-
Journal of the American Society of... Jan 2024Laser capture microdissection and mass spectrometry (LCM/MS) is a technique that involves dissection of glomeruli from paraffin-embedded biopsy tissue, followed by... (Review)
Review
Laser capture microdissection and mass spectrometry (LCM/MS) is a technique that involves dissection of glomeruli from paraffin-embedded biopsy tissue, followed by digestion of the dissected glomerular proteins by trypsin, and subsequently mass spectrometry to identify and semiquantitate the glomerular proteins. LCM/MS has played a crucial role in the identification of novel types of amyloidosis, biomarker discovery in fibrillary GN, and more recently discovery of novel target antigens in membranous nephropathy (MN). In addition, LCM/MS has also confirmed the role for complement proteins in glomerular diseases, including C3 glomerulopathy. LCM/MS is now widely used as a clinical test and considered the gold standard for diagnosis and typing amyloidosis. For the remaining glomerular diseases, LCM/MS has remained a research tool. In this review, we discuss the usefulness of LCM/MS in other glomerular diseases, particularly MN, deposition diseases, and diseases of complement pathways, and advocate more routine use of LCM/MS at the present time in at least certain diseases, such as MN, for target antigen detection. We also discuss the limitations of LCM/MS, particularly the difficulties faced from moving from a research-based technique to a clinical test. Nonetheless, the role of LCM/MS in glomerular diseases is expanding. Currently, LCM/MS may be used to identify the etiology in certain glomerular diseases, but in the future, LCM/MS can play a valuable role in determining pathways of complement activation, inflammation, and fibrosis.
Topics: Humans; Kidney Diseases; Kidney Glomerulus; Mass Spectrometry; Laser Capture Microdissection; Glomerulonephritis, Membranous; Amyloidosis
PubMed: 37749770
DOI: 10.1681/ASN.0000000000000221 -
Journal of the American Society For... Jul 2023Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed...
Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.
Topics: Humans; Workflow; Proteomics; High-Throughput Screening Assays; Peptides
PubMed: 37267530
DOI: 10.1021/jasms.3c00099 -
Comparative Cytogenetics 2023A brief overview of the current stage of the chromosome study of the insect order Hymenoptera is given. It is demonstrated that, in addition to routine staining and... (Review)
Review
A brief overview of the current stage of the chromosome study of the insect order Hymenoptera is given. It is demonstrated that, in addition to routine staining and other traditional techniques of chromosome research, karyotypes of an increasing number of hymenopterans are being studied using molecular methods, e.g., staining with base-specific fluorochromes and fluorescence hybridization (FISH), including microdissection and chromosome painting. Due to the advent of whole genome sequencing and other molecular techniques, together with the "big data" approach to the chromosomal data, the current stage of the chromosome research on Hymenoptera represents a transition from Hymenoptera cytogenetics to cytogenomics.
PubMed: 37953851
DOI: 10.3897/compcytogen.17.112332 -
Computers in Biology and Medicine Nov 2023In the era of immunotherapy, the suboptimal response rate and the development of acquired resistance among the initial beneficiaries continue to present significant...
Microdissection of cancer-associated fibroblast infiltration subtypes unveils the secreted SERPINE2 contributing to immunosuppressive microenvironment and immuotherapeutic resistance in gastric cancer: A large-scale study integrating bulk and single-cell transcriptome profiling.
In the era of immunotherapy, the suboptimal response rate and the development of acquired resistance among the initial beneficiaries continue to present significant challenges across multiple malignancies, including gastric cancer (GC). Considering that the interactions of tumor stroma, especially the cancer-associated fibroblasts (CAFs), with immune and tumor cells, play indispensable roles in tumor progression, tumor microenvironment remodeling and therapeutic responsiveness, in-depth exploration on the roles of CAFs and pivotal mediators of their functions may provide novel clues to increase the effectiveness of current immunotherapeutic drugs and further achieve synergistic antitumor response. Herein, through the consensus clustering of canonical biomarkers, three GC subclasses with different abundance of CAFs were virtually microdissected in four integrated bulk cohorts encompassing 2148 GC patients from 11 independent datasets. An extensive immunogenomic analysis revealed that tumors with high CAFs infiltration were characterized with unfavorable outcomes, aggressive phenotypes, decreased tumor immunogenicity, high risk of immune evasion and thus immunotherapeutic resistance. By leveraging large-scale single-cell transcriptomic profiling, a series of CAF-secreted proteins were identified, among which the SERPINE2 was confirmed to be restrictively enriched in stromal fibroblasts of GC tissues and contribute to promoting a protumor milieu and fostering an immunosuppressive microenvironment via bioinformatics computations and tissue microarray analysis. Moreover, pan-cancer investigations generalized the immunological roles of SERPINE2, especially in pan-gastrointestinal malignancies, with multiple real-world immunotherapy cohorts further confirming its implications on predicting immunotherapeutic efficacy. In conclusion, these findings suggest that the CAF-derived SERPINE2 is a promising immune-oncology target with therapeutic implications to further synergize the immunotherapeutic combinations.
