Laser Capture Microdissection Technology Research Articles

Abstract 3768: Optimization of digital pathology through laser capture microdissection with a 405 nm laser

Abstract Tumor cellular heterogeneity is a complex problem in cancer molecular diagnostics and personalized therapy. The tumor is a product of the different types and interactions of host and immune cells. Investigators have created a variety of methods to procure separate subpopulations of the tumor microenvironment for individual analysis. One of the most successful methods of this is laser capture microdissection (LCM). This method captures specific subpopulations of cells under direct microscopic visualization. This LCM technology has successfully been used for over 20 years, revealing a variety of insights into cancer pathogenesis and mechanisms of therapeutic response employed in numerous clinical trials. Nevertheless, there are two drawbacks to the current systems. For UV cutting (365 nm), the tissue is significantly damaged by the UV light energy. Infrared (808 nm) laser systems do not damage the tissue however, this method has low power and poor resolution. Our first hypothesis to be tested is that a new class of near UV 405 nm laser will have small capture areas approaching single cells while simultaneously preventing cellular damage. The second weakness of LCM is the requirement for an open-faced tissue section without a coverslip. Moreover, when visualizing the tissue of interest, the identification of regions of interest is obscured due to the refractive index mismatch between the laser microdissection cap and the tissue surface. We hypothesize that using a volatile organic compound with a refractive index similar to glass would elucidate the tissue region of interest to allow proper visualization. We successfully developed and tested a near 405 nm wavelength LCM system that utilizes a “liquid” coverslip to visualize the sample of interest with higher clarity, resolution, and contrast for immunohistochemistry, immuno-fluorescence, blood smear slides, and conventional hematoxylin-eosin tissue staining. Overall, these critical improvements LCM technology now allow for single-cell tissue capture, system automation, tissue visualization, and opens the door for artificial intelligence-assisted spatial profiling. Citation Format: Thomas Philipson, Marissa Howard, Kevin Johnson, Amanda Still, Furkat Yunusov, Emanuel Petricoin, Virginia Espina, Noel Gonzalez, Alan Carpino, Lance Liotta. Optimization of digital pathology through laser capture microdissection with a 405 nm laser [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3768.

Laser Capture Microdissection in the Spatial Analysis of Epigenetic Modifications in Skin: A Comprehensive Review

Each cell in the body contains an intricate regulation for the expression of its relevant DNA. While every cell in a multicellular organism contains identical DNA, each tissue-specific cell expresses a different set of active genes. This organizational property exists in a paradigm that is largely controlled by forces external to the DNA sequence via epigenetic regulation. DNA methylation and chromatin modifications represent some of the classical epigenetic modifications that control gene expression. Complex tissues like skin consist of heterogeneous cell types that are spatially distributed and mixed. Furthermore, each individual skin cell has a unique response to physiological and pathological cues. As such, it is difficult to classify skin tissue as homogenous across all cell types and across different environmental exposures. Therefore, it would be prudent to isolate targeted tissue elements prior to any molecular analysis to avoid a possibility of confounding the sample with unwanted cell types. Laser capture microdissection (LCM) is a powerful technique used to isolate a targeted cell group with extreme microscopic precision. LCM presents itself as a solution to tackling the problem of tissue heterogeneity in molecular analysis. This review will cover an overview of LCM technology, the principals surrounding its application, and benefits of its application to the newly defined field of epigenomics, in particular of cutaneous pathology. This presents a comprehensive review about LCM and its use in the spatial analysis of skin epigenetics. Within the realm of skin pathology, this ability to isolate tissues under specific environmental stresses, such as oxidative stress, allows a far more focused investigation.

