Combining Laser Capture Microdissection Research Articles

Advancing drug development by leveraging microdissection for proteogenomic analysis of histological sections.

e14660 Background: Combining laser capture microdissection (LCM) with proteogenomics in cancer research offers a targeted way to way to explore the intricate interactions between the tumor microenvironment and tumor cells proper. For immuno-oncology (IO) research, this is especially important, since improvements in the predictability of patient response to IO-based drugs is greatly needed. An improved understanding of the spatial relationships involving tumor cells, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: From fresh frozen tissue, LCM is used to isolate and obtain distinct histological cell types. Nucleic acids and proteins are extracted from captured cells for in-depth multi-modality molecular profiling assays. This includes multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing the analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, DNA sequencing can be performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. RNA-seq may be performed for gene expression. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. Our novel proteogenomics approach is focused around a customized enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: A proteogenomic approach is reported that is utilizing amount limited tissue quantities from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

#187 Vasorin (VASN) commands podocyte quiescence and glomerular homeostasis. A novel FSGS mechanism?

Abstract Background and Aims Podocyte dysfunction and loss of quiescence contribute to focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN). Here, we characterized the role of Vasorin (VASN) in podocytes. Method We combined laser capture microdissection and deep RNA sequencing of human glomeruli, RNAscope-based in situ hybridization, immunostaining and mouse models with Vasn-driven fluorescent reporter, targeted Vasn gene deletion in podocytes and cultures of human podocytes. Results VASN mRNA is strongly expressed in healthy human and mouse glomeruli with a podocyte-specific pattern. Both global and podocyte-specific, constitutive or inducible gene deletion of Vasn in mice induced a nephrotic syndrome and kidney failure after day 14, with podocyte dedifferentiation and loss and severe FSGS lesions, indicating a fundamental and non-developmental role of VASN in podocyte homeostasis. Furthermore, we measured markedly decreased VASN expression in podocytes of patients with FSGS and ANCA-associated CGN but not with Minimal Change Disease (MCD) and Membranous Nephropathy (MN). We thus wondered whether VASN abundance could be a disease modifier for glomerulopathies. Mice with podocyte-specific partial heterozygous deletion of Vasn showed no evident baseline renal abnormalities. Still, they showed significantly more podocyte loss, albuminuria and kidney failure than wild-type mice when challenged by type 1 diabetes with glomerular hyperfiltration or CGN experimental models. Human podocytes transduced with lentiviral VASN-targeting shRNA (shVASN) displayed increased focal adhesion dynamics, resistance to oscillating mechanical stretches, enhanced migratory capacity and epithelial-mesenchymal transition (EMT) markers. Moreover, shVASN podocytes had a higher proliferative capacity, increased cell-cycle reentry and a relative S-phase slow-down. Likewise, transcriptomics analysis found the up-regulation of genes involved in cell metabolism, adhesion and EMT and the down-regulation of genes involved in the S to G2/M phase transition. In vivo, we confirmed that VASN deficiency increased podocyte proliferation in situ and urinary shedding. Conclusion These findings highlight the novel role of VASN as an essential master protein that maintains podocyte quiescence and homeostasis during health and kidney diseases while offering a potential new therapeutic target. VASN control mechanisms driving podocyte cell-cycle reentry and detachment and FSGS. Precise mechanisms of VASN functions in podocytes need further insights into VASN molecular partners.

Novel TUBA4A variant causes congenital myopathy with focal myofibrillar disorganisation

BackgroundCongenital myopathies are a clinical, histopathological and genetic heterogeneous group of inherited muscle disorders that are defined on peculiar architectural abnormalities in the muscle fibres. Although there have been at...

