Toponome Mapping in Prostate Cancer: Detection of 2000 Cell Surface Protein Clusters in a Single Tissue Section and Cell Type Specific Annotation by Using a Three Symbol Code

Schubert, Walter; Gieseler, Anne; Krusche, A.; Hillert, Reyk

doi:10.1021/pr800944f

Cited by 30 publications

(43 citation statements)

References 40 publications

(149 reference statements)

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“…Multiplexed imaging has been demonstrated by eluting or stripping antibodies with low pH or denaturation (25)(26)(27). Multiepitope ligand cartography (MELC) is a photobleaching technique to achieve dye cycling (28)(29)(30). The process has demonstrated imaging of 100 antigens in a single sample.…”

Section: Discussionmentioning

confidence: 99%

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

Gerdes¹,

Sevinsky

Sood³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

641

761

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Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffinembedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.cancer diagnostics | high-content cellular analysis | image analysis | mTOR | multiplexing

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Section: Discussionmentioning

confidence: 99%

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

Gerdes¹,

Sevinsky

Sood³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

641

761

View full text Add to dashboard Cite

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“…Among these, Fourier-transform infrared and Raman microspectroscopy can provide biomolecular profile of individual cells in tissue sections, [81][82][83][84] and certain high-throughput multiplex immunocytochemical techniques can map a virtually unlimited number of proteins to individual cells in tissue sections (in situ proteome). [85][86][87] These technologies will enable taking advantage of the focal nature of the 4HNE immunostaining of mammary epithelial cells seen in most subjects. Being able to compare the molecular profiles of 4HNE+ and 4HNE-cells in tissue sections from the same subject will eliminate confounding variables of inter-individual differences in genetic background, environment/ lifestyle factors and hormonal status.…”

Section: Discussionmentioning

confidence: 99%

Identification of mammary epithelial cells subject to chronic oxidative stress in mammary epithelium of young women and teenagers living in USA

Weisz¹,

Shearer²,

Murata

et al. 2012

Cancer Biology & Therapy

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Current knowledge of changes in the mammary epithelium relevant to breast carcinogenesis is limited to when histological changes are already present because of a lack of biomarkers needed to identify where such molecular changes might be ongoing earlier during the decades-long latent stages of breast carcinogenesis. Breast reduction tissues from young women and teenagers, representative of the USA's high breast cancer incidence population, were studied using immunocytochemistry and a targeted PCR array in order to learn whether a marker of chronic oxidative stress [protein adducts of 4-hydroxy-2-nonenal (4HNE)] can identify where molecular changes relevant to carcinogenesis might be taking place prior to any histological changes. 4HNE-immunopositive (4HNE+) mammary epithelial cell-clusters were identified in breast tissue sections from most women and from many teenagers (ages 14-30 y) and, in tissues from women ages 17-27 y with many vs. few 4HNE+ cells, the expression of 30 of 84 oxidative stress associated genes represented in SA Bioscience RT2 Oxidative Stress and Antioxidant PCR array was decreased and only one was increased . 2-fold. This is in contrast to increased expression of many of these genes known to be elicited by acute oxidative stress. The findings validate using 4HNE-adducts to identify where molecular changes of potential relevance to carcinogenesis are taking place in histologically normal mammary epithelium and highlight differences between responses to acute vs. chronic oxidative stress. We posit that the altered gene expression in 4HNE+ tissues identified reflects adaptive responses to chronic oxidative stress that enable some cells to evade mechanisms that have evolved to prevent propagation of cells with oxidatively-damaged DNA and to accrue heritable changes needed to establish a cancer.

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“…We tested the frequencies of co-localization of pZAP70 and pSLP76 in synaptic areas that were also positive for pLAT and CD3 (pCD3[Y5]). The use of binarized imaging data resulting from the thresholding of fluorescent signals was not only useful in evaluating low-intensity signals of individual markers, but could also be used to generate and quantify CMP motifs (25,42,43), patterns of localizations of several markers within one spot (pixel). Although it is conceptually easier to think of a CMP motif as a protein complex, MELC analysis does not provide data on actual binding between proteins-only on shared locations.…”

Section: Distinct Phases Of Molecular Recruitment and Colocalization mentioning

confidence: 99%

Multimolecular Analysis of Stable Immunological Synapses Reveals Sustained Recruitment and Sequential Assembly of Signaling Clusters

Philipsen

Engels

Schilling

et al. 2013

Molecular & Cellular Proteomics

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The formation of the immunological synapse between T cells and antigen-presenting cells (APC) begins within minutes of contact and can take hours for full T-cell activation. Although early phases of the synapse have been extensively studied for a select number of proteins, later phases have not yet been examined in detail. We studied the signaling network in stable synapses by measuring the simultaneous localization of 25 signaling and structural molecules over 2 h at the level of individual synapses using multi-epitope ligand cartography (MELC). Signaling proteins including phospho(p)ZAP70, pSLP76, pCD3, and pLAT, along with proteins that influence synapse structure such as F-actin, tubulin, CD45, and ICAM-1, were localized in images of synapses and revealed the multidimensional construction of a mature synapse. The construction of the stable synapse included intense early TCR signaling, a phase of recruitment of structural proteins, and a sustained increase in signaling molecules and colocalization of TCR and pLAT signaling clusters in the center of the synapse. Consolidation of TCR and associated proteins resulted in formation of a small number of discrete synaptic microclusters. Development of synapses and cSMAC composition was greatly affected by the absence of Vav1, with an associated loss in PLC␥1 recruitment, pSLP76, and increased CXCR4. Adaptive immune responses are initiated by the meeting of a T cell and an antigen-presenting cell (APC) 1 bearing peptide-MHC (pMHC) complexes that are a specific fit for the T-cell receptor (TCR) on the T-cell surface. Within seconds, TCR signaling starts with a sequence of phosphorylation and de-phosphorylation events of membrane-proximal and -distal TCR-signaling molecules and their spatial reorganization into protein multiclusters (1). Together with the rearrangement of structural molecules at the cell-cell interface, these signals lead to the formation of a supramolecular structure termed the immunological synapse (1-3). The synapse can differ substantially in size and composition, but comprises several common structural motifs (4 -6). In the classical synapse, these structural motifs are organized in domains that form a target

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Toponome Mapping in Prostate Cancer: Detection of 2000 Cell Surface Protein Clusters in a Single Tissue Section and Cell Type Specific Annotation by Using a Three Symbol Code

Cited by 30 publications

References 40 publications

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

Identification of mammary epithelial cells subject to chronic oxidative stress in mammary epithelium of young women and teenagers living in USA

Multimolecular Analysis of Stable Immunological Synapses Reveals Sustained Recruitment and Sequential Assembly of Signaling Clusters

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