Protocol of Transcriptome Analysis of Decidual Placenta Cells

The advent of high-throughput sequencing technologies has expanded our understanding of the biological significance of non-coding regions of the genome. In recent years, more and more studies have been devoted to studying the role of noncoding RNAs in the development of diseases, as well as their participation in various cellular processes. Until now, all transcriptome studies of native placental tissue with the description of the noncoding RNA region were carried out without isolating individual cell populations. This approach, due to the high cellular heterogeneity of the placental tissue, significantly complicates the ability to determine the molecular-biological functions of individual cells and their role in the molecular pathogenesis of reproductive disorders. In this work, we propose a technique for obtaining total RNA from single decidual cells of frozen placental tissue obtained by laser-capture microdissection technology for transcriptome sequencing, including a cluster of noncoding RNAs. This technique can be successfully used to study the full-genome expression profile of other placental cell populations. The high accuracy of results on the transcriptome profiling of decidual cells obtained using the developed technique was additionally confirmed by an integrative analysis with the results of a 10x Genomics experiment.

Laser capture microdissection: techniques and applications in liver diseases.

Routine transcriptomic and proteomic analysis are usually performed at a whole organ or tissue level. These approaches provide an average readout of all cell types present within the tissue but do not allow differentiating the profile of specific cell populations. Laser capture microdissection (LCM) constitutes an excellent tool to isolate cell populations or areas of interest within a tissue. By direct visualization, the selected area is excised by a laser and can be further processed for a variety of downstream analyses. This technology has been widely used in the study of liver diseases, from DNA and RNA sequencing to mass spectrometry. However, LCM also has important limitations. To ensure the best integrity of the molecule of interest, optimal tissue preservation, careful tissue sectioning, and optimization of the staining procedure are required. The present review provides a description of the LCM technology, including tips and technical recommendations to perform the procedure, as well as an overview of studies using LCM technology in the field of liver disease.

The Isolation of Pure Populations of Neurons by Laser Capture Microdissection: Methods and Application in Neuroscience.

In mammals, the central nervous system (CNS) is constituted of various cellular elements, posing a challenge to isolating specific cell types to investigate their expression profile. As a result, tissue homogenization is not amenable to analyses of motor neurons profiling as these represent less than 10% of the total spinal cord cell population. One way to tackle the problem of tissue heterogeneity and obtain meaningful genomic, proteomic, and transcriptomic profiling is to use laser capture microdissection technology (LCM). In this chapter, we describe protocols for the capture of isolated populations of motor neurons from spinal cord tissue sections and for downstream transcriptomic analysis of motor neurons with RT-PCR. We have also included a protocol for the immunological confirmation that the captured neurons are indeed motor neurons. Although focused on spinal cord motor neurons, these protocols can be easily optimized for the isolation of any CNS neurons.

Using Laser Capture Microdissection to Isolate Cortical Laminae in Nonhuman Primate Brain.

Laser capture microdissection (LCM) is a technique that allows procurement of an enriched cell population from a heterogeneous tissue sample under direct microscopic visualization. Fundamentally, laser capture microdissection consists of three main steps: (1) visualizing the desired cell population by microscopy, (2) melting a thermolabile polymer onto the desired cell populations using infrared laser energy to form a polymer-cell composite (capture method) or photovolatizing a region of tissue using ultraviolet laser energy (cutting method), and (3) removing the desired cell population from the heterogeneous tissue. In this chapter, we discuss the infrared capture method only. LCM technology is compatible with a wide range of downstream applications such as mass spectrometry, DNA genotyping and RNA transcript profiling, cDNA library generation, proteomics discovery, and signal pathway mapping. This chapter profiles the ArcturusXT™ laser capture microdissection instrument, using isolation of specific cortical lamina from nonhuman primate brain regions, and sample preparation methods for downstream proteomic applications.

Exploring the potential of laser capture microdissection technology in integrated oral biosciences.

