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Conference Agenda

Overview and details of the sessions of this conference. Please select a date or room to show only sessions at that day or location. Please select a single session for detailed view (with abstracts and downloads if available).

 
Session Overview
Date: Wednesday, 12/Oct/2011
1:00pm - 2:30pmToolboxes in Stem Cell Research
Session Chair: Elmar Endl

Keynote Speaker: Oliver Brüstle, Institute of Reconstructive Neurobiology, University of Bonn, Germany


Lecture Hall 
 

Human Pluripotent Stem Cells as Tools for Modelling Neurodegeneration

Oliver Brüstle

University Bonn, Germany;

Human pluripotent stem cells and their neural derivatives open exciting avenues for modelling neurological disorders in vitro. A prerequisite for such studies are robust protocols that efficiently yield standardized populations of neural cell types.

Recently we have established protocols for the derivation of long-term self-renewing neuroepithelial stem (lt-NES) cells from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). Lt-NES represent a stable, rosette-forming population, which can still be patterned to generate distinct neural subpopulations enabling direct access to metabolic pathways and signalling cascades relevant for the pathogenesis of neurodegenerative disorders. Derived from disease-specific iPSC, lt-NES cells can be exploited for studying key pathogenic mechanisms such as protein aggregation in polyglutamine disorders directly in human neurons and without resorting to overexpression paradigms.

We applied this strategy to Machado-Joseph Disease (MJD; syn. spinocerebellar ataxia type 3; SCA3), a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene. Proteolytic cleavage of the MJD1 gene product, ataxin-3 (ATXN3), is believed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD.

Our data show that aggregate formation can be recapitulated in MJD iPS cell-derived neurons, and that this phenomenon can be used to dissect the molecular pathogenesis of pathological protein aggregation. These data indicate that developmentally early hESC and iPSC-derived neurons provide a unique window of opportunity to study pathogenic driver mechanisms underlying age-related neurodegenerative diseases in a pre-symptomatic phase preceding neuronal degeneration.


Amyloid generation and axonal tau pathology in human pluripotent stem cell-derived neurons expressing Alzheimer’s disease-associated mutant proteins

Jerome Mertens1, Philipp Koch1, Irfan Tamboli2, Kathrin Stüber1, Julia Ladewig1, Daniel Poppe1, Jonas Doerr1, Jochen Walter2, Oliver Brüstle1

1Institute of Reconstructive Neurobiology, Life&Brain Center, University of Bonn and Hertie Foundation, Germany; 2Institute for Neurology, Sigmund-Freud-Straße 25, D-53127 Bonn, Germany;

Alzheimer’s disease (AD) is the most frequent cause of dementia, and there is strong evidence that altered proteolytic generation of amyloid-beta peptides (Aβ) as well as aberrant behavior of the microtubule-associated protein tau play critical roles in AD pathogenesis. However, the effective mechanisms that conspire to trigger the degeneration of neurons remain obscure. Although amyloidogenesis and tau phosphorylation are known to be highly cell type and species-specific, most studies conducted so far have, due to the limited access to vital human neuronal tissue, been conducted using heterologous overexpression systems in a non-human background. We set out to explore whether mature human neurons generated from human pluripotent stem cell-derived neural stem cells (hNSC) could serve as in vitro model for AD. We found that neurons generated from differentiating hNSC express the neuron-specific APP695 isoform, β- and γ-secretases as well as the six tau isoforms typically found in the adult human brain. The neurons recapitulate key steps in proteolytic APP processing, exhibit a differentiation-dependent increase in Aβ secretion and BACE1 maturation and can be directly used to read out pharmacotherapeutic effects of compunds targeting γ-secretase including non-steroidal anti-inflammatory drugs (NSAIDs). Expression of mutant presenilin-1 (L166P) in these cells results in a partial loss of γ-secretase function and an increased Aβ42/40 ratio, which is resistant to γ-secretase modulation. Given the involvement of tau in cytoskeletal stabilization and axonal transport, we investigated whether an overload of normal tau or aberrantly pseudo-hyperphosphorylated tau influence axonal trafficking and resistance of neurons to oxidative stress. Comparative analyses of neurons expressing normal or pseudo-hyperphosphorylated tau revealed that only excess of aberrant tau but not normal tau leads to the pathological MC-1 conformation and suffices to impair axonal transport of mitochondria without inducing neuronal death. As a consequence, in non-redox-protected conditions, tau-induced malfunctions culminated in the development of axonal varicosities sequestering transported proteins and progressive neuronal cell death. Human pluripotent stem cell-derived neurons thus represent a unique cell-based tool for assessing cytopathological processes relevant for AD pathogenesis as well as for the evaluation of potential drugs directly in a human neuronal system.


TIMP-1 binds to CD63 and enhances murine hematopoietic stem and progenitor cell homing in vivo

C. Matthias Wilk1, Frank A. Schildberg2, Marcel A. Lauterbach3, Ron-Patrick Cadeddu1, Julia Fröbel1, Sebastian Buest1, Volker Westphal3, René H. Tolba4, Stefan W. Hell3, Akos Czibere5, Ingmar Bruns1, Rainer Haas1

1Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine-University, Düsseldorf, Germany; 2Institute for Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-University, Bonn, Germany; 3Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen, Germany; 4Institute for Laboratory Animal Science and Experimental Surgery, RWTH-Aachen University, Aachen, Germany; 5Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA;

Tissue Inhibitors of Metalloproteinases (TIMPs) like TIMP-1 are soluble serum proteins that are known for their dual functions. While TIMPs inhibit matrix metalloproteinases through their n-terminus, they also exert direct effects on different cell types such as on bone marrow derived cells. These cytotropic functions are mediated by a variety of cell surface proteins binding to the n-terminal region of TIMPs. The TIMP-1 protein is a recently identified tetraspanin interacting cell surface protein in the immortalized human breast epithelial cell line MCF10A. Tetraspanins like CD63 are proteins that consist of four transmembrane domains and are known to interact with integrins. Integrins play a crucial role in hematopoietic stem and progenitor cell (HSPC) homing and mobilization.

In this study we show that TIMP-1 co-localizes with the tetraspanin CD63 and beta-integrin on human CD34+ HSPCs. We found a functional interaction of TIMP-1 with this receptor complex resulting in a higher amount of activated beta-1 integrin on the cell surface. This leads to strengthened migratory capacities towards a SDF-1a gradient and to enhanced adhesion to fibronectin. As also described for other cell types, TIMP-1 prevents CD34+ HSPCs from apoptosis. This antiapoptotic effect stands in close relation to the finding that TIMP-1 stimulation of CD34+ HSPCs leads to increased BFU-E counts in clonogenic assays. In mice treated with TIMP-1 we found altered erythropoiesis parameters such as increased red blood cell counts and hemoglobin and higher numbers of reticulocytes. In a transplantation model based on congenic mice strains, we found strengthened homing parameters in host mice that received bone marrow transplants from previously TIMP-1 stimulated donor mice.

Taken together, these results reveal that TIMP-1 exerts direct effects on human and murine hematopoetic stem- and progenitor cells.


Mesenchymal Stem Cells from Osteoporotic Patients: A Novel Niche for In-vitro Disease Modelling

Thomas Martin Randau1, Frank Alexander Schildberg2, Margit Zweyer3, Sascha Gravius1, Robert Pflugmacher1, Dieter Christian Wirtz1, Dieter Swandulla3, El-Mustapha Haddouti1

1Department of Orthopedics and Trauma Surgery, University Clinic of Bonn, Germany; 2Institute of Molecular Medicine, University Clinic of Bonn, Germany; 3Institute of Physiology II, University Clinic of Bonn, Germany;

Osteoporosis is among the 10 most crucial global diseases. It is defined as a systemic deterioration of bone mass and micro-architecture, disturbing the delicate coupling of bone-forming (osteoblasts) and bone-resorbing cells (osteoclasts). Bone marrow harbours mesenchymal stem cells (MSCs), the progenitors of osteoblasts, but the vertebral body as a stem cell niche is quite poorly described. Whether dysfunction or deficiency of MSCs contributes to the pathogenesis of osteoporosis and the associated delayed bone healing is unclear. The aim of this project was to establish the first human cell culture model originating from the vertebral compression fracture. As the spine is the most commonly affected osteoporotic fracture, we wanted to prove that fractured vertebral bodies harbour a population of MSCs, and show how these cells might differ from those obtained from healthy donors.

