Immuno-laser Capture Microdissection Research Articles

The Microglial Transcriptome of Age-Associated Deep Subcortical White Matter Lesions Suggests a Neuroprotective Response to Blood-Brain Barrier Dysfunction.

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68+ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68+ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin-haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood-brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia.

Expression Profile of Myoepithelial Cells in DCIS: Do They Change From Protective Angels to Wicked Witches?

The mechanism of transition of ductal carcinoma in situ (DCIS) to invasive cancer is elusive but recently changes in the myoepithelial cells (MECs) have been implicated. The aim of this study is to investigate the changes in gene profile of MECs in DCIS that could compromise their tumor suppressor function leading to promotion of tumor progression. Immuno-laser capture microdissection (LCM) was used to isolate MECs from normal and DCIS breast tissues followed by whole genome expression profiling using Affymetrix HGU-133 plus2.0 arrays. The data were analyzed using Bioconductor packages then validated by using real-time quantitative polymerase chain reaction and immunohistochemistry. Ingenuity Pathways software analysis showed clustering of most of the altered genes in cancer and cell death networks, with the Wnt/B-catenin pathway as the top canonical pathway. Validation revealed a 71.4% correlation rate with the array results. Most dramatic was upregulation of Fibronectin 1 ( FN1 ) in DCIS-associated MECs. Immunohistochemistry analysis for FN1 on normal and DCIS tissues confirmed a strong correlation between FN1 protein expression by MECs and DCIS ( P <0.0001) and between high expression level and presence of invasion ( P =0.006) in DCIS. Other validated alterations in MEC expression profile included upregulation of Nephronectin and downregulation of parathyroid hormone like hormone ( PTHLH ), fibroblast growth factor receptor 2 ( FGFR2 ), ADAMTS5 , TGFBR3 , and CAV1 . In vitro experiments revealed downregulation of PTHLH in DCIS-modified MECs versus normal lines when cultured on Fibronectin matrix. This is the first study to use this in vivo technique to investigate molecular changes in MECs in DCIS. This study adds more evidences to the molecular deviations in MECs toward tumor progression in DCIS through upregulation of the tumor-promoting molecules that may lead to novel predictive and therapeutic targets.

Cell-type-specific gene expression profile by laser capture microdissection on mirror sections

Immuno-laser capture microdissection (Immuno-LCM) has been used to analyze cell-specific gene expression profiles. However, the usefulness of such a technique is frequently limited by RNA degradation. We, therefore, developed a rapid protocol of LCM on mirror sections, which allows for preserving RNA integrity. With such a procedure, we investigated cell-type-specific gene expression of γδ intraepithelial lymphocytes (IELs) in untreated celiac disease (CD).An increase in TGF-β mRNA expression levels was observed in γδ + IELs compared to intestinal enterocytes (IEs), whereas anti-inflammatory IL-10 mRNA production from γδ + IELs was lower compared to IEs.In untreated CD patients, the production of anti-inflammatory cytokines by γδ + IELs is suggestive of a regulatory function, thus playing a critical role in limiting inflammation. This work underscores the importance of LCM on mirror sections as a valuable tool to perform cell-type-specific molecular analysis in tissue.

Type 2 diabetes mellitus-associated transcriptome alterations in cortical neurones and associated neurovascular unit cells in the ageing brain

Type 2 diabetes mellitus (T2D), characterised by peripheral insulin resistance, is a risk factor for dementia. In addition to its contribution to small and large vessel disease, T2D may directly damage cells of the brain neurovascular unit. In this study, we investigated the transcriptomic changes in cortical neurones, and associated astrocytes and endothelial cells of the neurovascular unit, in the ageing brain. Neurone, astrocyte, and endothelial cell-enriched mRNA, obtained by immuno-laser capture microdissection of temporal cortex (Brodmann area 21/22) from 6 cases with self-reported T2D in the Cognitive Function and Ageing Study neuropathology cohort, and an equal number of age and sex-matched controls, was assessed by microarray analysis. Integrated Molecular Pathway Level Analysis was performed using the Kyoto Encyclopaedia of Genes and Genomes database on significantly differentially expressed genes, defined as P < 0.05 and fold-change ± 1.2. Hub genes identified from Weighted Gene Co-expression Network Analysis were validated in neurones using the NanoString nCounter platform. The expression and cellular localisation of proteins encoded by selected candidate genes were confirmed by immunohistochemistry. 912, 2202, and 1227 genes were significantly differentially expressed between cases with self-reported T2D and controls in neurones, astrocytes, and endothelial cells respectively. Changes in cortical neurones included alterations in insulin and other signalling pathways, cell cycle, cellular senescence, inflammatory mediators, and components of the mitochondrial respiratory electron transport chain. Impaired insulin signalling was shared by neurovascular unit cells with, additionally, apoptotic pathway changes in astrocytes and dysregulation of advanced glycation end-product signalling in endothelial cells. Transcriptomic analysis identified changes in key cellular pathways associated with T2D that may contribute to neuronal damage and dysfunction. These effects on brain cells potentially contribute to a diabetic dementia, and may provide novel approaches for therapeutic intervention.