The proteomic profile of Von Economo neurons supports their selective vulnerability in bvFTD

AbstractBackgroundVon Economo neurons (VENs) are long‐range projection neurons embedded in Layer 5 of the anterior cingulate gyrus (ACC) and frontoinsular cortex. VENs are selectively vulnerable in a number of diseases, most notably in those associated with behavioural, social, and cognitive changes, including the behavioural variant frontotemporal dementia (bvFTD). In particular, VENs of bvFTD donors with TDP43 or FUS pathology appear to be more vulnerable compared to donors with tau pathology. Unravelling the characteristics of VENs and the cause of their vulnerability in specific subtypes of FTD, will provide invaluable insight into disease mechanisms and potential avenues for drug therapeutics.MethodSingle‐cell laser capture microdissection (LCM) in combination with proteomics was used to analyse the proteome of VENs compared to neighbouring Layer 5 pyramidal neurons of the ACC (ACC‐PYR) and the superior frontal gyrus (SFG‐PYR), a region adjacent to the ACC. Immunohistochemistry (IHC) was performed on 10µm thick fresh frozen sections of the ACC and SFG from four neurologically healthy control donors with the neurofilament marker, SMI32. 1000 VENs and three times 1000 ACC‐PYR, and SFG‐PYR were then delineated and captured using LCM. After protein separation, all samples were analysed by label‐free mass spectrometry (LC‐MS/MS) in a data independent manner.ResultAn average 2660 proteins were detected per sample, which accumulated in a total detection of 4718 different proteins over all samples. 69 proteins had a significant change in abundance between VENs and SFG‐PYR, and 36 proteins when VENs were paired against ACC‐PYR. A number of proteins were validated with IHC. GO‐pathway analysis revealed RNA processing and splicing, neurofilament bundle assembly and cytoskeleton organization to be among top deregulated pathways, with many proteins localised to the ribonucleoprotein complex.ConclusionWe show a number proteins involved in RNA processing to be differentially expressed in VENs compared to neighbouring pyramidal neurons. Since TDP43 and FUS proteins are also involved in RNA processing we hypothesise that the selective vulnerability of VENs in specific FTD subtypes is through altered RNA metabolism.

Convergent evolution and B-cell recirculation in germinal centers in a human lymph node.

Germinal centers (GCs) play a central role in generating an effective immune response against infectious pathogens, and failures in their regulating mechanisms can lead to the development of autoimmune diseases and cancer. Although previous works study experimental systems of the immune response with mouse models that are immunized with specific antigens, our study focused on a real-life situation, with an ongoing GC response in a human lymph node (LN) involving multiple asynchronized GCs reacting simultaneously to unknown antigens. We combined laser capture microdissection of individual GCs from human LN with next-generation repertoire sequencing to characterize individual GCs as distinct evolutionary spaces. In line with well-characterized GC responses in mice, elicited by immunization with model antigens, we observe a heterogeneous clonal diversity across individual GCs from the same human LN. Still, we identify shared clones in several individual GCs, and phylogenetic tree analysis combined with paratope modeling suggest the re-engagement and rediversification of B-cell clones across GCs and expanded clones exhibiting shared antigen responses across distinct GCs, indicating convergent evolution of the GCs.

Microisolation of Spatially Characterized Single Populations of Neurons for RNA Sequencing from Mouse and Postmortem Human Brain Tissues.

Single-cell and single-population RNA sequencing (RNA-seq) is a rapidly evolving new field of intense investigation. Recent studies indicate unique transcriptomic profiles are derived based on the spatial localization of neurons within circuits and regions. Individual neuronal subtypes can have vastly different transcriptomic fingerprints, well beyond the basic excitatory neuron and inhibitory neuron designations. To study single-population gene expression profiles of spatially characterized neurons, we have developed a methodology combining laser capture microdissection (LCM), RNA purification of single populations of neurons, and subsequent library preparation for downstream applications, including RNA-seq. LCM provides the benefit of isolating single neurons characterized by morphology or via transmitter-identified and/or receptor immunoreactivity and enables spatial localization within the sample. We utilize unfixed human postmortem and mouse brain tissue that is frozen to preserve RNA quality in order to isolate the desired neurons of interest. Microisolated neurons are then pooled for RNA purification utilizing as few as 250 individual neurons from a tissue section, precluding extraneous nonspecific tissue contaminants. Library preparation is performed from picogram RNA quantities extracted from LCM-captured neurons. Single-population RNA-seq analysis demonstrates that microisolated neurons from both postmortem human and mouse brain tissues are viable for transcriptomic profiling, including differential gene expression assessment and bioinformatic pathway inquiry.

Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP)

A reoccurring issue in neuroepigenomic studies, especially in the context of neurodegenerative disease, is the use of (heterogeneous) bulk tissue, which generates noise during epigenetic profiling. A workable solution to this issue is to quantify epigenetic patterns in individually isolated neuronal cells using laser capture microdissection (LCM). For this purpose, we established a novel approach for targeted DNA methylation profiling of individual genes that relies on a combination of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). Using this approach, we determined cytosine-phosphate-guanine (CpG) methylation rates of single alleles derived from 50 neurons that were isolated from unfixed post-mortem brain tissue. In the present manuscript, we describe the general workflow and, as a showcase, demonstrate how targeted methylation analysis of various genes, in this case, RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be performed simultaneously. By doing so, we describe an adapted data analysis pipeline for LDBSP, allowing one to include and correct CpG methylation rates derived from multi-allele reactions. In addition, we show that the efficiency of LDBSP on DNA derived from LCM neurons is similar to the efficiency obtained in previously published studies using this technique on other cell types. Overall, the method described here provides the user with a more accurate estimation of the DNA methylation status of each target gene in the analyzed cell pools, thereby adding further validity to this approach.

Low-salt diet increases mRNA expression of aldosterone-regulated transporters in the intermediate portion of the endolymphatic sac.

The endolymphatic sac is a small sac-shaped organ at the end of the membranous labyrinth of the inner ear. The endolymphatic sac absorbs the endolymph, in which the ion balance is crucial for inner ear homeostasis. Of the three sections of the endolymphatic sac, the intermediate portion is the center of endolymph absorption, particularly sodium transport, and is thought to be regulated by aldosterone. Disorders of the endolymphatic sac may cause an excess of endolymph (endolymphatic hydrops), a histological observation in Meniere's disease. A low-salt diet is an effective treatment for Meniere's disease, and is based on the assumption that the absorption of endolymph in the endolymphatic sac abates endolymphatic hydrops through a physiological increase in aldosterone level. However, the molecular basis of endolymph absorption in each portion of the endolymphatic sac is largely unknown because of difficulties in gene expression analysis, resulting from its small size and intricate structure. The present study combined reverse transcription-quantitative polymerase chain reaction and laser capture microdissection techniques to analyze the difference of gene expression of the aldosterone-controlled epithelial Na+ channel, thiazide-sensitive Na+-Cl- cotransporter, and Na+, K+-ATPase genes in the three individual portions of the endolymphatic sac in a rat model. A low-salt diet increased the expression of aldosterone-controlled ion transporters, particularly in the intermediate portion of the endolymphatic sac. Our findings will contribute to the understanding of the physiological function of the endolymphatic sac and the pathophysiology of Meniere's disease.

Insight into the Molecular Characteristics of Langhans Giant Cell by Combination of Laser Capture Microdissection and RNA Sequencing.

PurposeThe presence of Langhans giant cell (LGC) is a hallmark of mycobacterium-induced granuloma. The molecular characteristics and functions of LGC remain unclear to date. The study aimed to systematically characterize the molecular characteristics of LGC and reveal the potential functions.MethodsHuman LGCs were purified through laser capture microdissection (LCM) in vitro. RNA sequencing and in-depth transcriptome analysis were performed for purified LGCs and macrophages in the same system. Skin samples from mycobacterial infection patients were used to confirm some of the transcriptional expression.ResultsHuman LGCs have different expression pattern from macrophages in the same in vitro system. A total of 967 differentially expressed genes were found. Bioinformatics analysis showed that LGCs are is characterized by active cell shape regulation, increased cytoskeletal components, weakened energy metabolism level, and reduced immune response. CCL7 may be a specific molecular for LGC to communicate with CCR1-expression cells in granuloma.ConclusionLGCs have unique molecular characteristics different from that of macrophages. They may play a role in maintaining the hemostasis in granuloma.

Spatiotemporal endometrial transcriptome analysis revealed the luminal epithelium as key player during initial maternal recognition of pregnancy in the mare

During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo’s signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10–13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level.

Multi-Omics Approach to Mitochondrial DNA Damage in Human Muscle Fibers.