Topics: Humans; Stomach Neoplasms; Cancer-Associated Fibroblasts; Tumor Microenvironment; Single-Cell Analysis; Drug Resistance, Neoplasm; Gene Expression Profiling; Transcriptome; Immunotherapy; Gene Expression Regulation, Neoplastic
PubMed: 37729702
DOI: 10.1016/j.compbiomed.2023.107406 -
Methods in Molecular Biology (Clifton,... 2024Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic...
Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic yeast Cryptococcus neoformans is essential because old yeast cells with age-related phenotypes accumulate during infection and are associated with increased virulence and antifungal tolerance. Microdissection and microfluidic devices are valuable tools for continuously tracking cells at the single-cell level. In this chapter, we describe the features of these two platforms and outline technical limitations and information to study aging mechanisms while assessing the lifespan of yeast cells.
Topics: Cryptococcus neoformans; Microdissection; Cellular Senescence; Lab-On-A-Chip Devices; Single-Cell Analysis; Cryptococcosis
PubMed: 38758331
DOI: 10.1007/978-1-0716-3722-7_25 -
High-quality single-cell transcriptomics from ovarian histological sections during folliculogenesis.Life Science Alliance Nov 2023High-quality, straightforward single-cell RNA sequencing (RNA-seq) with spatial resolution remains challenging. Here, we developed DRaqL (direct RNA recovery and...
High-quality, straightforward single-cell RNA sequencing (RNA-seq) with spatial resolution remains challenging. Here, we developed DRaqL (direct RNA recovery and quenching for laser capture microdissection), an experimental approach for efficient cell lysis of tissue sections, directly applicable to cDNA amplification. Single-cell RNA-seq combined with DRaqL allowed transcriptomic profiling from alcohol-fixed sections with efficiency comparable with that of profiling from freshly dissociated cells, together with effective exon-exon junction profiling. The combination of DRaqL with protease treatment enabled robust and efficient single-cell transcriptome analysis from formalin-fixed tissue sections. Applying this method to mouse ovarian sections, we were able to predict the transcriptome of oocytes by their size and identified an anomaly in the size-transcriptome relationship relevant to growth retardation of oocytes, in addition to detecting oocyte-specific splice isoforms. Furthermore, we identified differentially expressed genes in granulosa cells in association with their proximity to the oocytes, suggesting distinct epigenetic regulations and cell-cycle activities governing the germ-soma relationship. Thus, DRaqL is a versatile, efficient approach for high-quality single-cell RNA-seq from tissue sections, thereby revealing histological heterogeneity in folliculogenic transcriptome.
Topics: Female; Animals; Mice; Transcriptome; Ovary; Gene Expression Profiling; Oocytes; Cell Cycle
PubMed: 37722727
DOI: 10.26508/lsa.202301929 -
Cell Reports Jan 2024A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN...
A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases.
Topics: Humans; Amyotrophic Lateral Sclerosis; DNA-Binding Proteins; Motor Neurons; Proteome; Proteomics
PubMed: 38183652
DOI: 10.1016/j.celrep.2023.113636 -
Molecular Cancer Research : MCR Dec 2023Stage IA gastric adenocarcinoma, characterized by foci of intramucosal signet ring cells (SRC), is found in nearly all asymptomatic patients with germline pathogenic...