Abstract 2947: Towards advanced precision oncology assays - Selection of immunohistochemistry chromogen impacts microdissection and downstream molecular analysis of archival specimens

Abstract Introduction: Precision medicine promises to improve patient treatment outcomes based on the application of molecular profiling methodologies to pathological specimens. This approach enables the development of rationally derived therapy assignments for each individual patient's tumor (versus categorical treatment assignments). Since tumors are composed of heterogeneous cells forming clusters of subtypes, dissimilar molecular profiles may emerge depending on which region is sampled and analyzed. Additionally, a manual slide scrape may result in a reduced or nearly absent molecular signal (i.e. key driver). Clearly, for cancer there is a need for an efficient, upfront enrichment method to specifically isolate specific tumor cell populations. Immuno-Laser Capture Microdissection (immuno-LCM) is a technology based on immunohistochemistry (IHC) that can isolate specific cell types from a heterogeneous cellular population. We sought to determine the impact of various IHC chromogens on the efficacy of microdissection and downstream molecular analysis from immuno-LCM samples. Methods: Histological tissue specimens (clinical colorectal and lung cancer PDX) were processed using traditional IHC methods with a variety of chromogens, including: Vector Black, Impact Vector Red, 3-amino-9-ethylcarbazole (AEC), and 3,3'-diaminobenzidine (DAB). These chromogens were evaluated for staining quality, microdissection efficiency, biomolecular extraction, and evaluation of specific molecular targets. Results: Interestingly, DAB, the most commonly used chromogen, demonstrated a negative impact on the retrieval of biomolecules from immuno-LCM samples. However, chromogens such as Vector Black and Impact Vector Red showed much improved biomolecule extraction compared to DAB even though the yield of microdissected material was comparable. While other chromogens, such as AEC, demonstrated a greater efficiency of biomolecule extraction but showed incomplete immuno-LCM of specific IHC-stained cells. Conclusions: The data indicate that traditional IHC chromogens should be carefully considered when using immuno-LCM to analyze biomolecules. Citation Format: Sarah Laun, Adam Roberge, Robert Weigman, Aubrey Lewis, Valarie Greisman, Meeiyueh Liu, Owen Stephens, Erich Peterson, Ikjae Shin, Michael Emmert-Buck, Michael Tangrea, Donald J. Johann. Towards advanced precision oncology assays - Selection of immunohistochemistry chromogen impacts microdissection and downstream molecular analysis of archival specimens [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 2947.

Alternative transcription of a shorter, non-anti-angiogenic thrombospondin-2 variant in cancer-associated blood vessels

Thrombospondin-2 (TSP2) is an anti-angiogenic matricellular protein that inhibits tumor growth and angiogenesis. Tumor-associated blood vascular endothelial cells (BECs) were isolated from human invasive bladder cancers and from matched normal bladder tissue by immuno-laser capture microdissection. Exon expression profiling analyses revealed a particularly high expression of a short TSP2 transcript containing only the last 9 (3′) exons of the full-length TSP2 transcript. Using 5′ and 3′ RACE (rapid amplification of cDNA ends) and Sanger sequencing, we confirmed the existence of the shorter transcript of TSP2 (sTSP2) and determined its sequence which completely lacked the anti-angiogenic thrombospondin type 1 repeats domain. The largest open reading frame predicted within the transcript comprises 209 amino acids and matches almost completely the C-terminal lectin domain of full-length TSP2. We produced recombinant sTSP2 and found that unlike the full-length TSP2, sTSP2 did not inhibit vascular endothelial growth factor-A-induced proliferation of cultured human BECs, but in contrast when combined with TSP2 blocked the inhibitory effects of TSP2 on BEC proliferation. In vivo studies with stably transfected A431 squamous cell carcinoma cells revealed that full-length TSP2, but not sTSP2, inhibited tumor growth and angiogenesis. This study reveals that the transcriptional program of tumor stromal cells can change to transcribe a new version of an endogenous angiogenesis inhibitor that has lost its anti-angiogenic activity.