Bone marrow aspirate was taken from the vertebral bodies of osteoporotic and healthy donors undergoing spinal surgery. MSCs were isolated and expanded. All isolated cells were plastic adherent, proliferating, and showed a typical phenotype of MSCs. FACS analysis showed typical stem cell-like expression patterns of CD11b (neg), CD45 (neg), CD73 (pos) and CD106 (pos). MSCs were able to effectively suppress the proliferation of activated lymphocytes, and no differences were found between the groups. MSCs were successfully differentiated toward osteogenic, adipogenic and chondrogenic lineages as assessed by corresponding stainings and RT-PCR. Healthy and osteoporotic donors showed differences on the mRNA level for osteogenic differentiation markers. Interestingly, differential expression of regulatory receptors such as the endocannabinoid receptor 2 (CB2R) could be detected.

In summary, we were able to isolate, expand and characterize MSCs from vertebral bodies with osteoporotic compression fractures. These MSCs possess a similar phenotype and have similar characteristics, surface markers and immunomodulatory capacity compared to MSCs of healthy donors. Previous studies showed that the CB2R is a key player in the regulation of skeletal remodelling. As our results demonstrated clear differences in the expression of CB2R, this could explain the divergent differentiation potential of MSCs isolated from healthy and diseased donors.

 
2:30pm - 3:00pmCoffee break
 
3:00pm - 4:30pmStem Cells - Cancer - Cancer Stem Cells
Session Chair: Björn Scheffler
Session Chair: Leoni Kunz-Schughart
Keynote Speaker: Malcolm R. Alison, Barts and The London School of Medicine and Dentistry, London, UK
Lecture Hall 
 

A brief introduction to navigate through the foggy sea of cancer stem cell research

Leoni A. Kunz-Schughart

OncoRay - National Center for Radiation Research in Oncology, TU Dresden, Germany;

One of the most active and competitive areas in oncology research relates to the cancer stem cell paradigm. Unfortunately, communication in the field is hampered by subtle differences in semantics, and also the power of experimental data is often overinterpreted in the literature. An introduction will thus be given as a compass for the audience to navigate through the sometimes foggy sea of terminology in cancer stem cell research and to become aware of the potential and limitations of functional assays as well as current controversies in the use of biomarkers and theragnostic tools. Challenges of the cancer stem cell concept in light of phenomena such as phenotypic plasticity of stemness and dynamics of microenvironmental niches will briefly be stressed.


Recent developments in cancer stem cells: implications in the clinical setting

Malcolm R Alison

Barts and The London School of Medicine and Dentistry, United Kingdom;

Many if not all tumours contain a sub-population of self-renewing and expanding stem cells known as cancer stem cells (CSCs). The symmetric division of CSCs is one mechanism enabling expansion in their numbers as tumours grow, while epithelial mesenchymal transition (EMT) is another mechanism to generate further CSCs endowed with a more invasive and metastatic phenotype. Putative CSCs are prospectively isolated using methods based on either a surface marker (e.g. CD44, CD133) or an intracellular enzyme activity (e.g. ALDH, ABC transporter) [1,2]; subsequently assessed either by a ‘sphere-forming’ assay in non-adherent culture and/or by their ability to initiate new tumour growth when xenotransplanted into immunocompromised mice. Cell sub-populations enriched for tumour-initiating ability have also been found in murine tumours countering the argument that xenografting human cells merely selects human cells with an ability to grow in mice. Cancer progression can be viewed as an evolutionary process that generates new/multiple clones with a fresh identity; this may be a major obstacle to successful cancer stem cell eradication if treatment targets only a single type of stem cell [1].

In this lecture, I firstly briefly discuss evidence that cancer can originate from normal stem cells or closely related progenitors. I then outline the attributes of CSCs and review studies in which they have been identified in various cancers. Very recently it has become clear that the tumour stroma is not a mere bystander, being an important modulator of CSC behaviour. Finally, I discuss the implications of these findings for successful cancer therapies, concentrating on irradiation, the self-renewal pathways (Wnt, Notch, Hedgehog, PTEN/Akt) [3,4,5], aldehyde dehydrogenase activity, EMT, miRNAs and other epigenetic modifiers as potential targets for therapeutic manipulation. Many and varied therapeutic approaches to target CSCs are being evaluated and some have entered clinical trials.

1. Alison MR, Lim SM, Nicholson LJ. Cancer stem cells; problems for therapy? J Pathol 2011; 223: 147-161.

2. Alison MR, Guppy NJ, Lim SM, Nicholson LJ. Finding cancer stem cells: are aldehyde dehydrogenases fit for purpose? J Pathol. 2010; 222: 335-344.

3. Takebe N, Harris PJ, Warren RQ, Ivy SP. Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol. 2011;8(2):97-106.

4. Mas C, Ruiz i Altaba A. Small molecule modulation of HH-GLI signaling: current leads, trials and tribulations. Biochem Pharmacol. 2010;80(5):712-723.

5. Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D, Sarkar FH. Targeting Notch signaling pathway to overcome drug resistance for cancer therapy. Biochim Biophys Acta. 2010;1806(2):258-267.


ALDH1+ Glioblastoma cells mediate TMZ-resistance and recurrence

Daniel Trageser1, Martin Glas1, Roman Reinartz1, Anja Wieland1, Matthias Simon2, Frank van Landeghem3, Christel Herold-Mende4, Sabine Gogolock1, Franziska Lorbeer1, Ramona Eisenreich1, Heike Höfer1, Mihaela Keller1, Oliver Brüstle1, Björn Scheffler1

1Institute of Reconstructive Neurobiology, University of Bonn, Germany; 2Department of Neurosurgery, University of Bonn, Germany; 3Department of Neuropathology, University of Bonn, Germany; 4Division of Neurological Research, Department of Neurosurgery, University of Heidelberg, Germany;

One of the major implications of the recently coined cancer stem cell (CSC) hypothesis is that a circumscribed population of tumor cells is responsible for disease maintenance and therapy resistance. In this study, we present evidence for the occurrence of a distinct ALDH1+ subpopulation of CSCs within human glioblastoma (GBM) that shows a strong propensity to survive temozolomide (TMZ)-based chemotherapy regimens.

Using a tissue-microarray of 283 paraffin-embedded glioma specimens, we could demonstrate that the frequency of ALDH1+ cells correlates inversely with the PFS/OS of particularly those GBM patients that were treated with TMZ during recurrence of disease. We furthermore show that the frequency of ALDH1+ cells increases in recurrent GBM samples compared to tissue derived from the respective patient's primary manifestation. Using long-term TMZ application schemes, we could mimic this observation in vitro. ALDH1+ cells could be enriched from a series of previously characterized primary GBM cultures by a factor of 5-10x. These cells are tumorigenic and multipotent, show an increase in self-renewal, and their genomic profiles largely overlap with the original tumor. Various in vitro TMZ re-challenge paradigms confirmed their increased cellular resistance levels, which could be blocked irreversibly by application of DEAB, a selective ALDH-inhibitor. Our findings add creditability to the cancer stem cell hypothesis with a particular emphasis on therapy-related phenotype occurrence.


*Tumorigenicity of colorectal cancer cell lines with distinct CD133 and CD44 antigen pattern*

Julia Waurig, Lydia Kabus, Claudia Dittfeld, Yvette Garbe, Marit Wondrak, Leoni A. Kunz-Schughart

Tumor Pathophysiology, OncoRay – National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden, Germany;

Background & Aim

Various surface antigens, in particular CD133 and CD44, have been described as surrogate biomarkers to enrich colorectal cancer cell (CRC) subpopulations from primary tissue with enhanced tumorigenic potential. However, the relevance of these markers to define similar subpopulations in established CRC cell lines seems ambiguous. We showed earlier, that CD133 expression does not define a highly tumorigenic subpopulation in microsatellite-instable HCT-116 cells. In the present project we aimed to relate marker expression and tumorigenic capacity of microsatellite-stable, aneuploid CRC cell lines and to further evaluate the usefulness of CD133/CD44 antigen profiles for the identification of tumorigenic subpopulations.

Materials & Methods

Cell lines containing subpopulations with different CD133/CD44 antigen pattern were studied as verified by flow cytometry and Western blotting. Xenograft formation and growth were monitored after injection of CRC cell lines into the hind limbs of NMRI(nu/nu) mice with Matrigel. Cell numbers of 10,000, 500, 100 and as low as 10 cells were applied per injection site and mouse, respectively. Subpopulations from two cell lines were separated by fluorescence-activated cell sorting (FACS) and analyzed for their tumorigenic potential and marker expression in the resulting xenograft tumors.