Mitochondrial DNA deletions affect energy metabolism at tissue-specific and cell-specific threshold levels, but the pathophysiological mechanisms determining cell fate remain poorly understood. Chronic progressive external ophthalmoplegia (CPEO) is caused by mtDNA deletions and characterized by a mosaic distribution of muscle fibers with defective cytochrome oxidase (COX) activity, interspersed among fibers with retained functional respiratory chain. We used diagnostic histochemistry to distinguish COX-negative from COX-positive fibers in nine muscle biopsies from CPEO patients and performed laser capture microdissection (LCM) coupled to genome-wide gene expression analysis. To gain molecular insight into the pathogenesis, we applied network and pathway analysis to highlight molecular differences of the COX-positive and COX-negative fiber transcriptome. We then integrated our results with proteomics data that we previously obtained comparing COX-positive and COX-negative fiber sections from three other patients. By virtue of the combination of LCM and a multi-omics approach, we here provide a comprehensive resource to tackle the pathogenic changes leading to progressive respiratory chain deficiency and disease in mitochondrial deletion syndromes. Our data show that COX-negative fibers upregulate transcripts involved in translational elongation and protein synthesis. Furthermore, based on functional annotation analysis, we find that mitochondrial transcripts are the most enriched among those with significantly different expression between COX-positive and COX-negative fibers, indicating that our unbiased large-scale approach resolves the core of the pathogenic changes. Further enrichments include transcripts encoding LIM domain proteins, ubiquitin ligases, proteins involved in RNA turnover, and, interestingly, cell cycle arrest and cell death. These pathways may thus have a functional association to the molecular pathogenesis of the disease. Overall, the transcriptome and proteome show a low degree of correlation in CPEO patients, suggesting a relevant contribution of post-transcriptional mechanisms in shaping this disease phenotype.

ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIF-containing protein that controls root growth angle in barley and wheat.

The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain-containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.

Implications of zonal architecture on differential gene expression profiling and altered pathway expressions in mandibular condylar cartilage

Mandibular condylar cartilage (MCC) is a multi-zonal heterogeneous fibrocartilage containing different types of cells, but the factors/mechanisms governing the phenotypic transition across the zones have not been fully understood. The reliability of molecular studies heavily rely on the procurement of pure cell populations from the heterogeneous tissue. We used a combined laser-capture microdissection and microarray analysis approach which allowed identification of differential zone-specific gene expression profiling and altered pathways in the MCC of 5-week-old rats. The bioinformatics analysis demonstrated that the MCC cells clearly exhibited distinguishable phenotypes from the articular chondrocytes. Additionally, a set of genes has been determined as potential markers to identify each MCC zone individually; Crab1 gene showed the highest enrichment while Clec3a was the most downregulated gene at the superficial layer, which consists of fibrous (FZ) and proliferative zones (PZ). Ingenuity Pathway Analysis revealed numerous altered signaling pathways; Leukocyte extravasation signaling pathway was predicted to be activated at all MCC zones, in particular mature and hypertrophic chondrocytes zones (MZ&HZ), when compared with femoral condylar cartilage (FCC). Whereas Superpathway of Cholesterol Biosynthesis showed predicted activation in both FZ and PZ as compared with deep MCC zones and FCC. Determining novel zone-specific differences of large group of potential genes, upstream regulators and pathways in healthy MCC would improve our understanding of molecular mechanisms on regional (zonal) basis, and provide new insights for future therapeutic strategies.

Identification of Blood-Brain Barrier-Associated Proteins in the Human Brain.

The blood-brain barrier (BBB) is essential for cerebral homeostasis and controls the selective passage of molecules traveling in and out of the brain. Despite the crucial role of the BBB in a variety of brain diseases and its relevance for the development of drugs, there is little known about its molecular architecture. In particular, the composition of the basal lamina between the astrocytic end-feet and the endothelial cells is only partly known. Here, we present a proteomic analysis of the basal lamina of the human BBB. We combined laser capture microdissection with shotgun proteomics for selective enrichment and identification of specific proteins present in the cerebral microvasculature and arachnoidal vessels collected from normal human brain tissue specimens. Proteins found to be associated with the blood-brain barrier were validated by immunohistochemistry. Expression of membrane protein MLC1 was found in all brain barriers. Phosphoglucomutase-like protein 5 appeared to be variably present along the outer part of intracerebral vessels, and multidrug resistance protein 1 was identified in both intracerebral, as well as arachnoidal blood vessels. The results demonstrate the presence of so far unidentified proteins in the human BBB and illustrate topic differences in their expression. In conclusion, we showed that sample purification by microdissection followed by shotgun proteomics provides a list of proteins identified in the BBB. Subsequent immunohistochemistry detailed the respective expression sites of membrane protein MLC1 and phosphoglucomutase-related protein 5. The role of the identified proteins in the functioning of the BBB needs further investigations.

