Publications by authors named "Amin El-Heliebi"

33 Publications

Non-coding Natural Antisense Transcripts: Analysis and Application.

J Biotechnol 2021 Aug 6. Epub 2021 Aug 6.

Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center for Cell Signalling, Metabolism and Aging, Medical University of Graz, Neue Stiftingtalstraße 6/II, 8010 Graz, Austria; Christian Doppler Laboratory for innovative Pichia pastoris host and vector systems, Division of Cell Biology, Histology and Embryology, Medical University of Graz, Neue Stiftingtalstraße 6/II, 8010 Graz, Austria; BioTechMed-Graz, Mozartgasse 12/II, 8010 Graz, Austria. Electronic address:

Non-coding natural antisense transcripts (ncNATs) are regulatory RNA sequences that are transcribed in the opposite direction to protein-coding or non-coding transcripts. These transcripts are implicated in a broad variety of biological and pathological processes, including tumorigenesis and oncogenic progression. With this complex field still in its infancy, annotations, expression profiling and functional characterisations of ncNATs are far less comprehensive than those for protein-coding genes, pointing out substantial gaps in the analysis and characterisation of these regulatory transcripts. In this review, we discuss ncNATs from an analysis perspective, in particular regarding the use of high-throughput sequencing strategies, such as RNA-sequencing, and summarize the unique challenges of investigating the antisense transcriptome. Finally, we elaborate on their potential as biomarkers and future targets for treatment, focusing on cancer.
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http://dx.doi.org/10.1016/j.jbiotec.2021.08.005DOI Listing
August 2021

T-regulatory cells predict clinical outcome in soft tissue sarcoma patients: a clinico-pathological study.

Br J Cancer 2021 Aug 14;125(5):717-724. Epub 2021 Jun 14.

Division of Clinical Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria.

Background: Soft tissue sarcomas (STS) are generally considered non-immunogenic, although specific subtypes respond to immunotherapy. Antitumour response within the tumour microenvironment relies on a balance between inhibitory and activating signals for tumour-infiltrating lymphocytes (TILs). This study analysed TILs and immune checkpoint molecules in STS, and assessed their prognostic impact regarding local recurrence (LR), distant metastasis (DM), and overall survival (OS).

Methods: One-hundred and ninety-two surgically treated STS patients (median age: 63.5 years; 103 males [53.6%]) were retrospectively included. Tissue microarrays were constructed, immunohistochemistry for PD-1, PD-L1, FOXP3, CD3, CD4, and CD8 performed, and staining assessed with multispectral imaging. TIL phenotype abundance and immune checkpoint markers were correlated with clinical and outcome parameters (LR, DM, and OS).

Results: Significant differences between histology and all immune checkpoint markers except for FOXP3+ and CD3-PD-L1+ cell subpopulations were found. Higher levels of PD-L1, PD-1, and any TIL phenotype were found in myxofibrosarcoma as compared to leiomyosarcoma (all p < 0.05). The presence of regulatory T cells (Tregs) was associated with increased LR risk (p = 0.006), irrespective of margins. Other TILs or immune checkpoint markers had no significant impact on outcome parameters.

Conclusions: TIL and immune checkpoint marker levels are most abundant in myxofibrosarcoma. High Treg levels are independently associated with increased LR risk, irrespective of margins.
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http://dx.doi.org/10.1038/s41416-021-01456-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405702PMC
August 2021

Influence of tumor-infiltrating immune cells on local control rate, distant metastasis, and survival in patients with soft tissue sarcoma.

Oncoimmunology 2021 03 11;10(1):1896658. Epub 2021 Mar 11.

Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

Soft tissue sarcomas (STS) are considered non-immunogenic, although distinct entities respond to anti-tumor agents targeting the tumor microenvironment. This study's aims were to investigate relationships between tumor-infiltrating immune cells and patient/tumor-related factors, and assess their prognostic value for local recurrence (LR), distant metastasis (DM), and overall survival (OS). One-hundred-eighty-eight STS-patients (87 females [46.3%]; median age: 62.5 years) were retrospectively analyzed. Tissue microarrays (in total 1266 cores) were stained with multiplex immunohistochemistry and analyzed with multispectral imaging. Seven cell types were differentiated depending on marker profiles (CD3+, CD3+ CD4+ helper, CD3+ CD8+ cytotoxic, CD3+ CD4+ CD45RO+ helper memory, CD3+ CD8+ CD45RO+ cytotoxic memory T-cells; CD20 + B-cells; CD68+ macrophages). Correlations between phenotype abundance and variables were analyzed. Uni- and multivariate Fine&Gray and Cox-regression models were constructed to investigate prognostic variables. Model calibration was assessed with C-index. IHC-findings were validated with TCGA-SARC gene expression data of genes specific for macrophages, T- and B-cells. B-cell percentage was lower in patients older than 62.5 years ( = .013), whilst macrophage percentage was higher ( = .002). High B-cell ( = .035) and macrophage levels ( = .003) were associated with increased LR-risk in the univariate analysis. In the multivariate setting, high macrophage levels ( = .014) were associated with increased LR-risk, irrespective of margins, age, gender or B-cells. Other immune cells were not associated with outcome events. High macrophage levels were a poor prognostic factor for LR, irrespective of margins, B-cells, gender and age. Thus, anti-tumor, macrophage-targeting agents may be applied more frequently in tumors with enhanced macrophage infiltration.
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http://dx.doi.org/10.1080/2162402X.2021.1896658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954425PMC
March 2021