Results & Perspectives

HT29, SW620, SW480, LS513, LS1034 and SW837 CRC cells show different CD133/CD44 expression pattern in vitro but all produced xenograft tumors after injecting ≥ 500 cells per site. Cell injections of 10 cells led to tumor formation from both HT29 and SW620. In contrast to literature data, we did not observe any difference in tumor formation capacity of CD133-negative/low vs. CD133-positive HT29 populations and found CD133-negative cells to rapidly reexpress CD133. The same was true for SW620 cells which also contained a clear CD44-negative/low subfraction. Here, all subpopulations defined by the CD133/CD44 expression profile showed similar xenograft formation capacities. The conclusion that CD44 is as irrelevant for in vivo engraftment of CRC cell lines is not yet valid, because SW620 cells turned out to be negative in CD44 Western blot analysis. This phenomenon is under further investigation and the search for CRC cell lines with limited tumorigenic potential and respective subpopulations of cancer cells is ongoing.

This work was supported by DFG grant KU 971/7-1.

 
4:30pm - 5:00pmCoffee break
 
5:00pm - 6:30pmCancer Cells and their Microenvironment
Session Chair: Simone Diermeier-Daucher
Session Chair: Stephan Schmid
Keynote Speakers:
Klaus Elenius, MediCity Research Laboratory, University of Turku, Finland;
Nadia Harbeck, Clinic and Polyclinic for Gynecology and Obstetrics, University Hospital of Cologne, Germany;

Lecture Hall 
 

Tumor biology in breast cancer - what do we need to know clinically?

Nadia Harbeck

University Hospital of Cologne, Germany;

In order to understand how knowledge about tumor cell properties is utilized for clinical purposes, breast cancer constitutes a good example since in-depth understanding of breast cancer biology is already being used clinically in several ways. Breast cancer is the most common malignancy in women in industrialized countries with about 1 out of 8-10 women being affected during their life-time. The majority of all patients who present without manifest metastases, i.e. 70-80%, will be cured today by a multidisciplinary therapy concept. Yet, even at primary diagnosis, breast cancer is considered a systemic disease where single tumor cells may have already spread which cannot be detected by conventional technologies. Thus, systemic therapy is a key element in treating this disease.

The knowledge of tumor cell biology impacts clinical decision making in three different ways: prognostic factors allow forecasting the course of the disease, predictive factors enable assessment of response to therapy, and last but not least, individual biological properties of the tumor cells are utilized as therapy targets in breast cancer.

Next to conventional staging criteria such as tumor size, nodal involvement, and grade, steroid hormone receptor status (ER, PR) and HER2 status are the most important biological properties which are determined in every breast cancer. They also serve as therapy targets with anti-hormonal (endocrine) therapy being standard in hormone-receptor positive disease and anti-HER2 agents, trastuzumab and lapatinib being approved for HER2-positive disease. Usually, these markers are determined immunohistochemically in primary tumor tissue. However, single cell analysis is gaining clinical relevance, in early (eBC) as well as metastatic breast cancer (mBC).

Bone marrow (BM) micrometastases are a known prognostic factor in eBC with BM+ patients having a poor outcome. Yet, because of the additional clinical burden to patients, BM sampling has not been widely accepted as part of clinical routine. Moreover, proof of benefit from therapeutic interventions in patients with BM+ eBC is still lacking. More recently, circulating tumor cells (CTC) have been proposed as a prognostic marker in mBC and eBC. In particular, German investigators such as the SUCCESS study group or the DETECT consortium have been instrumental in integrating single cell analysis into clinical practice. Questions that are currently being addressed are not just the mere number of circulating cells as a prognostic marker but more importantly their biological properties as potential targets for therapy. Since immunohistochemically determined ER, PR, and HER2 status may differ in about 10-20% between primary tumor and metastasis, alternative techniques such as single cell analysis are currently been investigated in order to indicate targeted therapy in metastatic disease. As a proof of principle, the DETECT III trial will evaluate the benefit from anti-HER2 therapy in mBC with HER2-positive CTC but a HER2 negative primary tumor.

In conclusion, tumor heterogeneity makes in-depth understanding of tumor biology mandatory for clinical decision making. Properties of single tumor cells will thus become increasingly important, not just as prognostic or predictive markers, but more importantly for choosing targeted therapies and maybe also for elucidating therapy resistance.


Is ErbB4/HER4 a cancer drug target?

Klaus Elenius

University of Turku, Finland;

Our aim is to determine the role of the receptor tyrosine kinase ErbB4 as a factor regulating carcinogenesis and as a clinically relevant cancer drug target. ErbB4 is a member the ErbB subfamily of RTKs that serve as receptors for epidermal growth factor (EGF)-like ligands. There are four ErbBs of which the two first-discovered, EGFR (= ErbB1) and ErbB2, are well-documented human oncogenes and cancer drug targets. Indeed, several therapeutic antibodies and small molecular weight tyrosine kinase inhibitors that block EGFR and/or ErbB2 are currently in use in clinical practise. However, the role of ErbB4 in carcinogenesis and its potential as a therapeutic target have remained controversial and poorly understood.

We have identified four alternatively spliced isoforms ErbB4. Our recent in vitro and in vivo findings indicate that a specific ErbB4 isoform represents a promising cancer drug target. This isoform is capable of signaling via a novel mechanism involving proteolytic cleavage of the receptor and direct regulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) by an intracellular receptor fragment. Importantly, our data also indicate that inhibition of another, non-cleavable, ErbB4 isoform enhances growth. Thus, pharmaceutical inhibition of ErbB4 could be either beneficial or disadvantageous depending on the specific ErbB4 isoform present. Our hypothesis is that ErbB4 inhibitors should be developed to selectively block the function of the cleavable isoform. Determining the role of ErbB4 isoforms in cancer biology is relevant and necessary also as i) some of the kinase inhibitor drugs currently used in the clinic block ErbB4 activity as an off-target effect, and ii) pan-ErbB inhibitors targeted against all ErbBs (including ErbB4) are being developed by the pharmaceutical industry, while no solid information exists to predict the consequences of ErbB4 inhibition for the clinical outcome.


Multicolor flow cytometry in humanized tumor mice – A powerful method to study advanced cancer therapy in a novel in vivo model

Anja Kathrin Wege1, Wolfgang Ernst1, Judith Eckl2, Bernhard Frankenberger2, Alexander Kromer1, Gero Brockhoff1

1University of Regensburg, Germany; 2German Research Center for Environmental Health, Munich, Germany;

The immunological impact on antibody based anti-cancer therapies remains incompletely understood due to the lack of appropriate animal models for in-vivo analysis. Therefore we generated a novel humanized tumor mouse (HTM) model by concurrent transplantation of human hematopoietic stem cells and human breast cancer cells in neonatal NOD- scid IL2Rγnull (NSG) mice. This co-transplantation is an extension of the already existing humanized mouse model in which a fully functional human immune system develops upon CD34+ cell transplantation. These humanized mice served already as a powerful in-vivo tool for the study of a variety of human infectious diseases (e.g. in HIV, EBV, and Dengue fever), of human haematopoiesis or graft versus host disease (GvHD).

In HTM, five weeks after intrahepatic co-transplantation of human tumor cells and human hematopoietic cells, a functional human immune system in all organs and early tumor cell dissemination in bone marrow and lung had been analyzed using multi-color flow cytometry. Three months post transplant tumor cell effusions and macroscopic tumors associated to liver or spleen were found and in addition disseminated cells in different lymphoid and non-lymphoid organs were measurable.

Ongoing immune responses were determined by flow cytometry using a LSR-II flow cytometer (BD Biosciences), detecting specific T cell maturation and tumor cell specific T cell activation. In addition, Natural Killer cell accumulation and activation was observed in HTM which was further enhanced upon IL-15 treatment facilitating the possibility of immune cell modulation in, e.g. ADCC based immunotherapeutic approaches.

This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human hematopoietic stem cells resulting in a solid coexistence and interaction without evidence for rejection. Besides immunohistochemistry, quantitative PCR, and other molecular biological methods, flow cytometry is the major technique for characterizing of the ongoing immune response and the assessment of efficiency during cytokine manipulation.