MP68-09 INSULIN RECEPTOR IS OVEREXPRESSED IN TUMOR BLOOD VESSELS AND PREDICTS WORSE OUTCOME IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2015MP68-09 INSULIN RECEPTOR IS OVEREXPRESSED IN TUMOR BLOOD VESSELS AND PREDICTS WORSE OUTCOME IN BLADDER CANCER Cédric Poyet, Filip Roudnicky, Peter Wild, Lothar Dieterich, Lorenz Buser, Chien Ho, Tullio Sulser, Vivianne Otto, and Michael Detmar Cédric PoyetCédric Poyet More articles by this author , Filip RoudnickyFilip Roudnicky More articles by this author , Peter WildPeter Wild More articles by this author , Lothar DieterichLothar Dieterich More articles by this author , Lorenz BuserLorenz Buser More articles by this author , Chien HoChien Ho More articles by this author , Tullio SulserTullio Sulser More articles by this author , Vivianne OttoVivianne Otto More articles by this author , and Michael DetmarMichael Detmar More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2471AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Invasive bladder cancer (IBC) often leads to a metastatic and lethal disease, whereas non-invasive bladder cancer (NIBC) is characterized by high recurrence rates. Tumor angiogenesis provides the supply of oxygen and nutrients to the proliferating urothelial tumor cells. Tumor–associated blood vessels are believed to differ from normal vessels at the morphological and molecular level. Proteins that are only present on tumor vessels may thus serve as biomarkers and as therapeutic targets for inhibition of angiogenesis in bladder cancer. METHODS we have performed immuno-laser capture microdissection of blood vascular endothelial cells in conjunction with transcriptional profiling. RNA was extracted from isolated vascular blood endothelial Cultured blood vascular endothelial cells (BECs) were used. In addition tissue microarray and immunohistochemistry were performed. RESULTS Comparing the mRNA levels of insulin receptor of human IBC-associated BECs and of normal bladder BECs from 5 patients, we found that insulin receptor (INSR) was highly elevated on tumor vessels. This upregulation was confirmed at the protein level by immunohistochemical staining. Notably, strong INSR expression on bladder cancer-associated BECs was associated with significantly reduced progression-free survival (PFS) and significantly reduced overall survival (OS) in patients with IBC after radical cystectomy (n=66). Furthermore, strong INSR expression was associated with shorter recurrence-free survival (RFS) in patients with NIBC (n=90). In addition, several other carcinomas, sarcomas and lymphomas showed high level INSR expression of the blood vasculature as detected by immunohistochemical staining. To elucidate the mechanisms of INSR regulation, primary human dermal blood vascular endothelial cells (BECs) were incubated with growth/inflammatory factors (VEGF-A, FGF-2, HGF, TNF-α, TGF-β, IGF-1, IGF-2; all 20 ng/ml) or metabolic substrates/conditions (glucose – 30 mM, insulin - 10 μM, acidosis – pH 6.6, lactate – 10 mM, hypoxia – 1% O2) for 24 h. Hypoxia significantly upregulated INSR in BECs in vitro. CONCLUSIONS Our study provides evidence that INSR is upregulated on bladder cancer-associated blood vessels and is of prognostic relevance in bladder cancer. Moreover, it also indicates that INSR is upregulated by hypoxia and could have a role in tumor angiogenesis. The detailed role of INSR in tumor angiogenesis must be elucidated in future studies. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e861 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cédric Poyet More articles by this author Filip Roudnicky More articles by this author Peter Wild More articles by this author Lothar Dieterich More articles by this author Lorenz Buser More articles by this author Chien Ho More articles by this author Tullio Sulser More articles by this author Vivianne Otto More articles by this author Michael Detmar More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