Laser capture microdissection (LCM) is a high-end research and diagnostic technology that helps in obtaining pure cell populations for the purpose of cell- or lesion-specific genomic and proteomic analysis. Literature search on the application of LCM in oral tissues was made through PubMed. There is ample evidence to substantiate the utility of LCM in understanding the underlying molecular mechanism involving an array of oral physiological and pathological processes, including odontogenesis, taste perception, eruptive tooth movement, oral microbes, and cancers of the mouth and jaw tumors. This review is aimed at exploring the potential application of LCM in oral tissues as a high-throughput tool for integrated oral sciences. The indispensable application of LCM in the construction of lesion-specific genomic libraries with emphasis on some of the novel molecular markers thus discovered is also highlighted.

Editorial: Transport in Plant Microbe Interactions.

Editorial: Transport in Plant Microbe Interactions.

Identification of a core set of rhizobial infection genes using data from single cell-types.

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4′,4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

Laser capture microdissection: Big data from small samples.

Any tissue is made up of a heterogeneous mix of spatially distributed cell types. In response to any (patho) physiological cue, responses of each cell type in any given tissue may be unique and cannot be homogenized across cell-types and spatial co-ordinates. For example, in response to myocardial infarction, on one hand myocytes and fibroblasts of the heart tissue respond differently. On the other hand, myocytes in the infarct core respond differently compared to those in the peri-infarct zone. Therefore, isolation of pure targeted cells is an important and essential step for the molecular analysis of cells involved in the progression of disease. Laser capture microdissection (LCM) is powerful to obtain a pure targeted cell subgroup, or even a single cell, quickly and precisely under the microscope, successfully tackling the problem of tissue heterogeneity in molecular analysis. This review presents an overview of LCM technology, the principles, advantages and limitations and its down-stream applications in the fields of proteomics, genomics and transcriptomics. With powerful technologies and appropriate applications, this technique provides unprecedented insights into cell biology from cells grown in their natural tissue habitat as opposed to those cultured in artificial petri dish conditions.

Laser capture microdissection in Ectocarpus siliculosus: the pathway to cell-specific transcriptomics in brown algae.

Laser capture microdissection (LCM) facilitates the isolation of individual cells from tissue sections, and when combined with RNA amplification techniques, it is an extremely powerful tool for examining genome-wide expression profiles in specific cell-types. LCM has been widely used to address various biological questions in both animal and plant systems, however, no attempt has been made so far to transfer LCM technology to macroalgae. Macroalgae are a collection of widespread eukaryotes living in fresh and marine water. In line with the collective effort to promote molecular investigations of macroalgal biology, here we demonstrate the feasibility of using LCM and cell-specific transcriptomics to study development of the brown alga Ectocarpus siliculosus. We describe a workflow comprising cultivation and fixation of algae on glass slides, laser microdissection, and RNA amplification. To illustrate the effectiveness of the procedure, we show qPCR data and metrics obtained from cell-specific transcriptomes generated from both upright and prostrate filaments of Ectocarpus.

Laser‐Capture Microdissection

Laser‐Capture Microdissection

Laser capture microdissection for protein and NanoString RNA analysis.

Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are (a) visualization of cells via light microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810 nm), while in the cutting mode the laser is ultraviolet (355 nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes LCM using an Arcturus(XT) instrument for downstream protein sample analysis and using an mmi CellCut Plus® instrument for RNA analysis via NanoString technology.

Comparison and optimization of DNA recovery from sperm vs. epithelial cells using laser capture microdissection technology and an immunofluorescent staining system

Laser capture microdissection (LCM) is an accurate and robust tool for isolation and separation of pure cell populations from heterogeneous mixtures via direct visualization and collection of elements. In this study, the capability of the P.A.L.M. ® LCM for separation of sperm and epithelial cells, combined with immunofluorescent Sperm HY-LITER™ stain was assessed. LCM was evaluated for its sensitivity, specificity, and reproducibility using sperm–epithelial mixture slides with varying sperm counts and ages ranging 1–9 years. In addition, slides stained with immunofluorescent dyes were tested. Separation of sperm and epithelial cells using LCM is a robust method for processing of mixed samples and generates interpretable single source DNA profiles. Immunofluorescent staining method is compatible with the platform.