Overall, humanized tumor mice represent a novel, powerful in-vivo model that unprecedently permits the investigation of the human immune defense against cancer, in particular with respect to antibody based targeted therapies and might reveal new strategies to overcome therapy failure in non responder patients.


in-vivo Tissue Classification by Hyperspectral Imaging

Andreas OH Gerstner1, Wiebke Laffers1, Ron Martin2, Boris Thies2

1University of Bonn, Germany; 2University of Marburg, Germany;

Aim: To establish hyperspectral imaging for in vivo classification of human mucosal surfaces.

Material and methods: The larynx as a well-defined anatomical region was chosen as a prototypical surgical test area. The standard intra-operative setting for microlaryngoscopies was modified by using a polychromatic light source and a synchronous triggered monochromatic CCD-camera. Image stacks were analyzed by established software tools for principal component analysis and unsupervised hyperspectral classification.

Results: Sequential illumination of the same field of view by stepwise increasing wavelengths (390nm – 680nm, with 10nm steps) yielded a hyperspectral image datacube of the mucosa. These lambda-image stacks could be analyzed and classified by commercially available software. In principal component analysis, images at 590nm – 680nm loaded most onto the first principal component. Typically, the first principal component contained 95% of the total information. Unsupervised hyperspectral classification clustered the data thus highlighting areas of altered mucosa.

Conclusion: The technology of hyperspectral imaging can be applied to mucosal surfaces. This approach opens the way to analyze spectral characteristics of histologically different lesions in order to build up a spectral library and to allow non-touch optical biopsy.

 
6:30pm - 7:00pmCoffee break
 
7:00pm - 10:00pmEfficient Management of Core Facilities
Session Chair: Elmar Endl
Keynote Speaker: Derek Davies, London Research Institute, UK
Lecture Hall 
 

Does bigger mean better ? A Tale of Two Core Facilities

Derek Davies

Cancer Research UK, United Kingdom;

The major purpose of any core facility is to provide high quality hardware and staff knowledge and expertise which can be accessed by many research groups. Several recent publications have attempted to show the advantages of the core approach and how it can help foster collaboration between diverse groups who may not otherwise interact. There is however a critical mass of hardware and staff at which point a core would become viable in terms of the input it can have. But at what point could a core outgrow itself or need special treatment ?

In 2015 the London Research Institute will merge with the National Institute for Medical Research and move to a new building. Both Institutes currently have a flow core. The putative new flow core will serve over 100 research groups with different requirements to what each core is currently used to.

This presentation will address the concerns and approaches of the merger of two successful cores.


Instrumentation funding: influences on core facilities from a national funding agency's perspective

Johannes Janssen

Deutsche Forschungsgemeinschaft, Germany;

The presentation will reveal funding opportunities for flow cytometry and cell sorting equipment at German universities as provided by the DFG. A discussion of experts has resulted in a paper that outlines the boundary conditions for proposals and is provided to applicants and reviewers. Additionally, results from European discussions on effective use of equipments and core facilities will be presented as well as their impact on the development of national funding programmes.

 
Date: Thursday, 13/Oct/2011
9:00am - 10:30amNanosensors, Nanoantennas, Nanotools (Cytometry goes Nano reloaded)
Session Chair: Wolfgang Fritzsche
Session Chair: Ulrike Taylor
Keynote Speaker: Wolfgang Parak, Philipps-Universität, Marburg, Germany
Lecture Hall 
 

Colloidal Nano- and Microparticles: Towards Sensing Applications

Wolfgang Parak

Philipps Universität Marburg, Germany;

Nanomedicine nowadays is a popular key word in the media, though everyone seems to associate it with different visions, hopes, and even fears. From the point of view of a materials scientist it will be pointed out what new materials will be possible, how they will be designed, and which properties they could offer for diagnosis and treatment. It will be critically discussed that though sophisticated materials with advanced novel properties will be available in the future, they do not automatically match the requirements and demands of clinicians. The discussion is centred around one example, multifunctional polyelectrolyte capsules which might act as a "nano-submarine" for in vivo sensing and delivery, which is used to highlight promising interfaces between both disciplines.

_C. Röcker, M. Pötzl, F. Zhang, W. J. Parak, G. U. Nienhaus, "A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles", Nature Nanotechnology 4, 577-580 (2009).

P. Rivera Gil, S. de Koker, B. G. de Geest, W. J. Parak, "Intracellular processing of proteins mediated by biodegradable polyelectrolyte capsules", Nanoletters 9, 4398-4402 (2009).

J. Peteiro-Cartelle, M. Rodríguez-Pedreira, F. Zhang, P. Rivera Gil, L. L. del Mercato, W. J. Parak, "How colloidal nano- and microparticles could contribute to medicine - a personal perspective both from the eyes of physicians and materials scientists", Nanomedicine 4, 967-979 (2009)._


Interactions between cells and colloidal nanoparticles: From receptor-mediated interactions to laser hyperthermia.

Antonios Kanaras

University of Southampton, United Kingdom;

Manipulating the interactions of biological cells using nanotechnology is of great importance for the development of new diagnostic and therapy methods, drug delivery, and imaging. An important step to manipulate biological processes is to understand how the engineering, in terms of size, shape and functionality, of advanced colloidal nanoparticles can be employed to our benefit in order to control cellular functions.

In this presentation we demonstrate the interactions of a specific type of peptide-coated gold nanospheres with the angiogenic receptors VEGF and NRP-1 in the membrane of endothelial cells.[1-3] The particles containing the peptides bind to the receptors and promote an angiogenic response. We found that angiogenic genes are over-expressed in the case of pro-angiogenic-coated gold nanospheres and down-regulated in the case of anti-angiogenic-coated gold nanoparticles. The striking observation is that when the anti-angiogenic or pro-angiogenic molecules are attached to the gold nanospheres, the particle are 500 times more reactive in up-regulating or down-regulating the expression of angiogenic genes than the unbound angiogenic molecules.

Furthermore, we employ a low intensity laser-induced technique to manipulate the damage and repair of endothelial cells.[4] We will show that the degree of damage and repair of the cells is influenced by laser illumination in the presence of gold nanoparticles of various morphologies, which target the cellular membrane. Thus, we illustrate that plasmon-mediated mild laser hyperthermia, combined with specific targeting of cellular membranes, can enable new routes for controlling cell permeability and gene regulation in endothelial cells.

References:

1. Bartczak D.; Sanchez-Elsner T.; Louafi F., Millar T.; Kanaras, A. G. Small 2011, 7, 388–394.

2. Bartczak D.; Kanaras A. G. Langmuir 2011, 27 (16), 10119–10123

3. Kanaras, A. G.; Bartczak, D.; Millar, T. M.; Sanchez-Elsner, T.; Muskens, O. L. Proc. of SPIE 2011, Vol. 7909 79090J-1 doi: 10.1117/12.871602.

4. Bartczak, D.; Muskens, O. L.; Millar, T. M.; Sanchez-Elsner, T. Kanaras, A. G. NanoLett. 2011, 11, 1358–1363.


Internalization of gold nanoparticles in spermatozoa can be promoted by distinct bio-functionalization

Annette Barchanski1, Ulrike Taylor2, Csaba Laszlo Sajti1, Stephan Barcikowski3, Detlef Rath2

1Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover, Germany; 2Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Höltystrasse 10, 31535 Neustadt-Mariensee, Germany; 3University of Duisburg-Essen, Institute for Technical Chemistry, Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CeNIDE), Universitaetsstraße 7, 45141 Essen, Germany;

Directed targeting and strong labeling of specific cell populations with known phenotype, using long-term stable markers, is essential for their detection and separation from a heterogeneous cell suspension. Currently, an efficient method for cell sorting is based on high-speed flow cytometry, mostly after co-incubation of cells with fluorophore-tagged targeting moieties like DNA, antibodies or aptamers. However, photostability, cytotoxicity and membrane permeability are limiting fluorophore-specific attributes.

Gold nanoparticles (AuNP) as novel, fluorophore-alternative labeling markers will improve high-throughput screening, since the particles display high quantum yield without photo-bleaching and the possibility of non-destructive membrane penetration when coupled to penetrating agents additionally. Especially, AuNP fabricated by pulsed laser ablation in liquids are highly interesting for this application, due to an excellent biocompatibility, fabrication-dependent, tunable particle size and ease of efficient (bio)-functionalization in a one-step process with strong ligand binding.