IGFBP7, a novel tumor stroma marker, with growth-promoting effects in colon cancer through a paracrine tumor–stroma interaction

The activated tumor stroma participates in many processes that control tumorigenesis, including tumor cell growth, invasion and metastasis. Cancer-associated fibroblasts (CAFs) represent the major cellular component of the stroma and are the main source for connective tissue components of the extracellular matrix and various classes of proteolytic enzymes. The signaling pathways involved in the interactions between tumor and stromal cells and the molecular characteristics that distinguish normal 'resting' fibroblasts from cancer-associated or '-activated' fibroblasts remain poorly defined. Recent studies emphasized the prognostic and therapeutic significance of CAF-related molecular signatures and a number of those genes have been shown to serve as putative therapeutic targets. We have used immuno-laser capture microdissection and whole-genome Affymetrix GeneChip analysis to obtain transcriptional signatures from the activated tumor stroma of colon carcinomas that were compared with normal resting colonic fibroblasts. Several members of the Wnt-signaling pathway and gene sets related to hypoxia, epithelial-to-mesenchymal transition (EMT) and transforming growth factor-β (TGFβ) pathway activation were induced in CAFs. The putative TGFβ-target IGFBP7 was identified as a tumor stroma marker of epithelial cancers and as a tumor antigen in mesenchyme-derived sarcomas. We show here that in contrast to its tumor-suppressor function in epithelial cells, IGFPB7 can promote anchorage-independent growth in malignant mesenchymal cells and in epithelial cells with an EMT phenotype when IGFBP7 is expressed by the tumor cells themselves and can induce colony formation in colon cancer cells co-cultured with IGFBP7-expressing CAFs by a paracrine tumor-stroma interaction.

Target Proteomic Profiling of Frozen Pancreatic CD24+ Adenocarcinoma Tissues by Immuno-Laser Capture Microdissection and Nano-LC–MS/MS

Cellular heterogeneity of solid tumors represents a common problem in mass spectrometry (MS)-based analysis of tissue specimens. Combining immuno-laser capture microdissection (iLCM) and mass spectrometry (MS) provides a means to study proteins that are specific for pure cell subpopulations in complex tissues. CD24, as a cell surface marker for detecting pancreatic cancer stem cells (CSCs), is directly correlated with the development and metastasis of pancreatic cancer. Herein, we describe an in-depth proteomic profiling of frozen pancreatic CD24(+) adenocarcinoma cells from early stage tumors using iLCM and LC-MS/MS and a comparison with CD24(-) cells dissected from patient-matched adjacent normal tissues. Approximately 40 nL of tissue was procured from each specimen and subjected to tandem MS analysis in triplicate. A total of 2665 proteins were identified, with 375 proteins in common that were significantly differentially expressed in CD24(+) versus CD24(-) cells by at least a 2-fold change. The major groups of the differentially overexpressed proteins are involved in promoting tumor cell migration and invasion, immune escape, and tumor progression. Three selected candidates relevant to mediating immune escape, CD59, CD70, and CD74, and a tumor promoter, TGFBI, were further validated by immunohistochemistry analysis on tissue microarrays. These proteins showed significantly increased expression in a large group of clinical pancreatic adenocarcinomas but were negative in all normal pancreas samples. The significant coexpression of these proteins with CD24 suggests that they may play important roles in the progression of pancreatic cancer and could serve as promising prognosis markers and novel therapeutic targets for this deadly disease.

Abstract 3893: Endocan is upregulated on tumor vessels in invasive bladder cancer and mediates VEGF-A-induced angiogenesis.