UNLABELLED
Stage IA gastric adenocarcinoma, characterized by foci of intramucosal signet ring cells (SRC), is found in nearly all asymptomatic patients with germline pathogenic CDH1 variants and hereditary diffuse gastric cancer syndrome (HDGC). The molecular steps involved in initiating malignant transformation and promoting SRC dormancy in HDGC are unknown. Here, whole-exome bulk RNA sequencing (RNA-seq) of SRCs and adjacent non-SRC epithelium (NEP) was performed on laser-capture microdissected (LCM) regions of interest found in risk-reducing total gastrectomy specimens from patients with HDGC (Clinicaltrials.gov ID: NCT03030404). In total, 20 patients (6 male, 14 female) with confirmed HDGC were identified. Analysis of differentially expressed genes (DEG) demonstrated upregulation of certain individual EMT and proliferation genes. However, no oncogenic pathways were found to be upregulated in SRCs. Rather, SRC regions had significant enrichment in pathways involved in T-cell signaling. CIBERSORTx predicted significant increases in the presence of regulatory T cells (Treg) specific to SRC regions. IHC confirmed an increase in FOXP3+ cells in SRC foci, as well as elevations in CD4+ T cells and HLA-DR staining. In summary, the tumor immune microenvironment is microscopically inseparable from stage IA gastric SRCs using a granular isolation technique. An elevation in CD4+ T cells within SRC regions correlates with clinically observed SRC dormancy, while Treg upregulation represents a potential immune escape mechanism.
IMPLICATIONS
Characterization of the tumor-immune microenvironment in HDGC underscores the potential for the immune system to shape the transcriptional profile of the earliest tumors, which suggests immune-directed therapy as a potential cancer interception strategy in diffuse-type gastric cancer.
Topics: Humans; Male; Female; Stomach Neoplasms; Genetic Predisposition to Disease; Gastrectomy; Germ-Line Mutation; Carcinogenesis; Carcinoma, Signet Ring Cell; Adenocarcinoma; Cadherins; Tumor Microenvironment; Antigens, CD
PubMed: 37707375
DOI: 10.1158/1541-7786.MCR-23-0122 -
Genes Aug 2023Complex interactions between gene variants and environmental risk factors underlie the pathophysiological pathways in major psychiatric disorders. Autism Spectrum... (Review)
Review
Complex interactions between gene variants and environmental risk factors underlie the pathophysiological pathways in major psychiatric disorders. Autism Spectrum Disorder is a neuropsychiatric condition in which susceptible alleles along with epigenetic states contribute to the mutational landscape of the ailing brain. The present work reviews recent evolutionary, molecular, and epigenetic mechanisms potentially linked to the etiology of autism. First, we present a clinical vignette to describe clusters of maladaptive behaviors frequently diagnosed in autistic patients. Next, we microdissect brain regions pertinent to the nosology of autism, as well as cell networks from the bilateral body plan. Lastly, we catalog a number of pathogenic environments associated with disease risk factors. This set of perspectives provides emerging insights into the dynamic interplay between epigenetic and environmental variation in the development of Autism Spectrum Disorders.
Topics: Humans; Autism Spectrum Disorder; Autistic Disorder; Alleles; Biological Evolution; Epigenesis, Genetic
PubMed: 37761876
DOI: 10.3390/genes14091734 -
Clinical Kidney Journal Jan 2024Research on membranous nephropathy truly exploded in the last 15 years. This happened because of the application of new techniques (laser capture microdissection, mass... (Review)
Review
Research on membranous nephropathy truly exploded in the last 15 years. This happened because of the application of new techniques (laser capture microdissection, mass spectrometry, protein G immunoprecipitation, arrays) to the study of its pathogenesis. After the discovery of PLA2R as the major target antigen, many other antigens were identified and others are probably ongoing. Clinical and pathophysiology rebounds of new discoveries are relevant in terms of diagnosis and prognosis and it is time to make a first assessment of the innovative issues. In terms of classification, target antigens can be divided into: 'membrane antigens' and 'second wave' antigens. The first group consists of antigens constitutionally expressed on the podocyte membrane (as PLA2R) that may become a target of an autoimmune process because of perturbation of immune-tolerance. 'Second wave' antigens are antigens neo-expressed by the podocyte or by infiltrating cells after a stressing event: this allows the immune system to produce antibodies against them that intensify and maintain glomerular damage. With this abundance of target antigens it is not possible, at the moment, to test all antibodies at the bedside. In the absence of this possibility, the role of histological evaluation is still irreplaceable.
PubMed: 38213493
DOI: 10.1093/ckj/sfad228