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Journal of Genetics and Genomics = Yi... Sep 2023Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context, significantly enhancing our... (Review)
Review
Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context, significantly enhancing our understanding of the intricate and multifaceted biological system. With an increasing focus on spatial heterogeneity, there is a growing need for unbiased, spatially resolved omics technologies. Laser capture microdissection (LCM) is a cutting-edge method for acquiring spatial information that can quickly collect regions of interest (ROIs) from heterogeneous tissues, with resolutions ranging from single cells to cell populations. Thus, LCM has been widely used for studying the cellular and molecular mechanisms of diseases. This review focuses on the differences among four types of commonly used LCM technologies and their applications in omics and disease research. Key attributes of application cases are also highlighted, such as throughput and spatial resolution. In addition, we comprehensively discuss the existing challenges and the great potential of LCM in biomedical research, disease diagnosis, and targeted therapy from the perspective of high-throughput, multi-omics, and single-cell resolution.
Topics: Laser Capture Microdissection; Multiomics; Biomedical Research
PubMed: 37544594
DOI: 10.1016/j.jgg.2023.07.011 -
Kidney International Sep 2019Fibrillary glomerulonephritis is a glomerular disease historically defined by glomerular deposition of Congo red-negative, randomly oriented straight fibrils that lack a... (Review)
Review
Fibrillary glomerulonephritis is a glomerular disease historically defined by glomerular deposition of Congo red-negative, randomly oriented straight fibrils that lack a hollow center and stain with antisera to immunoglobulins. It was initially considered to be an idiopathic disease, but recent studies highlighted association in some cases with autoimmune disease, malignant neoplasm, or hepatitis C viral infection. Prognosis is poor with nearly half of patients progressing to end-stage renal disease within 4 years. There is currently no effective therapy, aside from kidney transplantation, which is associated with disease recurrence in a third of cases. The diagnosis has been hampered by the lack of biomarkers for the disease and the necessity of electron microscopy for diagnosis, which is not widely available. Recently, through the use of laser microdissection-assisted liquid chromatography-tandem mass spectrometry, a novel biomarker of fibrillary glomerulonephritis, DnaJ homolog subfamily B member 9, has been identified. Immunohistochemical studies confirmed the high sensitivity and specificity of DnaJ homolog subfamily B member 9 for this disease; dual immunofluorescence showed its colocalization with IgG in glomeruli; and immunoelectron microscopy revealed its localization to individual fibrils of fibrillary glomerulonephritis. The identification of this tissue biomarker has already entered clinical practice and undoubtingly will improve the diagnosis of this rare disease, particularly in developing countries where electron microscopy is less available. Future research is needed to determine whether DnaJ homolog subfamily B member 9 is an autoantigen or just an associated protein in fibrillary glomerulonephritis, whether it can serve as a noninvasive biomarker, and whether therapies that target this protein are effective in improving prognosis.
Topics: Biomarkers; Chromatography, High Pressure Liquid; Coloring Agents; Congo Red; Disease Progression; Endothelium, Vascular; Glomerulonephritis; HSP40 Heat-Shock Proteins; Humans; Kidney Failure, Chronic; Kidney Glomerulus; Kidney Transplantation; Laser Capture Microdissection; Membrane Proteins; Microscopy, Electron; Microscopy, Fluorescence; Microvessels; Molecular Chaperones; Prognosis; Renal Dialysis; Staining and Labeling; Tandem Mass Spectrometry
PubMed: 31227146
DOI: 10.1016/j.kint.2019.03.021 -
Laboratory Investigation; a Journal of... Jul 2023Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained...
Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.
Topics: MicroRNAs; Microdissection; Epithelial Cells; Formaldehyde; Gene Expression Profiling
PubMed: 36990152
DOI: 10.1016/j.labinv.2023.100133 -
Operative Neurosurgery (Hagerstown, Md.) Jan 2021The cerebellum is one of the most primitive and complex parts of the human brain. The fiber microdissection technique can be extremely useful for neurosurgeons to...
BACKGROUND
The cerebellum is one of the most primitive and complex parts of the human brain. The fiber microdissection technique can be extremely useful for neurosurgeons to understand the topographical organization of the cerebellum's important contents, such as the deep cerebellar nuclei and the cerebellar peduncles, and their relationship with the brain stem.
OBJECTIVE
To dissect the deep cerebellar nuclei and the cerebellar peduncles using the fiber microdissection technique.
METHODS
Under the operating microscope, 5 previously frozen, formalin-fixed human cerebellums and brain stems were dissected from the superior surface, and 5 were dissected from the inferior surface. Each stage of the process is described. The primary dissection tools were handmade, thin, wooden spatulas with tips of various sizes, toothpicks, and a fine regulated suction.
RESULTS
In 15 simplified dissection steps (6 for the superior surface and 9 for the inferior surface), the deep cerebellar nuclei (dentate, interpositus, and fastigial) and the cerebellar peduncles (inferior, middle, and superior) are delineated. Their anatomical relationships with each other and other neighboring structures are demonstrated.
CONCLUSION
The anatomy of the deep cerebellar nuclei and the cerebellar peduncles are clearly defined and understood through the use of the fiber microdissection technique. These stepwise dissections will guide the neurosurgeon in acquiring a topographical understanding of these complex and deep structures of the cerebellum. This knowledge, along with radiological information, can help in planning the most appropriate surgical strategy for various lesions of the cerebellum.