Abstract Tumor-associated blood vessels differ from normal vessels at the morphological and molecular level. Proteins that are only present on tumor vessels may serve as biomarkers and as therapeutic targets for inhibition of angiogenesis in cancer. To identify factors contributed by the tumor-associated endothelium, we performed immuno-laser capture microdissection (i-LCM) of blood vascular endothelial cells in conjunction with transcriptional profiling from surgically harvested cancer and normal matched bladder tissue of five patients with invasive bladder cancer. Comparing the transcriptional profiles of blood vascular endothelium from human invasive bladder cancer and from normal bladder tissue, we found that endocan (endothelial cell-specific molecule-1) was highly elevated on tumor vessels and associated with the filopodia of angiogenic endothelial tip cells in invasive bladder cancer. Notably, endocan expression on tumor vessels strongly correlated with the tumor stage and invasiveness and predicted a shorter recurrence-free survival time in non-invasive bladder cancers. Furthermore, both endocan and VEGF-A levels were significantly higher in plasma of patients with invasive bladder cancer (n=53) compared to healthy individuals (n=60). Using cultured blood vascular endothelial cells and an in vivo transgenic mouse model we found that endocan expression was strongly upregulated by VEGF-A activation of VEGFR-2. RNA interference-mediated knockdown of endocan in cultured blood vascular endothelial cells revealed that endocan was required for the promotion of cell migration and tube formation by VEGF-A. Furthermore, we found that endocan potentiates VEGFR-2 phosphorylation in response to VEGF-A. Therefore, blocking the interaction of endocan with either VEGFR-2 or VEGF-A might represent a promising approach for inhibiting tumor angiogenesis. Endocan might also serve as a novel biomarker for monitoring disease progression and the efficacy of VEGF-A-targeting therapies in patients with bladder cancer. Citation Format: Filip Roudnicky, Cedric Poyet, Peter J. Wild, Sarah Krampitz, Fabrizia Negrini, Reto Huggenberger, Anja Rogler, Robert Stöhr, Arndt Hartmann, Maurizio Provenzano, Vivianne I. Otto, Michael Detmar. Endocan is upregulated on tumor vessels in invasive bladder cancer and mediates VEGF-A-induced angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3893. doi:10.1158/1538-7445.AM2013-3893

Isolation of enriched glial populations from post-mortem human CNS material by immuno-laser capture microdissection

Isolating individual populations of cells from post-mortem (PM) central nervous system (CNS) tissue for transcriptomic analysis will provide important insights into the pathogenesis of neurodegenerative diseases. To date, research on individual CNS cell populations has been hindered by the availability of suitable PM material, unreliable sample preparation and difficulties obtaining individual cell populations. In this paper we report how rapid immunohistochemistry combined with laser capture microdissection (immuno-LCM) enables the isolation of specific cell populations from PM CNS tissue, thereby enabling the RNA profile of these individual cell types to be investigated. Specifically, we detail methods for isolating enriched glial populations (astrocytes, oligodendrocytes and microglia) and confirm this cell enrichment by polymerase chain reaction (PCR). In addition, the study details the numbers of each glial population required to obtain 50ng RNA, a suitable amount of starting material required to carry out microarray analysis that potentially may identify alterations of cell-specific genes and pathways associated with a range of neurodegenerative disorders.

Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan® Low Density Array

Immuno-laser capture microdissection (immuno-LCM) enables highly selective retrieval of designated cell populations from their in situ locations in complex tissue like the brain. However, the amount of tissue acquired by immuno-LCM is extremely limited, and the RNA purification, amplification and labeling steps necessary for expression analysis by hybridization microarray are tedious and time consuming. This report therefore describes a protocol in which these RNA steps are eliminated altogether, yet allows for global gene profiling. Specifically, immuno-LCM tissue was solubilized and the extract directly subjected to reverse transcription to generate cDNA. Pre-amplification of cDNA was performed next, and then relative expression of 96 different immune-related genes simultaneously determined by quantitative real-time PCR using a microfluidic card TaqMan® Low Density Array (TLDA). This protocol was highly reproducible and extremely sensitive, demonstrating high correlation of raw Ct values among both technical and biological replicate samples when using only 1/32 of total pre-amplified cDNA obtained from as little as 500 LCM ‘shots.’ As this abridged protocol takes only approximately 7h from LCM tissue acquisition to analysis by TLDA, it can prove a very effective tool for both screening and validation purposes when investigating gene regulation in health and disease of the nervous system and other tissues.

