm precisely positions a 40-micron-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.

Animals↗
PubMed2002

Telomerase activity in microdissected human breast cancer tissues: association with p53, p21 and outcome.

To evaluate the invasive potential of early tumorous lesions of the breast, especially DCIS, we have analyzed semiquantitative telomerase activity in well defined microdissected tissue areas from malignant or benign breast lesions from 145 patients. In order to prove the relationship to cell cycle defects, p53 and p21 proteins were analyzed in corresponding cryostat sections by immunohistochemistry. Telomerase activity was detected in 3 (33.3%) out of 9 benign breast lesions and 109 (80.1%) of 136 malignant tumors. Our study failed to demonstrate an association between telomerase activity, p21, established prognostic factors, such as lymph node status and metastasis, and clinical outcome. We found a high rate of cases (42.2%) with intratumoral heterogeneity concerning telomerase activity status. Telomerase heterogeneity was more obvious in samples with mixed tissue types, although we could not assign specifically the telomerase activity to lobular, ductal and intraductal tissue areas. Telomerase activity was detected in 81.8% of ductal carcinoma in situ (DCIS) which indicates telomerase reactivation as an early event in carcinogenesis. Fifteen (19.5%) out of 77 cases were positive for p53 protein in immunohistochemistry and found to be significantly more frequent in the subgroup with poor outcome. There was only a trend to an association of p53 with high level of telomerase. The prognostic relevance of telomerase activity for patients with benign breast lesions must be further investigated.

Adult↗
PubMed2003

Targeting hepatocytes from liver tissue by laser capture microdissection and proteomics expression profiling.

A tissue proteomics process is presented where hepatocyte cell isolation in combination with two-dimensional (2-D) gel electrophoresis and mass spectrometric identification were used to annotate the liver proteome. Laser microdissection of 8 microm liver tissue sections was performed and protein expression profiling was compared using a variety of quantities of input cells, and gel separation conditions. The 30 microm diameter laser generated the highest protein yields from the polymer coated caps following microsolubilization. We found that 6000 laser pulses (approximately 7200 hepatocytes) were required in order to generate high-resolution gel maps. Within homogeneous tissue samples, this could be accomplished in a total cycle time of 20 min using an automated dissection procedure. Close to 1000 high-quality gel annotations were generated from the corresponding 2-D gel expression profiles which matched closely the corresponding patterns of analytical-scale liver preparations detected by silver staining.

Animals↗
PubMed1998

Methylation of the HIC-1 candidate tumor suppressor gene in human breast cancer.

HIC-1 (hypermethylated in cancer) is a candidate tumor suppressor gene which is located at 17p13.3, a region which frequently undergoes allelic loss in breast and other human cancers. HIC-1 is proposed to be commonly inactivated in human cancers by hypermethylation of a normally unmethylated dense CpG island which encompasses the entire gene. To study whether HIC-1 inactivation may be important to the development of breast cancer, we first measured methylation of the HIC-1 gene in normal breast ductal tissues from microdissected frozen breast tissues and from epithelial cells purified from mammoplasty specimens. Surprisingly, in all normal breast ductal tissues we found approximately equal amounts of densely methylated HIC-1 and completely unmethylated HIC-1. This is in contrast to most normal tissues, in which all copies of HIC-1 are completely unmethylated. We then evaluated 39 primary breast cancer tissues and found virtually complete methylation of the HIC-1 gene in 26 (67%) of the cases. We also found loss of heterozygosity at the telomeric portion of chromosomal arm 17p in 22 of the 26 cases with strongly methylated HIC-1, suggesting that loss of an unmethylated HIC-1 allele may contribute to the inactivation of HIC-1 in cells with a pre-existing methylated allele. Finally, by RNase protection analysis, HIC-1 was found to be expressed in microdissected normal breast ductal tissues and unmethylated tumors but not in tumors with hypermethylation of the HIC-1 gene. These results indicate that hypermethylation of HIC-1 and associated loss of HIC-1 expression is common in primary breast cancer. Furthermore, the HIC-1 gene is densely methylated in approximately one-half of the alleles in normal breast epithelium, which may predispose this tissue to inactivation of this gene by loss of heterozygosity.

Breast Neoplasms↗
PubMed2002

Use of laser microdissection in complex tissue.

Concomitant with the rapid development in biomedical knowledge, including the methods of molecular biology and proteomics, and the manufacture of ever more precise optical instruments, powerful lasers, and sophisticated microcomputing hardware and software, laser microdissection systems have emerged which are now entering the field of routine research. Today, several devices are commercially available, congresses devoted to the latest advances in laser microdissection are now held on regular occasions, and the number of publications based on the use of these techniques has risen to over 250. With laser microdissection, histological treatment, such as chemical or immunological fixation and staining, can readily be combined with methods suitable for molecular biology or proteomics. As the optical, technical, and methodological resolution of polymerase chain reaction (PCR) and microdissection increases, genetic and phenotypic studies of biological material are possible even at the level of single cells and subcellular elements. Moreover, questions such as the paracrine interaction of cells within complex tissues, the development of cancer, and the role of single cells in tissue remodeling or development on the microscopic and molecular level can now be addressed precisely at the molecular level. This chapter reviewed the development of laser microdissection platforms, its potential impact on the future of research, and how, in particular, these technologies can be successfully integrated into modern research and routine histopathological studies of complex tissue.