Topics: Brain Stem; Cerebellar Nuclei; Cerebellum; Humans; Microdissection; White Matter
PubMed: 33047123
DOI: 10.1093/ons/opaa318 -
Current Protocols Jul 2023To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed...
To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique requires neither separation of cells by enzymatic digestion of any kind nor cell-specific reporter genes, and it allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capturing of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue Preparation for Laser Capture Microdissection Basic Protocol 2: Tissue Sectioning Basic Protocol 3: Laser Capture Microdissection of Embedded Tissue Basic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples.
Topics: Laser Capture Microdissection; RNA; Plants; Transcriptome; Paraffin Embedding
PubMed: 37486164
DOI: 10.1002/cpz1.844 -
Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine.Methods in Molecular Biology (Clifton,... 2022Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology...
Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.
Topics: Formaldehyde; Humans; Microdissection; Neoplasms; Paraffin Embedding; Polymerase Chain Reaction; Precision Medicine; Tissue Fixation
PubMed: 35094324
DOI: 10.1007/978-1-0716-1811-0_7 -
In Vitro Cellular & Developmental... Oct 2020Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which...
Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing.
Topics: Animal Shells; Animals; Cell Culture Techniques; Cell Proliferation; Cell Survival; Crustacea; Disinfection; Epidermal Cells; Microdissection; X-Ray Microtomography
PubMed: 33034828
DOI: 10.1007/s11626-020-00508-8 -
Biochimica Et Biophysica Acta.... Apr 2022Across a variety of systems, thousands of RNAs are localized to specific subcellular locations. However, for the vast majority of these RNAs, the mechanisms that... (Review)
Review
Across a variety of systems, thousands of RNAs are localized to specific subcellular locations. However, for the vast majority of these RNAs, the mechanisms that underlie their transport are unknown. Historically, these mechanisms were uncovered for a single transcript at a time by laboriously testing the ability of RNA fragments to direct transcript localization. Recently developed methods profile the content of subcellular transcriptomes using high-throughput sequencing, allowing the analysis of the localization of thousands of transcripts at once. By identifying commonalities shared among multiple localized transcripts, these methods have the potential to rapidly expand our understanding of RNA localization mechanisms.
Topics: Animals; Cell Culture Techniques; Embryo, Nonmammalian; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Laser Capture Microdissection; Neurons; RNA; RNA-Binding Proteins
PubMed: 34998919
DOI: 10.1016/j.bbamcr.2021.119202 -
The Journal of Craniofacial SurgeryTo obtain further understanding of the eyelid lymphatic anatomy.
OBJECTIVE
To obtain further understanding of the eyelid lymphatic anatomy.
METHOD
Thirty-two halves of eyelids from 16 fresh fetus cadavers were studied by microdissection using a mixture of 3% Prussian blue and chloroform to visualize the lymphatic vessels.
RESULTS
Three layers of lymphatic plexuses were demonstrated in the eyelids: a superficial or preorbicularis muscle plexus; a pretarsal or postorbicular muscle plexus; and a deep or posttarsal plexus. Furthermore, communicating branches among these plexuses were also spotted.
CONCLUSIONS
The study results demonstrated the topographic distribution of the eyelid lymphatic vessels and confirmed the existence of communicating branches. This discovery will be conducive to understanding the route and mechanism by which inflammation of the eyelid spreads and cancer disseminates. It also provides anatomical insights to apply during eyelid surgery with regard to the prevention of possible eyelid lymphatic injury.
Topics: Cadaver; Eyelids; Humans; Lymphatic System; Lymphatic Vessels; Microdissection
PubMed: 34224459
DOI: 10.1097/SCS.0000000000007759 -
Journal of Translational Medicine Feb 2024Combined small-cell lung carcinoma (cSCLC) represents a rare subtype of SCLC, the mechanisms governing the evolution of cancer genomes and their impact on the tumor...
BACKGROUND
Combined small-cell lung carcinoma (cSCLC) represents a rare subtype of SCLC, the mechanisms governing the evolution of cancer genomes and their impact on the tumor immune microenvironment (TIME) within distinct components of cSCLC remain elusive.
METHODS
Here, we conducted whole-exome and RNA sequencing on 32 samples from 16 cSCLC cases.
RESULTS
We found striking similarities between two components of cSCLC-LCC/LCNEC (SCLC combined with large-cell carcinoma/neuroendocrine) in terms of tumor mutation burden (TMB), tumor neoantigen burden (TNB), clonality structure, chromosomal instability (CIN), and low levels of immune cell infiltration. In contrast, the two components of cSCLC-ADC/SCC (SCLC combined with adenocarcinoma/squamous-cell carcinoma) exhibited a high level of tumor heterogeneity. Our investigation revealed that cSCLC originated from a monoclonal source, with two potential transformation modes: from SCLC to SCC (mode 1) and from ADC to SCLC (mode 2). Therefore, cSCLC might represent an intermediate state, potentially evolving into another histological tumor morphology through interactions between tumor and TIME surrounding it. Intriguingly, RB1 inactivation emerged as a factor influencing TIME heterogeneity in cSCLC, possibly through neoantigen depletion.
CONCLUSIONS
Together, these findings delved into the clonal origin and TIME heterogeneity of different components in cSCLC, shedding new light on the evolutionary processes underlying this enigmatic subtype.
Topics: Humans; Lung Neoplasms; Microdissection; Small Cell Lung Carcinoma; Adenocarcinoma; Carcinoma, Large Cell; Genomics; Tumor Microenvironment
PubMed: 38383412
DOI: 10.1186/s12967-024-04968-4