Effect of Immunohistochemistry on Molecular Analysis of Tissue Samples

Laser-based tissue microdissection is an important tool for the molecular evaluation of histological sections. The technology has continued to advance since its initial commercialization in the 1990s, with improvements in many aspects of the process. More recent developments are tailored toward an automated, operator-independent mode that relies on antibodies as targeting probes, such as immuno-laser capture microdissection or expression microdissection (xMD). Central to the utility of expression-based dissection techniques is the effect of the staining process on the biomolecules in histological sections. To investigate this issue, the authors analyzed DNA, RNA, and protein in immunostained, microdissected samples. DNA was the most robust molecule, exhibiting no significant change in quality after immunostaining but a variable 50% to 75% decrease in the total yield. In contrast, RNA in frozen and ethanol-fixed, paraffin-embedded samples was susceptible to hydrolysis and digestion by endogenous RNases during the initial steps of staining. Proteins from immunostained tissues were successfully analyzed by one-dimensional electrophoresis and mass spectrometry but were less amenable to solution phase assays. Overall, the results suggest investigators can use immunoguided microdissection methods for important analytic techniques; however, continued improvements in staining protocols and molecular extraction methods are key to further advancing the capability of these methods.

Proteomic analysis of prolactinoma cells by immuno-laser capture microdissection combined with online two-dimensional nano-scale liquid chromatography/mass spectrometry

BackgroundPituitary adenomas, the third most common intracranial tumor, comprise nearly 16.7% of intracranial neoplasm and 25%-44% of pituitary adenomas are prolactinomas. Prolactinoma represents a complex heterogeneous mixture of cells including prolactin (PRL), endothelial cells, fibroblasts, and other stromal cells, making it difficult to dissect the molecular and cellular mechanisms of prolactin cells in pituitary tumorigenesis through high-throughout-omics analysis. Our newly developed immuno-laser capture microdissection (LCM) method would permit rapid and reliable procurement of prolactin cells from this heterogeneous tissue. Thus, prolactin cell specific molecular events involved in pituitary tumorigenesis and cell signaling can be approached by proteomic analysis.ResultsProteins from immuno-LCM captured prolactin cells were digested; resulting peptides were separated by two dimensional-nanoscale liquid chromatography (2D-nanoLC/MS) and characterized by tandem mass spectrometry. All MS/MS spectrums were analyzed by SEQUEST against the human International Protein Index database and a specific prolactinoma proteome consisting of 2243 proteins was identified. This collection of identified proteins by far represents the largest and the most comprehensive database of proteome for prolactinoma. Category analysis of the proteome revealed a widely unbiased access to various proteins with diverse functional characteristics.ConclusionsThis manuscript described a more comprehensive proteomic profile of prolactinomas compared to other previous published reports. Thanks to the application of immuno-LCM combined with online two-dimensional nano-scale liquid chromatography here permitted identification of more proteins and, to our best knowledge, generated the largest prolactinoma proteome. This enlarged proteome would contribute significantly to further understanding of prolactinoma tumorigenesis which is crucial to the management of prolactinomas.

Endothelial cell heterogeneity of blood‐brain barrier gene expression along the cerebral microvasculature

The blood-brain barrier (BBB) refers to the network of microvessels that selectively restricts the passage of substances between the circulation and the central nervous system (CNS). This microvascular network is comprised of arterioles, capillaries and venules, yet the respective contribution of each of these to the BBB awaits clarification. In this regard, it has been postulated that brain microvascular endothelial cells (BMEC) from these different tributaries might exhibit considerable heterogeneity in form and function, with such diversity underlying unique roles in physiological and pathophysiological processes. Means to begin exploring such endothelial differences in situ, free from caveats associated with cell isolation and culturing procedures, are crucial to comprehending the nature and treatment of CNS diseases with vascular involvement. Here, the recently validated approach of immuno-laser capture microdissection (immuno-LCM) coupled to quantitative real-time PCR (qRT-PCR) was used to analyze gene expression patterns of BMEC retrieved in situ from either capillaries or venules. From profiling 87 genes known to play a role in BBB function and/or be enriched in isolated brain microvessels, results imply that most BBB properties reside in both segments, but that capillaries preferentially express some genes related to solute transport, while venules tend toward higher expression of an assortment of genes involved in inflammatory-related tasks. Fuller appreciation of such heterogeneity will be critical for efficient therapeutic targeting of the endothelium and the management of CNS disease.

Microenvironmental Changes in DCIS: Myoepithelial Cells Change from Tumour Suppressor to Tumour Promoter.