We have recently demonstrated the penetration of bi-functionalized, laser-generated AuNP loaded with a targeting DNA fragment and various membrane-penetrating agents in distinct combinations and ligand ratios into sperm. A clear trend was observed, starting with sporadic cell membrane attachment of the conjugates, via membrane penetration and accumulation inside sperm up to explicit nucleus internalization, depending on attached cargo and nanoparticle size.

Moreover, first results on AuNP online detection by advanced flow cytometry were obtained, supporting the high potential of specific cell population labeling with gold nanoparticles for improved high-throughput sorting.


Exposure to gold nanoparticles affects sperm functionality and early embryogenesis

Ulrike Taylor1, Annette Barchanski2, Stephan Barcikowski3, Wilfried Kues1, Detlef Rath1

1Institute of Farm Animal Genetics, Friedrich Loeffler Institut, Germany; 2Laserzentrum Hannover e.V., Hannover, Germany; 3University of Duisburg-Essen, Institute for Technical Chemistry, Technical Chemistry I and Center for Nanointegration Duisburg-Essen (CeNIDE), Essen, Germany;

Due to their unique optical properties and easy modification with functional molecules, gold nanoparticles (AuNP) are considered to be valuable new tools for bio-imaging and therapeutic applications. However, it has been shown that nanoparticles can cross the blood-testicular-barrier. Therefore, the present work aimed to investigate the effect of AuNP on the fertilising ability of spermatozoa, as well as on the developmental capacity of the resulting embryo. Nanoparticles were produced by laser ablation in water and coincubated (1 and 10 µg/ml final concentration) with bovine spermatozoa for 2 hours. In parallel a negative control was run. Subsequently nanoparticles were removed via centrifugation. Immediately after treatment, the spermatozoa were used for in vitro fertilisation of in vitro matured bovine oocytes. For the experiments a total of 1406 oocytes was equally divided between treatment groups. In vitro culture was stopped either after 19h to investigate sperm fertilizing ability by assessment of pronucleus formation, or after 8 days to measure blastocyst development. Both parameters were significantly influenced by the presence of AuNP at a concentration of 10 µg/ml. Pronucleus formation dropped from 66.6±7.5% (untreated control) to 36.7±9.3% (p<0.05) (treated group), while blastocyst development decreased from 32.6±1.0% to 21.1±1.1% (p<0.001), respectively. In conclusion, the results show that low doses of gold nanoparticles (1µg/ml) left the spermatozoa unimpaired. However, concentrations of 10 μg/ml AuNP resulted already after a exposition time of 2 hours in detrimentral effects on sperm function and early embryogenesis. These results request a more detailed analysis on the interaction of nanoparticles with germ cells and early embryos. Further research will focus on the understanding of the mechanisms behind the observed phenomenon.

 
10:30am - 11:00amCoffee break
 
11:00am - 12:30pmAdvanced Microscopy
Session Chair: Thomas Kroneis
Session Chair: Dieter G. Weiss

Keynote Speaker:

Raluca Niesner, Deutsches Rheuma-Forschungszentrum, Berlin, Germany

Martin Fuhrmann, German Center for Neuredegenerative Diseases, Bonn, Germany


Lecture Hall 
 

Advances in dynamic intravital two-photon microscopy - focus on optical performance and molecular specificity

Raluca Niesner1,2, Jan-Leo Rinnenthal1,3, Karolin Pollok1,2, Volker Andresen4, Christian Börnchen5, Martin Behne5, Helena Radbruch1,6, Anja Hauser1

1Deutsches Rheuma-Forschungszentrum Berlin, Germany; 2Neuroimmunology in ECRC, Charité – University Hospital, Berlin; 3Institute for Neuropathology, Charité – University Hospital, Berlin; 4LaVision Biotec GmbH, Bielefeld; 5Dermatology, Eppendorf University of Medicine, Hamburg; 6BSRT and Laboratory for Molecular Psychiatry, CC15, Charité – University Hospital, Berlin;

Understanding physiological and pathological mechanisms in hardly accessible organs necessary, for instance, to develop novel selective therapy strategies, implies the investigation of cellular dynamics, cell-cell interactions and cellular function in genuine environment: the organism. Two-photon laser-scanning microscopy best enables highly-resolved visualization of cellular motility in the living organism, i.e. intravitally. However, limitations of standard TPLSM based on Ti:Sa laser excitation and photomultiplier detection, which are related to still reduced penetration depth, depth-dependent deterioration of the 3D spatial resolution, photobleaching of the cromophores, photodamage of the tissue and unspecific autofluorescence, prevents us from unequivocally monitoring and quantifying cellular communication intravitally. The benefits of two technological improvements of TPLSM for dynamic intravital imaging are discussed here: an infrared (IR) excitation source, i.e. the optical parametric oscillator, and a structured-illumination-like laser-scanning technique, i.e. striped-illumination multi-beam TPLSM. Using these technical advancements, the communication between antigen-carrying units on the surface of follicular dendritic cells and B-cells within the germinal center of the popliteal lymph node in living mice could be imaged. Moreover, the cellular function, which is the key of tissue and organ function, is still a hardly to access information for the most interesting studies, i.e. dynamic, highly-resolved studies in the living organism. Concretely referring to the neuronal function, it is reliably given by the intracellular Ca-level, which can be measured in CerTN L15 mice expressing a FRET-based Ca-biosensor in neurons. Here we present for the first time truly quantitative and dynamic Ca-level monitoring with diffraction-limited resolution in the brain of living mice by means of FRET-Fluorescence Lifetime Imaging (FLIM). The FLIM technique is based on a synchronized multichannel multi-detector TCSPC device. Using this method we are able to monitor the changes in the neuronal function, which build the roots of neurodegeneration.


Two photon in vivo imaging in neurodegenerative diseases

Martin Fuhrmann

German Center for Neurodegenerative Diseases, Germany;

Due to increasing age and lifespan the incidence of dementia is rising. Neurodegenerative diseases like Alzheimer's and Prion disease are characterized by the deposition of malfolded aggregated proteins. Hallmarks of these diseases represent neuron and synapse loss leading ultimately to cognitive decline. Moreover, neurodegenerative diseases exhibit a strong neuroinflammatory component that influences disease progression. Repetitive two photon in vivo imaging over the progression of disease allows to analyze kinetics of disease specific details like synapse and neuron loss. Moreover, the use of transgenic and knockout animal models enables to recapitulate and to interfere with neurodegenerative diseases.


Optogenetics in cardiovascular research: a new tool for light-induced depolarization of cardiomyocytes and vascular smooth muscle cells in vitro and in vivo

Philipp Sasse

University Bonn, Germany;

Purpose: Electrical stimulation is commonly used for activation of myocytes in clinics as well as in cardiac research. However, this has considerable side effects, lacks cell specificity and longer lasting or sub-threshold depolarization are not possible. In addition, electrical stimulation is problematic in loosely coupled cells such as vascular smooth muscle cells. We aimed to overcome these limitations by expression of channelrhodopsin 2 (ChR2), a light-gated cation channel, in muscle cells in order to stimulate these using light.

Methods: Embryonic stem cells were transfected with a plasmid encoding a ChR2-EYFP fusion protein under control of the chicken β-actin promoter and were used for generation of transgenic mice by diploid aggregation. Functional expression of ChR2 was analyzed in cardiomyocytes and transfected A7R5 VSMCs using patch clamp experiments, in the heart in vivo using ECG-recordings and in the aorta using isometric force measurements of aortic rings with a wire-myograph.

Results: Membrane-bound ChR2-EYFP was detected in cardiomyocytes of the whole heart and in smooth muscle cells of the abdominal aorta. Isolated ventricular cardiomyocytes from the adult heart and ChR2-expressing A7R5 vascular smooth muscle cells showed large inward currents upon illumination with blue light, and brief pulses of light evoked free running action potentials. Light pulses on the atrium or ventricle of the ChR2 mouse in vivo induced atrial or ventricular pacing in the ECG. Efficient stimulation was observed even using illumination areas as small as 0.05mm² (corresponding to ~50 cardiomyocytes). Continuous illumination of ventricular areas led to spontaneous arrhythmic extrabeats. Aortic rings from the ChR2 mouse contracted upon illumination and the force was similar to noradrenalin-induced contractions with fast on- (< 800 ms) and off-kinetics (< 2.5 s). Contractions could be maintained up to 10 min by constant illumination.

Conclusions: Light induced depolarization of cardiomyocytes and smooth muscle cells expressing ChR2 allows non-contact stimulation of the heart and the aorta.