Methylation Profiling of Mediastinal Gray Zone Lymphoma Reveals a Distinctive Signature with Elements Shared by Classical Hodgkin's Lymphoma and Mediastinal Large B-Cell Lymphoma

AbstractAbstract 747 Background:Mediastinal gray zone lymphoma (MGZL) is a newly recognized entity demonstrating transitional morphologic and immunophenotypic features between classical Hodgkin's lymphoma, nodular sclerosis subtype (CHLNS) and primary mediastinal large B-cell lymphoma (PMLBCL). CHLNS and PMLBCL differ in morphology, immunophenotype, and therapeutic consequences. MGZLs present a challenge both to the pathologist and clinician, as the criteria to distinguish MGZL from CHLNS and PMLBCL are still imprecise, and the optimal treatment approach is as yet undetermined. Purpose of Study:Epigenetic changes have been implicated in the loss of the B-cell program in CHL, and might provide a basis for the immunophenotypic alterations seen in MGZL. Thus, we performed a large scale DNA methylation array of MGZL, CHLNS, and PMLBCL as well as diffuse large B-cell lymphoma (DLBCL) to investigate the biological underpinnings of MGZL and how it corresponds to the two related entities CHLNS and PMLBCL and the less related entity DLBCL. Microdissection of tumor cells was performed to identify changes in the tumor cell population, and allow comparison with the background inflammatory and stromal milieu. Results:Principal component analysis (PCA) demonstrated that MGZLs have a distinct epigenetic profile intermediate between CHLNS and PMLBCL but clearly different from that of DLBCL. Analysis of common hypo- and hypermethylated CpG targets in MGZL, CHLNS, PMLBCL and DLBCL was performed. MGZL showed great overlap with CHLNS (49 common targets) and PMLBCL (50 common targets). In contrast, MGZL shared only two common targets with DLBCL. However, using the epigenetic profiles we were able to establish class prediction models that could distinguish between MGZL, CHLNS and PMLBCL with a final combined prediction of 100%. Pyrosequencing for selected CpG sites from different genes were performed and confirmed the high accuracy of methylation array results. Conclusions:MGZLs share several clinical and pathological features with CHLNS and PMLBCL. Our findings further underscore the close biological relationship between MGZL, CHLNS and PMLBCL, and ready distinction from DLBCL. However, MGZL has a distinct epigenetic identity that shares elements of both parent disorders. As the first biological study on MGZL, our results provide novel insights into MGZL pathogenesis, and its relationship to CHLNS and PMLBCL. Disclosures:Emmert-Buck:NIH: Michael Emmert-Buck is an inventor on all NIH patents covering laser capture microdissection technology and receives royalty payments through the NIH technology transfer program., Patents & Royalties.

BioSpotlight

BioSpotlight

Laser Capture Microdissection in Dentistry

Laser capture microdissection (LCM) allows for the microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. According to the recent development of the LCM technologies and methodologies, the LCM has been used in various kinds of tissue specimens in dental research. For example, the real-time polymerase-chain reaction (PCR) can be performed from the formaldehyde-fixed, paraffin-embedded, and immunostained sections. Thus, the advance of immuno-LCM method allows us to improve the validity of molecular biological analysis and to get more accurate diagnosis in pathological field in contrast to conventional LCM. This paper is focused on the presentation and discussion of the existing literature that covers the fields of RNA analysis following LCM in dentistry.