Personalized medicine in Austria: expectations and limitations.

Per Med 2020 11 7;17(6):423-428. Epub 2020 Oct 7.

Department of Political Science, University of Vienna, Vienna 1010, Austria.

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http://dx.doi.org/10.2217/pme-2020-0061DOI Listing
November 2020

A Multi-Analyte Approach for Improved Sensitivity of Liquid Biopsies in Prostate Cancer.

Cancers (Basel) 2020 Aug 11;12(8). Epub 2020 Aug 11.

Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, Austria.

Novel androgen receptor (AR) signaling inhibitors have improved the treatment of castration-resistant prostate cancer (CRPC). Nonetheless, the effect of these drugs is often time-limited and eventually most patients become resistant due to various AR alterations. Although liquid biopsy approaches are powerful tools for early detection of such therapy resistances, most assays investigate only a single resistance mechanism. In combination with the typically low abundance of circulating biomarkers, liquid biopsy assays are therefore informative only in a subset of patients. In this pilot study, we aimed to increase overall sensitivity for tumor-related information by combining three liquid biopsy approaches into a multi-analyte approach. In a cohort of 19 CRPC patients, we (1) enumerated and characterized circulating tumor cells (CTCs) by mRNA-based in situ padlock probe analysis, (2) used RT-qPCR to detect cancer-associated transcripts (e.g., and -splice variant 7) in lysed whole blood, and (3) conducted shallow whole-genome plasma sequencing to detect amplification. Although 44-53% of patient samples were informative for each assay, a combination of all three approaches led to improved diagnostic sensitivity, providing tumor-related information in 89% of patients. Additionally, distinct resistance mechanisms co-occurred in two patients, further reinforcing the implementation of multi-analyte liquid biopsy approaches.
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http://dx.doi.org/10.3390/cancers12082247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465186PMC
August 2020

Distribution and prognostic significance of gluconeogenesis and glycolysis in lung cancer.

Mol Oncol 2020 11 1;14(11):2853-2867. Epub 2020 Sep 1.

Division of Pulmonology, Department of Internal Medicine, Medical University of Graz, Austria.

Inhibition of glycolysis has been considered as a therapeutic approach in aggressive cancers including lung cancer. Abbreviated gluconeogenesis, mediated by phosphoenolpyruvate carboxykinase (PEPCK), was recently discovered to partially circumvent the need for glycolysis in lung cancer cells. However, the interplay of glycolysis and gluconeogenesis in lung cancer is still poorly understood. Here, we analyzed the expression of GLUT1, the prime glucose transporter, and of PCK1 and PCK2, the cytoplasmic and mitochondrial isoforms of PEPCK, in 450 samples of non-small cell lung cancer (NSCLC) and in 54 NSCLC metastases using tissue microarrays and whole tumor sections. Spatial distribution was assessed by automated image analysis. Additionally, glycolytic and gluconeogenic gene expression was inferred from The Cancer Genome Atlas (TCGA) datasets. We found that PCK2 was preferentially expressed in the lung adenocarcinoma subtype, while GLUT1 expression was higher in squamous cell carcinoma. GLUT1 and PCK2 were inversely correlated, GLUT1 showing elevated expression in larger tumors while PCK2 was highest in smaller tumors. However, a mixed phenotype showing the presence of both, glycolytic and gluconeogenic cancer cells was frequent. In lung adenocarcinoma, PCK2 expression was associated with significantly improved overall survival, while the opposite was found for GLUT1. The metabolic tumor microenvironment and the 3-dimensional context play an important role in modulating both pathways, since PCK2 expression preferentially occurred at the tumor margin and hypoxia regulated both, glycolysis and gluconeogenesis, in NSCLC cells in vitro, albeit in opposite directions. PCK1/2 expression was enhanced in metastases compared to primary tumors, possibly related to the different environment. The results of this study show that glycolysis and gluconeogenesis are activated in NSCLC in a tumor size and oxygenation modulated manner and differentially correlate with outcome. The frequent co-activation of gluconeogenesis and glycolysis in NSCLC should be considered in potential future therapeutic strategies targeting cancer cell metabolism.
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http://dx.doi.org/10.1002/1878-0261.12780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607181PMC
November 2020

Using In Situ Padlock Probe Technology to Detect mRNA Splice Variants in Tumor Cells.