High-Content Cell-Based Phenotypic Screening: Applications of Automated Confocal Microscopy in Cell Biology

Eugenio Fava

German Center for Neurodegenerative Diseases, Germany;

With the advent of automated microscopy and sophisticated automated image analysis softwares the high-throughput screening world is facing an exciting challenge. A new cell-based perspective for high-throughput assay has become possible in alternative to classic homogeneous assays.

The possibility to quantitatively relate multiple parameters to each other, on a single cell based level, within a large cell population is opening new pathways for target discovery and the understanding of complex intracellular mechanisms. Here we show the results of the application of automated microscopy to an high content screening in a genome wide loss of function analysis for endocytosis function.

Finally we will discuss the future development of automated microscopy and the use in cell-based phenotypic assays

 
12:30pm - 2:30pmLunch / Poster Session

Poster viewing by poster award commitee will start at 1:00 pm.

1:30pm - 2pm (Lecture Hall)

Life Technologies Presentation: “The Attune® Acoustic Focusing Cytometer – no more either/or - never sacrifice throughput for sensitivity again”


 
 

Cytomics in LIFE Project: Prediction of myocardial infarction and escalating immune response syndrome by polychromatic immunophenotyping.

Jozsef Bocsi, Aniko Szabo, Attila Tarnok

University of Leipzig, Germany;

Myocardial infarction (MI) is one of the leading causes of death in the western hemisphere. Activation of leukocyte adhesion molecules occurs after coronary intervention and the level of activation correlates to restenosis (Cytometry B Clin Cytom 2003;53:63). Surface expression changes of leukocytes assessed by flow cytometry (FCM) proved to be predictor of restenosis after stenting and advanced coronary artery disease. Therefore, we speculated that a complex immunophenotyping based on polychromatic FCM may be a useful approach to predict adverse cardiac events including MI in adults and risk for escalating immune response in children with congenital heart disease. In this presentation the development and quality control of a 10-color 13 antibody polychromatic FCM will be reported in detail. This panel combines the identification of all major leukocyte subsets with activation marker expression.

As proof of concept, we analyzed patients obtained through the cluster of excellence project “Leipzig Research Center for Civilization Diseases – LIFE” (www.uni-leipzig-life.de/) who suffered from angina pectoris without MI (n=24) and MI patients who underwent cardiac catheterization and stenting (n=25) that activated the immune system. By multivariate statistical comparison and classification both groups could be clearly discriminated based on neutrophil and monocyte count and activation marker expression.

We will apply this panel to determine the normal values in healthy individuals from LIFE-CHILD and LIFE-ADULT for prospective prediction of cardiac events according to the Human Cytome concept (J Biol Regul Homeost Agents. 2004;18:87).

Finantial support:

European Fond for Regional Development, EFRE, Bruxelles Landesexzellenzinitiative LIFE).

Sächsische Aufbaubank (SAB), Dresden (11624/1845 )


Triggering cancer cell death using light-inactivation of the Ki-67 protein

Ramtin Rahmanzadeh1, Gereon Hüttmann1, Johannes Gerdes2, Tayyaba Hasan3

1University of Luebeck, Germany; 2Leibniz Research Center Borstel; 3Harvard Medical School;

OBJECTIVE: Ki-67 is highly expressed in all proliferating cells and antibodies against this protein are widely used as prognostic tools in tumor diagnosis. Photo inactivation after microinjection of anti-Ki-67 antibodies showed a crucial role of Ki-67 in the synthesis of rRNA. With a nanotechnology based approach we show the delivery of antibodies to the cell nucleus and selective cell death after laser irradiation.

MATERIALS AND METHODS: Anti-Ki-67 antibodies were first conjugated to fluorescein-isothiocyanate (FITC) and then microinjected into cells or encapsulated into liposomes. Ongoing RNA-synthesis after microinjection and irradiation was pulse labeled with 5-fluorouridine. The efficacy of Ki-67 targeting on cell viability after liposomal delivery and irradiation was shown in vitro ovarian cancer models - monolayer cultures as well as 3D cultures.

RESULTS: Photo inactivation after microinjection resulted in inhibition of nucleolar rRNA synthesis. After incubation with the liposomal constructs, localization of the FITC-labeled antibodies in the nucleoli of the cells was confirmed by confocal microscopy. Irradiation with a 488 nm laser led to a significant loss of cell viability – in monolayer and in the 3D culture model. The observed cell killing was epitope specific. Only treatment with the antibody TuBB-9, let to inhibition of rRNA synthesis and cell death, while treatment with the antibody MIB-1 showed no effect.

CONCLUSIONS: Our findings showed the first evidence for the biological role of the Ki-67 protein in rRNA-synthesis and suggest that photo inactivation of pKi-67 is an attractive novel therapeutic approach for cancer treatment.


Anti-CD4 antibodies prevent Graft-versus-Host-Disease after hematopoietic stem cell therapy in mice

Ulrich Sack1, Nadja Hilger2, Christopher Oelkrug2, Manuela Ackermann1, Christian Fricke3, Dietger Niederwieser1, Frank Emmrich2, Stephan Fricke2

1Universität Leipzig, Germany; 2Fraunhofer IZI, Leipzig, Germany; 3Medizinische Dienste SRO, Langenthal, Switzerland;

Despite highly sophisticated therapeutic approaches, HSCT is still associated with a considerable number of complications such as graft versus host disease (GvHD). For treatment of GvHD, the standard immunosuppressive drugs can be associated with less long-term success and toxicity. Improved therapeutic approaches are still in need.

We developed a full MHC-mismatch in vivo hematopoietic transplantation model to test anti-human CD4 antibodies directly in mice. Using cells from transgenic human CD4+, murine CD4-, human HLA+ (TTG-C57Bl/6) mice, a unique transplantation model (human CD4+ to BALB/c) was established. The therapeutic effects (survival, leukocyte subset recovery, organ repair, chimerism, GvHD development) after transplantation compared with donor cells from C57Bl/6 wild-type mice were analyzed for 60 days. Engraftment and distribution of donor TTG cells in recipient mice was confirmed by flow cytometry, histology and immunohistology.

After transplantation, the survival rate was significantly increased in mice transplanted with TTG donor cells and receiving the anti-CD4 antibodies (0 to 83%, p<0.001, n=12). The observed effect is specific for transplantation of TTG in Balb/c mice and could not be repeated using C57Bl/6 wild-type mice (survival 0%, p<0.001, n=12). Mice transplanted without antibodies developed a severe GvHD at day 12 after transplantation and showed a full donor (TTG) hematopoiesis confirmed by expression of human CD4, TTG-H2Kb, and HLA-DR. Without antibody treatment, GvHD mice died within 19 days. Antibody receiving mice also showed a full donor (TTG) hematopoiesis but no signs of GvHD occurred and the GvHD score was significantly lower (p<0.001).

We developed a unique stable murine GvHD transplantation model using TTG mice as donors. By using specially developed anti-CD4 antibodies we were able to prevent GvHD development in a full MHC-mismatch transplantation model.


Investigation on the Optical Stabilization of Nanorods by Silica Shell Layer for Cell Elimination Purposes

Florian Rudnitzki, Gereon Hüttmann

Universität zu Lübeck, Germany;

Pulsed laser irradiated gold nanoparticles can mediate manipulation or elimination of cells and proteins by thermal and mechanical effects. In contrast to spherical particles nanorods are presently not as suitable due to laser induced changes of their absorption characteristics. A possibility for optical stabilization is investigated and discussed in this work. The physical effects and its implications are clarified in this work by means of computational models and experimental.


A2b Receptor as a Marker for a Selectable Mesenchymal Stem Cell Character Specific for Different Body Regions

Julia Delling, Dilaware Khan, Martin Lange, Emilio Giorgi, Yu Zhang, Andreas Pansky, Edda Tobiasch

Hochschule Bonn Rhein Sieg, Department of Natural Science, von-Liebig-Str. 20, 53359 Rheinbach,Germany;

Purinergic receptors are evolutionary very old and found in a variety of cells throughout the body. Purinergic receptors are grouped in P1 and P2; the former ones are subdivided into A1, A2a, A2b and A3 with adenosine as ligand. They are G-protein coupled ion channels. It has been shown that P2 receptors have an influence on mesenchymal stem cell (MSCs) fate. Therefore it was investigated if P1 receptors MSC fate and its character as well.