Abstract Background: The mechanism of transition of ductal carcinoma in-situ (DCIS) to invasive cancer is poorly understood but recently changes in the microenvironment have been implicated. A unique feature of the DCIS microenvironment is the presence of a myoepithelial population. Normal myoepithelial cells (MECs) have a broad tumour suppressor function but these cells show changes in gene expression and phenotype in DCIS.Hypothesis: We hypothesised that during the evolution of DCIS breast myoepithelial cells undergo a switch in gene expression profile that compromises their suppressor function and may lead to promotion of tumour progression.Materials and methods: To investigate this, Immuno-Laser Capture Microdissection was used to isolate MECs from normal and DCIS breast tissues followed by whole genome expression profiling using Affymetrix HGU-133 plus2.0 arrays. The data were analysed using Bioconductor (www.bioconductor.org) packages within the open source R statistical environment (www.r-project.org). We set a double threshold for significant changes in gene expression with a False Discovery Rate (FDR) &amp;lt;= 0.05 and an absolute fold change ≥4.5.Results: Ingenuity Pathways software analysis showed clustering of most of the altered genes in cancer, cell death and cell-cell signalling networks, with the integrin pathway as the top canonical pathway. Validation using QRT-PCR and immunohistochemistry (IHC) revealed a 71.4% correlation rate with the array results. Most dramatic was up-regulation of Fibronectin-1 (FN1) in DCIS MEC. IHC analysis for FN1 on normal (n=88) and DCIS tissues with and without associated invasion (n=87) confirmed a strong correlation between FN1 protein expression by MEC and DCIS (p&amp;lt;0.0001). We set cut off point of 50% for percentage of ducts showing positive staining for FN1 by MECs in pure DCIS cases and DCIS cases that have progressed to IDC and found a statistically significant correlation between high expression level (&amp;gt;50%) and presence of invasion (p=0.006). Moreover, culture of normal and DCIS-modified MEC lines on a Fibronectin matrix revealed that FN1 modifies MEC function and abrogates their tumour suppressor activity.Other key alterations identified and validated include up-regulation of the recently characterised Nephronectin and down-regulation of PTHLH, FGFR2, ADAMTS5, TGFBR3, and CAV2, each of which has been implicated in suppressing tumour growth and invasion or inhibition of angiogenesis.Conclusion and significance: This is the first study to use this in-vivo technique to investigate molecular changes in MEC in DCIS and to reveal that DCIS associated MECs show up-regulation of the tumour-promoting molecule FN1. Our data support the hypothesis that there is switch in MEC function from suppressor to potential promoter activity that can influence the progression of in-situ to invasive disease and may lead to novel predictive and therapeutic targets. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4161.

Novel markers for enterochromaffin cells and gastrointestinal neuroendocrine carcinomas

The gene expression profile of metastasizing serotonin-producing neuroendocrine carcinomas, which arise from enterochromaffin cells in the jejunum and ileum, is still largely unknown. The aim of this study was to identify genes and proteins, which are preferentially expressed by neuroendocrine carcinoma and enterochromaffin cells and therefore potential novel biomarkers and/or therapeutic targets. Six carcinoma specimens and six normal ileal mucosas were profiled by Affymetrix microarrays. Advanced bioinformatics identified differentially and specifically expressed genes, which were validated by quantitative real-time-PCR on tumor cells extracted by laser capture microdissection and normal enterochromaffin cells extracted by immunolaser capture microdissection. We identified six novel marker genes for neuroendocrine carcinoma cells: paraneoplastic antigen Ma2 (PNMA2), testican-1 precursor (SPOCK1), serpin A10 (SERPINA10), glutamate receptor ionotropic AMPA 2 (GRIA2), G protein-coupled receptor 112 (GPR112) and olfactory receptor family 51 subfamily E member 1 (OR51E1). GRIA2 is specifically expressed by neuroendocrine carcinoma cells whereas the others are also expressed by normal enterochromaffin cells. GPR112 and OR51E1 encode proteins associated with the plasma membrane and may therefore become targets for antibody-based diagnosis and therapy. Hierarchical clustering shows high similarity between primary lesions and liver metastases. However, chemokine C-X-C motif ligand 14 (CXCL14) and NK2 transcription factor related locus 3 Drosophila (NKX2-3) are expressed to a lower level in liver metastases than in primary tumors and normal enterochromaffin cells, which implies a role in neuroendocrine carcinoma differentiation. In conclusion, this study provides a list of genes, which possess relatively specific expression to enterochromaffin and neuroendocrine carcinoma cells and genes with differential expression between primary tumors and metastases. We verified six novel marker genes that may be developed as biomarkers and/or therapeutic targets.