Methods Mol Biol 2020 ;2148:361-378

Division of Cell Biology, Histology & Embryology, Gottfried Schatz Research Center, Medical University Graz, Graz, Austria.

Advanced prostate cancer (PC) patients commonly receive anti-hormonal drugs targeting the androgen receptor (AR) signaling pathways. However, almost all patients acquire therapy resistance that can be caused by AR amplification or expression of AR splice variant 7 (AR-V7). Therefore, AR-V7 and AR expression are potential biomarkers for early detection of therapy resistance. Here, we present our padlock probe (PLP)-based approach for the in situ detection of AR full length, AR-V7, and prostate-specific transcripts in PC cell lines, which is applicable for circulating tumor cells (CTCs) isolated from cancer patients. First, PC cell lines are seeded on glass slides. Then, cDNA is created using target-specific reverse transcription primers. PLPs are hybridized to the cDNA and ligated to form circular single-stranded DNA molecules. The PLP sequence is ligated and amplified by rolling circle amplification and the resulting rolling circle products can be detected using fluorescently labeled probes. Quantification can be automated using the image analysis software CellProfiler.
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http://dx.doi.org/10.1007/978-1-0716-0623-0_23DOI Listing
March 2021

In Vivo Detection of Circulating Tumor Cells in High-Risk Non-Metastatic Prostate Cancer Patients Undergoing Radiotherapy.

Cancers (Basel) 2019 Jul 3;11(7). Epub 2019 Jul 3.

Department of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, Austria.

High-risk non-metastatic prostate cancer (PCa) has the potential to progress into lethal disease. Treatment options are manifold but, given a lack of surrogate biomarkers, it remains unclear which treatment offers the best results. Several studies have reported circulating tumor cells (CTCs) to be a prognostic biomarker in metastatic PCa. However, few reports on CTCs in high-risk non-metastatic PCa are available. Herein, we evaluated CTC detection in high-risk non-metastatic PCa patients using the in vivo CellCollector CANCER01 (DC01) and CellSearch system. CTC counts were analyzed and compared before and after radiotherapy (two sampling time points) in 51 high-risk non-metastatic PCa patients and were further compared according to isolation technique; further, CTC counts were correlated to clinical features. Use of DC01 resulted in a significantly higher percentage of CTC-positive samples compared to CellSearch (33.7% vs. 18.6%; = 0.024) and yielded significantly higher CTC numbers (range: 0-15 vs. 0-5; = 0.006). Matched pair analysis of samples between two sampling time points showed no difference in CTC counts determined by both techniques. CTC counts were not correlated with clinicopathological features. In vivo enrichment using DC01 has the potential to detect CTC at a higher efficiency compared to CellSearch, suggesting that CTC is a suitable biomarker in high-risk non-metastatic PCa.
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http://dx.doi.org/10.3390/cancers11070933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678903PMC
July 2019

Histological processing of un-/cellularized thermosensitive electrospun scaffolds.

Histochem Cell Biol 2019 Apr 17;151(4):343-356. Epub 2018 Dec 17.

Department of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6, 8010, Graz, Austria.

Histological processing of thermosensitive electrospun poly(ε-caprolactone)/poly(L-lactide) (PCL/PLA) scaffolds fails, as poly(ε-caprolactone) (PCL) is characterized by its low-melting temperature (Tm = 60 °C). Here, we present an optimized low-temperature preparation method for the histological processing of un-/cellularized thermosensitive PCL/PLA scaffolds.Our study is aimed at the establishment of an optimized dehydration and low-melting-point paraffin-embedding method of electrospun PCL/PLA scaffolds (un-/cellularized). Furthermore, we compared this method with (a) automatized dehydration and standard paraffin embedding, (b) gelatin embedding followed by automatized dehydration and standard paraffin embedding, (c) cryofixation, and (d) acrylic resin embedding methods. We investigated pepsin and proteinase K antigen retrieval for their efficiency in epitope demasking at low temperatures and evaluated protocols for immunohistochemistry and immunofluorescence for cytokeratin 7 (CK7) and in situ padlock probe technology for beta actin (ACTB). Optimized dehydration and low-melting-point paraffin embedding preserved the PCL/PLA scaffold, as the diameter and structure of its fibers were unchanged. Cells attached to the PCL/PLA scaffolds showed limited alterations in size and morphology compared to control. Epitope demasking by enzymatic pepsin digestion and immunostaining of CK7 displayed an invasion of attached cells into the scaffold. Expression of ACTB and CK7 was shown by a combination of mRNA-based in situ padlock probe technology and immunofluorescence. In contrast, gelatin stabilization followed by standard paraffin embedding led to an overall shrinkage and melting of fibers, and therefore, no further analysis was possible. Acrylic resin embedding and cyrofixation caused fiber structures that were nearly unchanged in size and diameter. However, acrylic resin-embedded scaffolds are limited to 3 µm sections, whereas cyrofixation led to a reduction of the cell size by 14% compared to low-melting paraffin embedding. The combination of low-melting-point paraffin embedding and pepsin digestion as an antigen retrieval method offers a successful opportunity for histological investigations in thermosensitive specimens.
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http://dx.doi.org/10.1007/s00418-018-1757-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469612PMC
April 2019