Molecular Characterization of Testicular Germ Cell Tumors Using Tissue Microdissection.

Testicular germ cell tumors are among the most common malignancies seen in children and young adults. Genomic studies have identified characteristic molecular profiles in testicular cancer, which are associated with histologic subtypes and may predict clinical behavior including treatment responses. Emerging molecular technologies analyzing tumor genomics, transcriptomics, and proteomics may now guide precision management of testicular tumors. Laser-assisted microdissection methods such as laser capture microdissection efficiently isolate selected tumor cells from routine pathology specimens, avoiding contamination from nontarget cell populations. Laser capture microdissection in combination with next generation sequencing makes precise high throughput genetic evaluation effective and efficient. The use of laser capture microdissection (LCM) for molecular testing may translate into great benefits for the clinical management of patients with testicular cancers. This review discusses application protocols for laser-assisted microdissection to investigate testicular germ cell tumors.

Abstract 2499: Genomic spatial profiling of archived pre-malignant lesions of the breast epithelium

Abstract The widespread adoption of breast cancer screening has increased the detection of pre-malignant lesions such as ductal carcinoma in situ (DCIS) but there are currently no reliable biomarkers to stratify DCIS and avoid over-treatment. Importantly, the identification of novel biomarkers in DCIS is notoriously difficult. The rare and late recurrence to subsequent breast cancer (10% at 10 years) poses challenges in experimental design that only a few established, retrospective studies can solve. Most tissue specimens in the associated registries are small, fixed and archived in paraffin (FFPE), precluding their characterization via standard genomic assays. Besides, pure DCIS often present multi-focal heterogenous histology of unclear mutational make-up. Here, we present an approach combining laser capture microdissection with state-of-the-art genomic library preparation methods to enable molecular profiling from minute amounts of damaged nucleic acids and demonstrate its validity for the spatial molecular characterization of DCIS specimen. Targeted DNA sequencing was performed on limited dilutions of a test DNA from archived breast tissue. Three library preparation strategies were evaluated, included adaptation of classic A-tailing (AT), alternate single-strand (SS) or blunt-end (BE) adapter ligations. The use of an AT protocol resulted less than 10% of targeted base pairs covered at more than 20x (Cov20) from lower than 50ng of DNA and suggested the adapter ligation as initial bottleneck. In contrast, SS or BE adapter ligation from 3ng of DNA reduced the fraction of duplicate reads, increasing the Cov20 to 49% and 100% respectively. Artifactual C to T substitutions, a signature of formalin damage, were restricted to variants of low allelic fraction (<5%). As a result, the use of ensemble variant calling and heuristic filtering to eliminate artifacts and germline variants, led to exome-wide mutational profile of 89% precision and 88% recall from 3ng input, comparable to replicate deep sequencing from a mirrored fresh frozen specimen. The presence of an ERBB2 amplification was validated in all tested conditions. The BE approach for whole exomes was validated on DCIS regions (N=8) and normal epithelium (N=1) dissected from 3 FFPE DCIS blocks. The normal epithelium was unaltered and copy number alterations both consistent and inconsistent within specimen were identified, including FGFR1 and ERBB2 gains or TP53 loss restricted to high-grade lesions. Short variants and indels were rare (~123 per region), mostly shared within each specimen and unlikely to represent driver mutations. The proposed approach can support the spatial mutational profiling of pre-malignant lesions. Compatible with a limited quantity of tissue from archived specimens, the approach sets a new paradigm for the retrospective molecular characterization of specimen with longitudinal follow-up and associated long-term outcome. Citation Format: Daniela Nachmanson, Joseph Steward, Taeyong Kim, Huazhen Yao, Eliza Jeong, Thomas O'Keefe, Gillian L. Hirst, Laura Esserman, Alexander Borowsky, Farnaz Hasteh, Kristen Jepsen, Olivier Harismendy. Genomic spatial profiling of archived pre-malignant lesions of the breast epithelium [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2499.

Laser capture microdissection in combination with mass spectrometry: Approach to characterization of tissue-specific proteomes of Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

Lgr5+\xa0telocytes are a signaling source at the intestinal villus tip

The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis.