Expression of immunoreactivity and genetic mutation in eosinophilic and ciliated metaplastic changes of endometrial glandular and stromal breakdown: cytodiagnostic implications

Various metaplastic changes may be present in endometrium, in which also cellular atypias may often be observed. Particularly, eosinophilic and ciliated changes (ECCs) occur in both nonneoplastic and neoplastic endometrium. This may cause confusion in the cytodiagnosis. This study was enterprised to investigate the possible help of immunocytochemical and cytogenetic study in the diagnostic and biologic assessment of ECC cells. In immunocytochemistry for p53 protein, Ki-67, and cyclin A, the material consists of 40 cases of cytologic smears examined by direct sampling of the endometrial cavity comprising 30 cases of ECC in endometrial glandular and stromal breakdown (EGBD) and 10 cases of well-differentiated adenocarcinoma (G1). After cytodiagnosis, immunostaining for p53 protein, Ki-67, and cyclin A was performed on multiple wet-fixed slides from each single case to evaluate the immunoreactivity, intensity of nuclear staining, and nuclear labeling index (N-LI). The intensity of nuclear staining was scored as negative (0), weak (1), moderate (2), or strong (3), and the N-LI was scored as less than 10% (0), from 10% to 25% (1), from 26% to 50% (2), or more than 50% (3), and the final score was calculated by adding both partial scores. A statistical significance test was performed by using Mann-Whitney U test, and the result was judged as significant when the P value was less than .05. For genetic mutation analysis of p53, the material comprised 6 cases of EGBD in which a high score was measured with immunocytochemistry for p53 protein, and the presence of ECC was confirmed on the hematoxylin and eosin. The ECC cells on paraffin-embedded specimens were captured using laser capture microdissection technology. Mutations in p53 gene (exons 5-8) were examined using DNA sequencing analysis. In immunocytochemistry for p53 protein, Ki-67, and cyclin A, the proportions of immunoreactive cells for p53 were statistically higher in ECC compared with those of G1 (P < .05). The proportions of the immunoreactive cells for Ki-67 and cyclin A were statistically higher in G1 compared with those of ECC (P < .05). (2) In genetic mutation analysis of p53, DNA sequencing of p53 in 6 cases revealed no mutations. The percentage of immunoreactive cells for p53 protein were higher in ECC than in G1, but the mutation point was not confirmed in genetic mutation analysis. The differential expression of these biologic parameters in ECC cells could be considered of possible relevance to the cytopathologic diagnosis in the future.

Laser-capture Microdissection and Protein Extraction for Protein Fingerprint of OSCC and OLK

To find new biomarkers and establish histopathology protein fingerprint models for early detection of oral squamous cell carcinoma (OSCC), laser capture microdissection (LCM) technology was utilized in 21 OSCC tissues and 7 oral leukoplaque (OLK) tissues as well as their adjacent normal tissues. Each sample was then detected by SELDI-TOF-MS technology and CM10 protein chip as well as bioinformatics tools. Three proteomic biomarker patterns were identified. Pattern 1 distinguishes patients with OLK from healthy individuals. Pattern 2 distinguishes patients with OSCC from healthy individuals. Pattern 3 distinguishes patients with OSCC from patients with OLK. The analysis yielded both a specificity and a sensitivity of 90.48% for pattern 1, a specificity of 100.00% and a sensitivity of 85.71% for pattern 2, and a specificity of 100.00% and a sensitivity of 85.71% for pattern 3. Proteome mass/charge 3714, 3515, and 4944 built the distinguished protein peaks between the OSCC tumor and adjacent normal tissues. The accuracy of the blind prediction was 90.48% (38/42). M/Z 15122 and 7569 built the distinguished protein peaks between the OLK and adjacent normal tissues. M/Z 3738 and 11366 built the distinguished protein peaks between the OSCC and the OLK. By employing LCM technology combined with SELDI-TOF-MS technology and bioinformatics approaches, histopathology would not only facilitate the discovery of better biomarkers for OSCC and OLK, but also provide a useful tool for molecular diagnosis by potential biomarker.

Laser capture microdissection technology

Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions through molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser capture microdissection is a method to procure subpopulations of tissue cells under direct microscopic visualization. Laser capture microdissection technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, mass spectrometry proteomics discovery and signal pathway profiling.