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.

J Vis Exp 2018 05 15(135). Epub 2018 May 15.

Institute of Cell Biology, Histology and Embryology, Medical University of Graz.

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs). A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell's amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (<10 - >50%) and needs some further attention. This device is not cleared for the use in patients.
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http://dx.doi.org/10.3791/56394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101176PMC
May 2018

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and Point Mutations in Circulating Tumor Cells.

Clin Chem 2018 03 4;64(3):536-546. Epub 2018 Jan 4.

Institute of Cell Biology, Histology and Embryology, Medical University Graz, Austria.

Background: Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms.

Methods: We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients.

Results: In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or mut transcripts.

Conclusions: Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.
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http://dx.doi.org/10.1373/clinchem.2017.281295DOI Listing
March 2018

Separation of low and high grade colon and rectum carcinoma by eukaryotic translation initiation factors 1, 5 and 6.

Oncotarget 2017 Nov 5;8(60):101224-101243. Epub 2017 Sep 5.

Institute of Pathology, Medical University of Graz, Graz, Austria.

Colorectal cancer (CRC) is the third most common cause of cancer related death worldwide. Furthermore, with more than 1.2 million cases registered per year, it constitutes the third most frequent diagnosed cancer entity worldwide. Deregulation of protein synthesis has received considerable attention as a major step in cancer development and progression. Eukaryotic translation initiation factors (eIFs) are involved in the regulation of protein synthesis and are functionally linked to the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. The identification of factors accounting for colorectal carcinoma (CRC) development is a major gap in the field. Besides the importance of eIF3 subunits and the eIF4 complex, eIF1, eIF5 and eIF6 were found to be altered in primary and metastatic CRC. We observed significant difference in the expression profile between low and high grade CRC. eIF1, eIF5 and eIF6 are involved in translational control in CRC. Our findings also indicate a probable clinical impact when separating them into low and high grade colon and rectum carcinoma. eIF and mTOR expression were analysed on protein and mRNA level in primary low and high grade colon carcinoma (CC) and rectum carcinoma (RC) samples in comparison to non-neoplastic tissue without any disease-related pathology. To assess the therapeutic potential of targeting eIF1, eIF5 and eIF6 siRNA knockdown in HCT116 and HT29 cells was performed. We evaluated the eIF knockdown efficacy on protein and mRNA level and investigated proliferation, apoptosis, invasion, as well as colony forming and polysome associated fractions. These results indicate that eIFs, in particular eIF1, eIF5 and eIF6 play a major role in translational control in colon and rectum cancer.
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http://dx.doi.org/10.18632/oncotarget.20642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5731869PMC
November 2017

Downregulation of p53 drives autophagy during human trophoblast differentiation.

Cell Mol Life Sci 2018 05 27;75(10):1839-1855. Epub 2017 Oct 27.

Institute of Cell Biology, Histology and Embryology, Medical University Graz, Neue Stiftingtalstraße 6, F/03/38, 8010, Graz, Austria.

The placental barrier is crucial for the supply of nutrients and oxygen to the developing fetus and is maintained by differentiation and fusion of mononucleated cytotrophoblasts into the syncytiotrophoblast, a process only partially understood. Here transcriptome and pathway analyses during differentiation and fusion of cultured trophoblasts yielded p53 signaling as negative upstream regulator and indicated an upregulation of autophagy-related genes. We further showed p53 mRNA and protein levels decreased during trophoblast differentiation. Reciprocally, autophagic flux increased and cytoplasmic LC3B-GFP puncta became more abundant, indicating enhanced autophagic activity. In line, in human first trimester placenta p53 protein mainly localized to the cytotrophoblast, while autophagy marker LC3B as well as late autophagic compartments were predominantly detectable in the syncytiotrophoblast. Importantly, ectopic overexpression of p53 reduced levels of LC3B-II, supporting a negative regulatory role on autophagy in differentiating trophoblasts. This was also shown in primary trophoblasts and human first trimester placental explants, where pharmacological stabilization of p53 decreased LC3B-II levels. In summary our data suggest that differentiation-dependent downregulation of p53 is a prerequisite for activating autophagy in the syncytiotrophoblast.
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http://dx.doi.org/10.1007/s00018-017-2695-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910494PMC
May 2018

Unravelling the biological secrets of microchimerism by single-cell analysis.