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence.

Profound transcriptional heterogeneity in anatomically adjacent single cells suggests that robust tissue functionality may be achieved by cellular phenotype diversity. Single-cell experiments investigating the network dynamics of biological systems demonstrate cellular and tissue responses to various conditions at biologically meaningful resolution. Herein, we explain our methods for gathering single cells from anatomically specific locations and accurately measuring a subset of their gene expression profiles. We combine laser capture microdissection (LCM) with microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). We also use this microfluidic RT-qPCR platform to measure the microbial abundance of gut contents.

Abstract P4-05-05: A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions

Abstract Background: The widespread adoption of breast cancer screening has increased the detection of pre-malignant lesions such as ductal carcinoma in situ (DCIS) but has not translated into increased survival rates. There are currently no reliable biomarkers to stratify DCIS into those which would not develop into an aggressive invasive cancer from those that do, and avoid over-treatment. The interaction with the DCIS microenvironment has likely been under-appreciated or not studied in the adequate clinical context. Importantly, the identification of novel biomarkers in DCIS is notoriously difficult. The rare and late recurrence to subsequent breast cancer (10% at 10 years) poses challenges in experimental design that only a few established, retrospective studies can solve. Most tissue specimens in the associated registries are small, fixed and archived in paraffin (FFPE), precluding their characterization via standard genomic assays. Here, we present an approach combining laser capture microdissection (LCM) with state-of-the-art genomic library preparation methods to enable molecular profiling from minute amounts of damaged nucleic acids and demonstrate its validity for the spatial molecular characterization of DCIS specimen. Methods: Targeted DNA sequencing (whole exome and cancer gene panel) was performed on limited dilutions (200, 50, 10,3 ng) of a test DNA from archived breast tissue, or validation DNA from 10 LCM regions from 3 DCIS specimens. Three library preparation strategies were evaluated, included adaptation of classic A-tailing (AT), or alternate single-strand (SS) or blunt-end (BE) adapter ligations. The expression profiling was performed using SMART-3SEQ protocol on 15 LCM regions from 8 DCIS specimen. Results: The use of an FFPE-optimized hybrid-capture AT protocol resulted in poor coverage performance (7% of targeted base pairs covered at ≥20x) from test DNA inputs lower than 50 ng and suggested the ligation of the sequencing adapter as the initial bottleneck. Alternatively steps using SS or BE adapter ligation from 3 ng of test DNA (~500 cells) increased the library complexity, reducing the fraction of duplicate reads and increasing the fraction of targeted base pairs covered at 20x to 49% and 100% respectively. Artifactual cytosine to tyrosine substitutions, a signature of formalin damage, were restricted to variants of low allelic fraction (<5%). As a result, the use of molecular barcodes, ensemble variant calling and heuristic variant filtering, resulted in 98% precision and 96% recall in variant calling accuracy from 3ng input, when compared to 200 ng input from a fresh frozen mirrored specimen. Similarly, the presence of an ERBB2 amplification was validated in all tested conditions. The validation of the BE approach on 10 LCM regions from 3 DCIS showed decreasing levels of clonal relationships when comparing regions from adjacent sections, then from matching distinct regions, then from distinct specimen. The heterogeneity of TP53 loss of heterozygosity within a specimen was consistent with high-grade. Furthermore, gene expression profiling of 15 regions from 8 archived DCIS specimen differentiated expression profiles of stromal and epithelial regions, within and between specimen, highlighting sets of genes associated with DCIS subtype and the immune make-up of the micro-environment. Conclusion: The proposed approach - from coordinated specimen preparation to mutational and expression profiling - was therefore validated to support the spatial characterization of pre-cancerous lesions and the microenvironment in which they evolve. Compatible with a limited quantity of tissue from archived specimens, the approach sets a new paradigm for the retrospective molecular characterization of specimen with longitudinal follow-up and associated long-term outcome. Citation Format: Daniela Nachamanson, Joseph Steward, Taeyong Kim, Huazhen Yao, Eliza Jeong, Thomas O'Keefe, Gillian L. Hirst, Farnaz Hasteh, Kristen Jepsen, Laura J. Esserman, Alexander Borowsky, Olivier Harismendy. A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-05.