Animals↗
PubMedDate not supplied

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.

Biopsy↗
PubMed1994

Clonality analysis of defined B-cell populations in archival tissue sections using microdissection and the polymerase chain reaction.

A simple microdissection technique involving the use of a drawn-out glass pipette was developed for isolation of defined cell subsets from tissue sections. Using this technique and the polymerase chain reaction (PCR), clonally rearranged immunoglobulin (Ig) heavy chain genes were reliably amplified in single neoplastic follicles or few hundreds of tumour cells isolated from archival haematoxylin and eosin or immunostained sections of B-cell lymphomas. A polyclonal nature was consistently demonstrated in reactive lymphoid follicles or interfollicular reactive B-cells within the same lymphoma sections. Microdissection of lymphoma cells from within foci of chronic inflammation improved the resolution of tumour-specific PCR products by reducing amplification of background polyclonal B-cell sequences. The combination of microdissection and PCR techniques, therefore, provides an important tool for the investigation of B-cell lymphomas and also allows simple and specific access for other molecular genetic analyses of different cell subsets on tissue sections.

B-Lymphocytes↗
PubMed1979

Micropreparation techniques in quantitative histochemistry - density gradient centrifugation, manual microdissection and laser microbeam preparation of tissue.

Each quantitative histochemical problem needs its specific method for tissue preparation. In this connection two of the most important preparation methods, density gradient centrifugation and microdissection of freeze-dried tissue slices, are described. Density gradient centrifugation is a very effective procedure for preparative separation of cell particles such as cell nuclei. The details of the preparation of glial and neuronal cell nuclei are described. The in vitro phosphorylation of histone in the chromatin in relation to age is given as a practical example of the quantitative histochemical application to a preparation of cell nuclei. Other techniques of tissue preparation are the manual tissue microdissection according to Lowry and the Laser microbeam preparation. Advantages and disadvantages of both methods are compared. It is shown, that the introduction of Laser microbeam dissection technique, as alternative to manual microdissection, add new dimensions to Lowry's ultramicrochemical methods. One has greater freedom in the choice of the sample size and the number of samples dissected from the same slice. Furthermore, the need for a well-trained person for the preparation is eliminated. The preparation is also considerably less time consuming and easier to perform than the manual free hand preparation. Two quantitative histochemical methods used for the investigation of microdissected tissue samples are described: the gas-chromatography-massfragmentography (GC/MS)-method for determination of transmitters and its metabolites as well as the enzymatic cycling technique of Lowry. The GC/MS-method is explained with an example of noradrenaline and dopamine determination. The enzymatic cycling technique is demonstrated in combination with the Oil-Well-Technique for determination of the NADP-cycle.

Cell Fractionation↗
PubMed1994

Subtraction hybridization cloning of RNA amplified from different cell populations microdissected from cryostat tissue sections.

We describe a generally applicable and easily reproducible method for the cloning of differentially expressed RNA, amplified from small numbers of enriched cell populations, obtained by microdissection from single cryostat sections. The procedure involves homopolymeric A tailing of cDNA synthesized from released RNA using an anchored (NN)T12 primer. Subsequent entire cDNA population polymerase chain reaction amplification was carried out using a biotinylated (X)nT16 primer-adaptor in the presence of biotin-dATP. This biotinylated driver cDNA was then twice hybridized in 50-fold excess to heterologous target cDNA made with nonbiotinylated (Y)nT16 primer; common hybrids and excess driver cDNA were magnetically removed following the addition of streptavidin-coated magnetospheres which bound biotinylated strands, leaving enriched target population sequences. These were then directly amplified through the tails using a primer containing only the target-specific (Y)n sequence. Insertion into a lambda-phage vector was facilitated by means of an EcoR1 site incorporated in the (Y)n primer. Subsequent packaging and transformation into Escherichia coli NM522 resulted in cDNA libraries containing approximately 5 x 10(3)-10(4) pfu. Screening of these primary libraries with cDNA derived from the starting populations yielded a large number of differentially hybridizing clones which are currently under analysis.

Base Sequence↗
PubMed2002

cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue.

Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to approximately 10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs ( approximately 500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from normoxic lungs. Validation by an alternative method was obtained by linking microdissection with real-time polymerase chain reaction (PCR). As suggested by the array data, nine selected genes with different factors of up-regulation were fully confirmed by the PCR technique. Thus, a rapid protocol is presented combining microdissection and array profiling that demands low quantities of initial RNA to assess reliably cell-type-specific gene regulation even within nonneoplastic complex tissues.

Animals↗