Brief Funct Genomics 2018 07;17(4):255-264

Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, Gothenburg, Sweden.

The presence of microchimeric cells is known for >100 years and well documented since decades. Earlier, microchimeric cells were mainly used for cell-based non-invasive prenatal diagnostics during early pregnancy. Microchimeric cells are also present beyond delivery and are associated to various autoimmune diseases, tissue repair, cancer and immune tolerance. All these findings were based on low complexity studies and occasionally accompanied by artefacts not allowing the biological functions of microchimerism to be determined. However, with the recent developments in single-cell analysis, new means to identify and characterize microchimeric cells are available. Cell labelling techniques in combination with single-cell analysis provide a new toolbox to decipher the biology of microchimeric cells at molecular and cellular level. In this review, we discuss how recent developments in single-cell analysis can be applied to determine the role and function of microchimeric cells.
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http://dx.doi.org/10.1093/bfgp/elx027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063264PMC
July 2018

Biological and Molecular Characterization of Circulating Tumor Cells: A Creative Strategy for Precision Medicine?

Adv Clin Chem 2017;82:71-103. Epub 2017 Jul 15.

Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Graz, Austria.

Circulating tumor cells (CTCs) are a group of rare cells disseminated from either primary or metastatic tumors into the blood stream. CTCs are considered to be the precursor of cancer metastasis. As a critical component of liquid biopsies, CTCs are a unique tool to understand the formation of metastasis and a valuable source of information on intratumor heterogeneity. Much effort has been invested in technologies for the detection of CTCs because they are rare cells among the vast number of blood cells. Studies in various cancers have repeatedly demonstrated that increased CTC counts prior to or during treatment are significantly associated with poor outcomes. In the new era of precision medicine, the study of CTCs reaches far beyond detection and counting. The rapidly growing field of analytical platforms for rare-cell analysis allows in-depth characterization of CTCs at the bulk cell and single-cell level. Genetic profiling of CTCs may provide an insight into the real-time tumor status, may allow the monitoring and evaluation of treatment response in clinical routine, and may lead to the development of novel therapeutic targets as well.
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http://dx.doi.org/10.1016/bs.acc.2017.06.001DOI Listing
April 2019

Visualization of tumor heterogeneity by in situ padlock probe technology in colorectal cancer.

Histochem Cell Biol 2017 Aug 20;148(2):105-115. Epub 2017 Mar 20.

Institute of Pathology, Medical University of Graz, Graz, Austria.

Tumor heterogeneity is considered a major cause for therapy resistance in colorectal cancer. Sub-populations of cells with different genetic alterations may exist in spatially distinct areas. Upon therapy, resistant sub-clones may enrich and ultimately lead to disease progression. Although ample data are available on tumors which are heterogeneous on a morphological level, only little is known about morphologically homogeneous tumors. We aimed to investigate if morphologically homogeneous colorectal cancer can harbor a heterogeneous genetic landscape. We chose to microdissect six morphologically homogeneous colorectal carcinomas into several areas and performed next-generation sequencing (NGS) to identify tumors with genetic heterogeneity. We then applied an mRNA-based in situ mutation detection technology based on padlock probes to localize and visualize mutations directly in the tumor tissue. In three out of six tumors, NGS revealed a high rate of variability of mutations between different tumor areas. We selected two cases for in situ mutation detection to visualize genetic heterogeneity. In situ mutation detection confirmed differences in mutant allele frequencies between different tumor areas of morphological homogeneous tumors. We conclude that genetic heterogeneity in morphologically homogeneous colorectal cancer is an observable, but underreported event. Our results illustrate the power of in situ mutation analysis to visualize genetic heterogeneity directly in tumor tissue.
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http://dx.doi.org/10.1007/s00418-017-1557-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508037PMC
August 2017

Catch and Release: rare cell analysis from a functionalised medical wire.

Sci Rep 2017 02 24;7:43424. Epub 2017 Feb 24.

Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria.

Enumeration and especially molecular characterization of circulating tumour cells (CTCs) holds great promise for cancer management. We tested a modified type of an in vivo enrichment device (Catch&Release) for its ability to bind and detach cancer cells for the purpose of single-cell molecular downstream analysis in vitro. The evaluation showed that single-cell analysis using array comparative genome hybridization (array-CGH) and next generation sequencing (NGS) is feasible. We found array-CGH to be less noisy when whole genome amplification (WGA) was performed with Ampli1 as compared to GenomePlex (DLRS values 0.65 vs. 1.39). Moreover, Ampli1-processed cells allowed detection of smaller aberrations (median 14.0 vs. 49.9 Mb). Single-cell NGS data obtained from Ampli1-processed samples showed the expected non-synonymous mutations (deletion/SNP) according to bulk DNA. We conclude that clinical application of this refined in vivo enrichment device allows CTC enumeration and characterization, thus, representing a promising tool for personalized medicine.
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http://dx.doi.org/10.1038/srep43424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5324062PMC
February 2017

KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients.