Validation of immuno-laser capture microdissection coupled with quantitative RT-PCR to probe blood–brain barrier gene expression in situ

Laser capture microdissection (LCM) holds great potential for analyzing gene expression profiles in situ. Most recently, this laboratory employed a novel immunostain-based LCM protocol (immuno-LCM) to selectively retrieve brain microvascular endothelial cells (BMEC) from intimately associated perivascular cells. However, before this protocol can be confidently coupled to downstream analytical platforms, it must be demonstrated that any variability associated with it is minimal, so as not to obscure data interpretation. As various factors could contribute to variability, this study focused on determining whether technical inconsistency and/or biological diversity of sample populations, played such a role. Specifically, two separate immuno-LCM-derived BMEC samples derived from adjacent tissue sections of a single mouse (to detect only technical variability), and from analogous tissue sections of three different mice (to detect technical and biological variability) were compared for their relative expression of 16 genes, using quantitative-RT-PCR (qRT-PCR). Both significant linear and rank-order correlations were observed between different sections from the same animal, underscoring lack of technical variability in this LCM application. Furthermore, a three-dimensional scatter plot of gene expression profiles from the three animals was linear, and ANOVA showed absence of statistically significant differences between any of the animals, confirming lack of biological variability. These findings argue that immuno-LCM coupled to qRT-PCR affords a reproducible means to assay gene expression in situ.

Analysis of mouse brain microvascular endothelium using immuno‐laser capture microdissection coupled to a hybrid linear ion trap with Fourier transform‐mass spectrometry proteomics platform

The purpose of this study was to identify the protein profile of the mouse brain microvascular endothelium in situ. This involved coupling of a double-label, immuno-laser capture microdissection (LCM) process with LTQ-FT mass spectrometry to perform the in situ proteomic analysis. LCM was utilized to isolate cells from frozen mouse brain tissue sections. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and LC-MS analysis. Processed samples were subsequently analyzed using a linear IT coupled with a Fourier transform mass spectrometer (LTQ-FT MS). Overall, in this study, 881 proteins were identified from a specific cell category using immuno-guided LCM to probe these cell types along the entirety of the cerebral microvascular tree. The identification of sufficient numbers of proteins with high biological interest should allow us to study protein expression by specific cell types - as defined by certain cell markers - in complex tissues.

IgVH Mutational Status and Clonality Analysis of Richter's Transformation

Approximately 5% of B-cell chronic lymphocytic leukemia (B-CLL) patients develop a secondary aggressive lymphoma, usually of diffuse large B-cell type (DLBCL), termed Richter's transformation (RT). Rarely, classic Hodgkin lymphoma (HL) is observed. Published small series suggest that tumor cells in DLBCL and HL can be clonally identical to the B-CLL clone or arise as an independent, secondary lymphoma. We describe the morphology, immunophenotype, and clinical features of 34 classic RT patients with DLBCL, 6 cases of B-CLL with HL, and 8 cases with scattered CD30-positive Hodgkin and Reed-Sternberg (HRS)-like cells. The clonal relationship of the 2 components was analyzed using sequencing analysis of immunoglobulin heavy chain variable region (IgVH) genes. In classic RT, 18/23 B-CLL cases (78%) showed clonal progression to DLBCL with identical IgVH sequences in both lymphoma components, whereas in 5 cases (22%) the DLBCL was clonally unrelated. Among clonally related RT samples, 73% carried unmutated IgVH genes, whereas 4/5 unrelated cases were mutated. Immunophenotypically, most cases of DLBCL irrespective of clonal relatedness showed significant differences in phenotype compared with the B-CLL, with common loss of CD5 and CD23. Using immuno-laser capture microdissection, sequencing of the IgVH CDR3 region of isolated HRS cells showed that 2/2 cases with HL were clonally unrelated, whereas they were clonally identical in 1/2 cases of B-CLL with scattered HRS-like cells. HRS or HRS-like cells in all 3 unrelated cases showed evidence of Epstein-Barr virus infection. Of interest, 5/6 cases of B-CLL with HL, and 5/6 cases of B-CLL with HRS cells showed mutated IgVH genes.