MSCs are a broadly used source of adult multipotent stem cells due to their potential of patient specific treatment and only minor ethical problems. They can be isolated from various regions like bone marrow, umbilical cord, and adipose tissue. Those assessed from adipose tissue are of high interest, because they can be harvested easily and in a high number without additional pain. They can be differentiated towards a variety of different lineages such as adipogenic, osteogenic and chondrogenic lineage. It has been shown that MSCs derived from head regions have a different lineage commitment capacity as MSCs from the body. Therefore it was investigated if a different commitment in various body regions can be linked to a different genetic expression pattern of P1 receptors.

This hypothesis was explored by investigating the P1A2b receptor expression in MSCs derived from belly, hip and thigh. The differentiation potential of thigh derived cells towards the osteogenic lineage was higher if compared to belly and hip. Interestingly that capacity could be linked to the expression of adenosine receptor A2b which was upregulated in thigh derived cells compared to those derived from hip or belly.

Here we show for the first time that MSCs derived from various body regions possess a different stem cell character as proven via the different differentiation capacity. The osteogenic character can be specifically evaluated by P1A2b. A finding, which might be of interest for future regenerative medicine approaches because a higher lineage commitment is connected to a lower tumorigenic potential and thus it is one step further towards a more safe future application.


Clustering and discrimination of pediatric patients undergoing open heart surgery with and without methylprednisolone treatment by cellular immune parameters

Attila Tarnok, Jozsef Bocsi, Anja Mittag, Arkadiusz Pierzchalski, Pavel Osmancik, Ingo Dähnert

University of Leipzig, Germany;

Methylprednisolone (MP) is frequently preoperatively administered in children undergoing open heart surgery. Aim of this medication is to inhibit overshooting immune responses. Cellular and humoral immunological changes in pediatric patients were compared for heart surgeries with and without prior MP administration. Pre and postoperative values were compared. Cluster analysis was applied for identification of suitable parameters characterizing both groups. Aim was to identify applicable parameters for prediction of operation outcome and as decision criterion for MP administration.

Blood samples were analyzed from two aged matched groups with surgical correction of septum defects. Group without MP treatment consisted of 10 patients; MP was administered to 23 patients (median dose: 11mg/kg) before cardiopulmonary bypass (CPB). Blood was taken 24h preoperatively, at the end of CPB and 4h postoperatively and analyzed by clinical chemistry and flow cytometry. Nine four color immunophenotype panels were used including phenotypic as well as functional/activation markers. Humoral response as well as changes in differential blood count, lymphocyte subsets and cellular activation was monitored.

More than 200 parameters were obtained from the analysis of each blood sample. Cross-validation revealed several parameters able to discriminate between MP treated and untreated group and to identify specific immune modulation by CPB. MP administration resulted in a delayed activation of monocytes, reduced CD4+ and CD8+ T-lymphocyte and increased B-cell counts. Cluster analysis by genes@work demonstrated that classification of patients is possible based on the identified cellular and humoral parameters. An analysis of these parameters prior to surgery might be taken as decision criterion for MP administration.

Financial support:

Deutsche Stiftung für Herzforschung, Frankfurt, Germany.

German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0313909)

MSM-0021620817, Charles University Prague, Czech Republic


COMPARATIVE COMPOUND SCREENING OF TUMOR SPHERES AND ADHERENT CULTURES FROM MALIGNANT PRIMARY AND SECONDARY BRAIN TUMORS

Roman Reinartz1, Martin Glas1, Anja Schramme1, Daniel Trageser1, Dennis Plenker1, Sabine Gogolok1, Marius Küpper1, Ramona Eisenreich1, Mihaela Keller1, Heike Höfer1, Anke Leinhaas1, Matthias Simon2, Oliver Brüstle1, Björn Scheffler1

1Institute of Reconstructive Neurobiology, University of Bonn, Germany; 2Department of Neurosurgery, University of Bonn, Germany;

Despite great efforts in the field of drug discovery for the last few decades, radiation and chemotherapy paradigms do not prevail for most patients suffering from malignant brain tumors. It remains largely unclear what cellular mechanisms or what cellular identities can be made responsible for the remarkable resistance to therapeutic efforts. Recent studies exposed tumor cell heterogeneity and the identification of various tumor-initiating subpopulations as a crucial element in the development of new therapeutic approaches. It was shown that primary cell cultures maintained under stem cell conditions could recapitulate cellular heterogeneity in vitro and in vivo. These conditions generally imply the use of defined serum-free media and a continuous supply of growth factors for the maintenance of cells in vitro. It would be reasonable to assume that these methods are ideally suited for the application of high throughput drug screening assays. Here, we present data acquired from a primary compound-screening assay using the Killer Collection library that encompasses 160 synthetic and natural toxic substances. We investigated primary cultures of glioblastoma multiforme (WHO IV) and melanoma brain metastases in adhesive vs. anchorage-independent settings. Data and pattern analysis implied increased resistances of tumor cells cultured under anchorage independent conditions. These findings were present in primary and secondary brain tumor samples and could not be explained based on sample variance alone. Our data rather suggest that the influences of cell-cell and cell-matrix interactions on the results of primary drug screening assays are currently underestimated.

(Supported by the Lichtenberg Program of the VW Foundation)


Continuous phasing – a synchronization technique for recombinant yeast Pichia pastoris

Maria Lisa Socher, Felix Krujatz, Florian Schmieder, Judith Greulich, Jost Weber, Thomas Bley

TU Dresden, Germany;

In order to understand the heterogeneity within cell populations various cell cycle synchronization techniques have been developed, because the properties of a population are determined by the properties of their individuals. Synchronous populations allow the investigation of cell cycle dependent events and hence help to improve the understanding of a fermentation process significantly.

The continuous phasing technique (cpt_words in italics_) is a powerful tool to synchronize cells under physiological conditions without addition of blocking factors or the use of organisms with genetically engineered cell cycle regulation. The cpt_words in italics_ achieves this by sequential changes of nutrition and starving. Asynchronous growing cells are forced into a defined limitation whereon half of the fermentation broth is removed and the remaining cells are replenished with fresh medium. The recurring shift of nutrition and starving adjusts the cells into the same cell cycle stage.

A few species of yeasts e.g. Candida utilis_words in italics_ have been synchronized successfully with cpt_word in italics_, however, so far no recombinant organism has been investigated by using this method.

The presented study shows the capability of cpt_words in italics_ to synchronize a recombinant strain of the yeast Pichia pastoris_words in italics_. To achieve this, a precise control of both the feed and draw pump was implemented by balancing the broth waste bottle and the fresh feed reservoir. The determination of cell concentration and DNA content via flow cytometry are important parameters to define the degree of synchronization.

The analysis of these properties turned out to be a difficult task, as the chosen strain showed a tendency to flocculation. Dissociation of the cell clusters via EDTA and medium optimization were tested.


Analysis of Transgenic Fluorophore Protein-Loaded Boar Spermatozoa

Wiebke Garrels1, Stephanie Holler1, Christina Struckmann1, Ulrike Taylor1, Doris Herrmann1, Brigitte Barg-Kues1, Sabine Klein1, Christine Ehling1, Detlef Rath1, Heiner Niemann1, Zoltan Ivics2, Wilfried Kues1

1Institute of Farm Animal Genetics, Friedrich Loeffler Institut, Germany; 2Max-Delbrück-Center for Molecular Medicine, Berlin, German;

Recently, we employed a non-autonomous Sleeping Beauty (SB) transposon to generate transgenic pigs with an ubiquitous expression of the Venus fluorophore protein in somatic cells (Garrels et al., 2011). Here, we analyzed Venus expression in germ cells of founder animals and animals of F1-generation. The spermatozoa were Venus positive as determined by fluorescence microscopy and flow cytometry (FACScan; BD Bioscience, Heidelberg, Germany; argon laser [488 nm; 15 mW], filter for green fluorescence [530/30 nm]). Motility parameters of spermatozoa were measured with a computer-assisted sperm analyser (CASA; Hamilton Thorne Bioscience-IVOS, Beverly, USA). The measured parameters were as follows: percentage of total motile sperm, percentage of progressive motile sperm, progressive velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s) and linearity of track, (VSL/VCL). Spermatozoa from transgenic boars had the same motility parameters as non-transgenic controls. Sorting of sperm cells into X- and Y-chromosome bearing populations, did not reveal any differences in Venus-fluorescence with respect to sex chromosomes. Molecular analysis revealed 3 monomeric integrations of the transposon. Interestingly, all spermatozoa were uniformly Venus-positive and gave a distinct fluorescence peak in flow-cytometric measurements, but did not contain Venus transcripts. The monomeric transgenes segregated, and approximately 10 % of the offspring were non-transgenic. These data suggest a genotype independent load with Venus protein and a non-genetic transgenerational transmission to zygotes.