Oncotarget 2016 Dec;7(51):84705-84717

Research Unit on Ion Channels and Cancer Biology, Medical University of Graz, Austria.

Numerous studies showed abnormal expression of ion channels in different cancer types. Amongst these, the potassium channel gene KCNJ3 (encoding for GIRK1 proteins) has been reported to be upregulated in tumors of patients with breast cancer and to correlate with positive lymph node status. We aimed to study KCNJ3 levels in different breast cancer subtypes using gene expression data from the TCGA, to validate our findings using RNA in situ hybridization in a validation cohort (GEO ID GSE17705), and to study the prognostic value of KCNJ3using survival analysis. In a total of > 1000 breast cancer patients of two independent data sets we showed a) that KCNJ3 expression is upregulated in tumor tissue compared to corresponding normal tissue (p < 0.001), b) that KCNJ3 expression is associated with estrogen receptor (ER) positive tumors (p < 0.001), but that KCNJ3 expression is variable within this group, and c) that ER positive patients with high KCNJ3 levels have worse overall (p < 0.05) and disease free survival probabilities (p < 0.01), whereby KCNJ3 is an independent prognostic factor (p <0.05). In conclusion, our data suggest that patients with ER positive breast cancer might be stratified into high risk and low risk groups based on the KCNJ3 levels in the tumor.
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http://dx.doi.org/10.18632/oncotarget.13224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5356693PMC
December 2016

Detection of Fetal Sex, Aneuploidy and a Microdeletion from Single Placental Syncytial Nuclear Aggregates.

Fetal Diagn Ther 2017 27;41(1):32-40. Epub 2016 Apr 27.

School of Medical Science, Menzies Health Institute Queensland, Gold Coast Campus, Griffith University, Southport, Qld., Australia.

Objectives: A key problem in prenatal screening using extra-embryonic cells is the feasibility of extracting usable DNA from a small number of cells. Syncytial nuclear aggregates (SNAs) are multinucleated structures shed from the placenta. This study assesses the potential of SNAs as a source of fetal DNA for the detection of genetic abnormalities.

Methods: SNAs were collected in vitro. Whole-genome amplification was used to amplify DNA from single SNAs, and DNA quality and quantity was assessed by spectrophotometry and PCR. Confocal microscopy was used to count nuclei within SNAs, determine metabolic activity and investigate DNA damage. Fetal sex and chromosomal/genetic abnormalities were investigated with array-comparative genomic hybridization (aCGH).

Results: DNA was amplified from 81% of the individual SNAs. A mean of 61 ± 43 nuclei were found per SNA. DNA strand breaks were found in 76% of the SNAs. Seventy-five percent of SNAs yielded whole-genome-amplified DNA of sufficient quality for aCGH after storage and shipping. Individual SNAs from the same pregnancy reliably gave the same chromosomal profile, and fetal sex and trisomies could be detected. A microdeletion was detected in one pregnancy.

Conclusion: SNAs could provide a source of extra-embryonic DNA for the prenatal screening/diagnosis of fetal sex and chromosomal and sub-chromosomal genetic abnormalities.
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http://dx.doi.org/10.1159/000445112DOI Listing
February 2017

TNF-α alters the inflammatory secretion profile of human first trimester placenta.

Lab Invest 2016 Apr 11;96(4):428-38. Epub 2016 Jan 11.

Institute of Cell Biology, Histology and Embryology, Medical University Graz, Graz, Austria.

Implantation and subsequent placental development depend on a well-orchestrated interaction between fetal and maternal tissues, involving a fine balanced synergistic cross-talk of inflammatory and immune-modulating factors. Tumor necrosis factor (TNF)-α has been increasingly recognized as pivotal factor for successful pregnancy, although high maternal TNF-α levels are associated with a number of adverse pregnancy conditions including gestational hypertension and gestational diabetes mellitus. This study describes effects of exogenously applied TNF-α, mimicking increased maternal TNF-α levels, on the secretion profile of inflammation associated factors in human first trimester villous placenta. Conditioned culture media from first trimester villous placental explants were analyzed by inflammation antibody arrays and ELISA after 48 h culture in the presence or absence of TNF-α. Inflammation antibody arrays identified interleukin (IL)-6, IL-8, chemokine (C-C motif) ligand 2 (CCL2), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as the most abundantly secreted inflammation-associated factors under basal culture conditions. In the presence of TNF-α, secretion of GM-CSF, CCL5, and IL-10 increased, whereas IL-4 and macrophage CSF levels decreased compared with controls. ELISA analysis verified antibody arrays by showing significantly increased synthesis and release of GM-CSF and CCL5 by placental explants in response to TNF-α. Immunohistochemistry localized GM-CSF in the villous trophoblast compartment, whereas CCL5 was detected in maternal platelets adhering to perivillous fibrin deposits on the villous surface. mRNA-based in situ padlock probe approach localized GM-CSF and CCL5 transcripts in the villous trophoblast layer and the villous stroma. Results from this study suggest that the inflammatory secretion profile of human first trimester placenta shifts towards increased levels of GM-CSF, CCL5, and IL10 in response to elevated maternal TNF-α levels, whereas IL-6 and IL-8 remain unaffected. This shift may represent a protective mechanism by human first trimester villous placenta to sustain trophoblast function and dampen inflammatory processes in the intervillous space.
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http://dx.doi.org/10.1038/labinvest.2015.159DOI Listing
April 2016

Low-Volume On-Chip Single-Cell Whole Genome Amplification for Multiple Subsequent Analyses.

Methods Mol Biol 2015 ;1347:245-61

Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria.

Multiple analyses such as DNA profiling, sequencing, or comparative genome hybridization (CGH) done on the single-cell level long for pre-amplification due to the diploid human genome. Isothermal whole genome amplification allows amplification of long DNA templates from single cells. When analysis needs to be performed under rare cell conditions additional care needs to be taken due to the fact that, even after pre-enrichment, few candidate target cells are still dispersed among an overwhelming number of non-target background cells. Here, we describe a protocol where we define a population of candidate target cells based on specific staining. Candidate cells are then isolated by laser microdissection and pressure catapulting (LMPC) and transferred onto a microliter reaction slide. This slide allows monitoring the single-cell isolation process and isothermal whole genome amplification in less than 2 μL. The amplification products obtained from single cells can be forwarded to multiple analyses.
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http://dx.doi.org/10.1007/978-1-4939-2990-0_17DOI Listing
June 2016

Whole Genome Amplification of Labeled Viable Single Cells Suited for Array-Comparative Genomic Hybridization.

Methods Mol Biol 2015 ;1347:233-43

Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria.

Understanding details of a complex biological system makes it necessary to dismantle it down to its components. Immunostaining techniques allow identification of several distinct cell types thereby giving an inside view of intercellular heterogeneity. Often staining reveals that the most remarkable cells are the rarest. To further characterize the target cells on a molecular level, single cell techniques are necessary. Here, we describe the immunostaining, micromanipulation, and whole genome amplification of single cells for the purpose of genomic characterization. First, we exemplify the preparation of cell suspensions from cultured cells as well as the isolation of peripheral mononucleated cells from blood. The target cell population is then subjected to immunostaining. After cytocentrifugation target cells are isolated by micromanipulation and forwarded to whole genome amplification. For whole genome amplification, we use GenomePlex(®) technology allowing downstream genomic analysis such as array-comparative genomic hybridization.
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http://dx.doi.org/10.1007/978-1-4939-2990-0_16DOI Listing
June 2016

Quality Control of Isothermal Amplified DNA Based on Short Tandem Repeat Analysis.

Methods Mol Biol 2015 ;1347:129-40

Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, 8010, Graz, Austria.

This protocol describes the use of a 16plex PCR for the purpose assessing DNA quality after isothermal whole genome amplification (WGA). In short, DNA products, generated by amplification multiple displacement amplification, are forwarded to PCR targeting 15 short tandem repeats (STR) as well as amelogenin generating up to 32 different PCR products. After amplification, the PCR products are separated via capillary electrophoresis and analyzed based on the obtained DNA profiles. Isothermal WGA products of good DNA quality will result in DNA profiles with efficiencies of >90 % of the full DNA profile.
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http://dx.doi.org/10.1007/978-1-4939-2990-0_10DOI Listing
June 2016

Using Multiplex PCR for Assessing the Quality of Whole Genome Amplified DNA.

Methods Mol Biol 2015 ;1347:119-28

Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria.

This chapter describes a simple and inexpensive multiplex PCR-based method to assess the quality of whole genome amplification (WGA) products generated from heat-induced random fragmented DNA. A set of four primer pairs is used to amplify DNA sequences of WGA products in and downstream of GAPDH gene in yielding 100, 200, 300, and 400 bp fragments. PCR products are analyzed by agarose gel electrophoresis and the respective WGA quality is classified according to the number of obtained PCR bands. WGA products that yield three or four PCR bands are considered to be of high quality and yield good results when analyzed by means of array comparative genome hybridization (CGH).
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http://dx.doi.org/10.1007/978-1-4939-2990-0_9DOI Listing
June 2016

Whole Genome Amplification by Isothermal Multiple Strand Displacement Using Phi29 DNA Polymerase.

Methods Mol Biol 2015 ;1347:111-7

Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria.

The here described method of isothermal whole genome amplification (iWGA) uses a Phi29 DNA polymerase-based kit (Illustra GenomiPhi V2 DNA Amplification Kit) that amplifies minute quantities of DNA by multiple strand displacement upon random hexamer primer binding. Starting from genomic DNA or single cells this amplification yields up to 5 μg of iWGA product with fragment lengths of 10 kb and longer. As this amplification lacks the need of fragmenting DNA, its products are well suited for many downstream applications (e.g. sequencing and DNA profiling). On the contrary, degraded DNA samples are not supported by the nature of the amplification and are not well suited.
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http://dx.doi.org/10.1007/978-1-4939-2990-0_8DOI Listing
June 2016

Heat-Induced Fragmentation and Adapter-Assisted Whole Genome Amplification Using GenomePlex® Single-Cell Whole Genome Amplification Kit (WGA4).

Methods Mol Biol 2015 ;1347:101-9

Research Unit for Single Cell Analysis, Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Harrachgasse 21, Graz, 8010, Austria.

Whole genome amplification (WGA) is a widely used technique allowing multiplying picogram amounts of target DNA by several orders of magnitude. The technique described here is based on heat-induced random fragmentation yielding DNA strands mainly ranging from 0.1 to 1 kb in length. The fragmented DNA is then subjected to library generation by annealing of adaptor sequences to both ends of the DNA fragments. Using primers hybridizing to the adapter sequences, the DNA is amplified by thermal cycling. This amplification typically yields > 2 mg DNA from a single cell, is suited for amplifying DNA isolated from (partly) degraded samples [e.g. formalin-fixed paraffin-embedded (FFPE) material] and works well when used for array-comparative genome hybridization (array-CGH).
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http://dx.doi.org/10.1007/978-1-4939-2990-0_7DOI Listing
June 2016

Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

PLoS One 2014 5;9(12):e114251. Epub 2014 Dec 5.

Center for Medical Research, Medical University of Graz, Graz, Austria.

Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114251PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257693PMC
July 2015

High-fat diet triggers Mallory-Denk body formation through misfolding and crosslinking of excess keratin 8.

Hepatology 2014 Jul 27;60(1):169-78. Epub 2014 May 27.

Department of Internal Medicine I, Center for Internal Medicine, University Medical Center Ulm, Ulm, Germany.

Unlabelled: Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated keratins 8/18 (K8/K18). MDBs are characteristic of alcoholic and nonalcoholic steatohepatitis (NASH) and discriminate between the relatively benign simple steatosis and the more aggressive NASH. Given the emerging evidence for a genetic predisposition to MDB formation and NASH development in general, we studied whether high-fat (HF) diet triggers MDB formation and liver injury in susceptible animals. Mice were fed a high-fat (HF) or low-fat (LF) diet plus a cofactor for MDB development, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Additionally, we fed nontransgenic and K8 overexpressing mice (K8tg) with the HF diet. The presence of MDB and extent of liver injury was evaluated using biochemical markers, histological staining, and immunofluorescence microscopy. In DDC-fed animals, an HF diet resulted in greater liver injury and up-regulation of inflammation-related genes. As a potential mechanism, K8/K18 accumulation and increased ecto-5'-nucleotidase (CD73) levels were noted. In the genetically susceptible K8tg mice, HF diet triggered hepatocellular injury, ballooning, apoptosis, inflammation, and MDB development by way of 1) decreased expression of the major stress-inducible chaperone Hsp72 with appearance of misfolded keratins; 2) elevated levels of the transglutaminase 2 (TG2); 3) increased K8 phosphorylation at S74 with subsequent TG2-mediated crosslinking of phosphorylated K8; and 4) higher production of the MDB-modifier gene CD73.

Conclusion: Our data demonstrate that HF diet triggers aggregate formation and development of liver injury in susceptible individuals through misfolding and crosslinking of excess K8.
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http://dx.doi.org/10.1002/hep.27068DOI Listing
July 2014

Resolving tumor heterogeneity: genes involved in chordoma cell development identified by low-template analysis of morphologically distinct cells.

PLoS One 2014 4;9(2):e87663. Epub 2014 Feb 4.

Center for Medical Research, Medical University of Graz, Graz, Austria.

The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. UCHL3, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold) and U-CH1 (3.7-fold) cells. The mannosyltransferase ALG11 (695-fold) and the phosphatase subunit PPP2CB (18.6-fold) were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. TMEM144, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087663PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3913634PMC
December 2014
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