Estimate of the mobility of oligosaccharide chains in glycogen molecule

Anna Yurievna Chestnova, Natalia Nikolaevna Bezborodkina, Grigory Israilovich Stein

Institute of Cytology Russian Academy of Sciences, Russian Federation;

Fluorescence (or Förster) resonance energy transfer (FRET) is one of the few techniques that are capable of giving dynamic information about the nanometer-range proximity between molecules.

In our work we have analyzed the structure of glycogen in individual hepatocytes of normal liver after glucose administration to fasting rats by FRET method. To mark labile (LF) and stable (SF) fractions of glycogen were employed fluorescent Schiff-type reagents auramine-SO2 (donor) and ethidiumbromide-SO2 (acceptor). The maximum wavelength of the donor emission and acceptor absorption 526 and 546 nm. Registration FRET between the donor and the acceptor was carried out on a LSCM Leica TCS SР5 with the application of FRET AB (acceptor photobleaching) procedure. For the donor excitation was employed laser 405 nm. Acceptor bleaching performed argon laser at a wavelength of 514 nm. Objective HCX PL APO 40x/0.70 imm was applied. Enclosing medium - immersion oil 518 F. The distance between the glucose residues in the glycogen molecule was determined by efficiency of FRET: Е = (Dpost – Dpre) / Dpost, where Dpre and Dpost are donor fluorescence intensity before and after acceptor’s photobleaching.

As a result, photobleaching of the acceptor in the light intensity was reduced on average by 50%. FRET efficiency in hepatocytes in various stages of refeeding of glucose to rats after administration ranged from 3 to 30% and averaged 10-14%. We assume that the structure of glycogen significantly affect the value of this indicator.

As the Ferster's distance R0 for a pair of auramine-ethidiumbromide is about 3.2 nm (the calculations used the values of the quantum yield of 0.3 auramine and extinction coefficient of EtBr 5680 M-1cm-1) and the possibility of energy transfer from D to A is reduced by increasing the distance between them in the sixth degree: E = 1 / (1 + (r/R0)6), such fluctuations FRET suggest that the distance between D and A in the particle glycogen, on average, can be changed at 0.37 nm. Based on the fact that the diameter of the glucose molecule is 0.7 nm, the change to 0.37 nm R0 means that a fluctuation of oligosaccharide chains per molecule of glycogen can reach 53% of the diameter of the glucose residues.

Thus, the results of the study suggest that the glycogen molecule possess a labile structure.

The work was supported by Russian Found of Basic Research (RFBR № 08-04-00971).


Fluorescence half live of EGFP in _Schizosaccharomyces pombe_

Jost Weber, Susann Kurtz, Kirsten Kottmeier, Gerhard Rodel, Thomas Bley, Kai Ostermann

TU Dresden, Germany;

Heterologous protein expression is a powerful tool to produce relevant amounts of proteins. The fission yeast Schizosaccharomyces (S.) pombe is far under represented as a host cell in the production of recombinant proteins. Vectors harbouring the strong thiamine-repressible nmt1-promoter allow efficient expression of target proteins in S. pombe. To meet the optimal expression the genetic design of the S. pombe expression strains has to be adjusted to the protein of interest. To achieve these objectives, methods must be available to rapidly monitor the expression of the target gene. Green fluorescent protein (GFP) turned out to be an extremely powerful tool as a fluorescent marker. By employing recombinant DNA techniques the GFP gene can be fused to target genes, and the expression can be easily monitored by following the fluorescence. Flow cytometry allows to simply quantify the expression of GFP or of GFP-fusion proteins. Though strategies for efficient expression were developed for S. pombe, one major problem for the efficient production of the protein of interest is the proteolytic degradation of the recombinant gene product by host- specific proteases. This decreases protein yields. One possibility of reducing the impact of host- specific proteases is the deletion of the proteases of S. pombe.

In order to evaluate the effect of cleavage of the protein of interest half life periods of the ORF33r protein from Parapoxvirus ovis (an immune stimulator) fused to EGFP and EGFP alone expressed by S. pombe were investigated. Furthermore the effect protease deficiency on the half life period was investigated. To achieve this, the decrease of the average fluorescence intensity was monitored after the down-regulation of the nmt promoter by addition of thiamine. The results show that the fluorescence half live of the EGFP-ORF fusion protein and the EGFP are 2.6 h and 4.6 h, respectively, for the non protease deficient strain of the fission yeast. The protease deficiency effects the half live of the EGFP. It increases by about 17 % to 5.6 h. For the fusion protein the effect is more pronounced. The fluorescence half life increases by 47 % to 5.1 h. Though these measurements cannot give allow conclusion on the degradation of the non fluorescent part of the protein, the figures show that protease deficient strains of S. pombe may help to increase the productivities of heterologously expressed protein.


HER4 coexpression is associated with improved recurrence free survival in HER2-positve, Herceptin treated patients

Gero Brockhoff1, Anna Machleidt1, Gerhard Piendl1, Silvia Seegers2, Andrea Sassen2, Stefan Buchholz1, Simone Diermeier-Daucher1, Katharina Schardt1, Olaf Ortmann2

1Dpt. of Gynecology and Obstetrics, University of Regensburg, Germany; 2Inst. of Pathology, University of Regensburg, Germany;

Background: Individual therapy efficiency of HER2-positive metastatic and pre-metastatic breast cancer patients varies significantly and spans from effectual responsiveness over acquired insensitivity to complete resistance from the outset. Thus no predictive information can be deduced from HER2 diagnostics so that molecular biomarkers indicative for sensitivity / resistance to Herceptin are needed to be identified. The HER2 related HER4-receptor has been shown to have ambivalent (pro-apoptotic or pro-proliferative) activity and consequently represents a prime candidate to affect HER2 activity under Herceptin treatment. We retrospectively analyzed potential her4 gene amplification and HER4 protein expression in HER2-positive, Herceptin treated patients. Patient’s overall and recurrence free survival was evaluated as a function of HER2/HER4 expression.

Methods: Using dual color Fluorescence in-situ Hybridization (FISH probes, Zytovision, Bremerhaven, Germany) and qPCR (LC480, Roche, Penzberg, Germany) we quantitatively investigated primary breast cancer tissues from nearly 50 (FISH) and 160 (PCR) patients who received Herceptin treatment. We quantified the her4 gene copy numbers and evaluated the protein expression profile of all four known HER4 isotypes (JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, JM-b/CYT2).

Results: FISH analysis revealed a positive and independent prognostic marker in Herceptin treated breast cancer patients with respect to overall survival. Moreover by quantitative PCR analysis we found a significant variability of HER4 protein expression (JM-a/CYT1 and JM-a/CYT2; no JM-b isotypes) in HER2 positive breast cancer tissues, whereas HER2/HER4 positive patients show a significant better recurrence free survival compared to HER2 positive but HER4 negative patients (p = 0,003).

Conclusions: HER4 has been demonstrated to potentially exert tumor suppressing activity and in turn to have a favourable impact on the course of breast cancer disease. We show here that HER4 expression prolongs in particular recurrence free survival of Herceptin treated patients which indicates a functional integration of HER4 into anti-HER2 targeting. Complementing functional studies allowing for isotype specific function of HER4 will elucidate the special role of this receptor tyrosine kinase in the context of Herceptin treatment and might facilitate individualized anti-ErbB-receptor targeting with higher efficiency.


High-throughput screening identifies new candidate compounds for glioblastoma treatment

Anja Wieland1, Martin Glas1, Roman Reinartz1, Daniel Trageser1, Matthias Simon2, Heike Höfer1, Mihaela Keller1, Ramona Eisenreich1, Sabine Gogolok1, Björn Scheffler1

1Stem Cell Pathologies, Institute of Reconstructive Neurobiology, University of Bonn, Germany; 2Department of Neurosurgery, University of Bonn, Germany;

Despite combined and optimized surgical, radio- and chemotherapeutic measures in patients suffering from glioblastoma (GBM), recurrence of disease occurs always, leading to median overall survival of 14.6 months. The resistance to standard therapy may be a consequence of cancer stem cells (CSCs) initiating the disease and /or driving the progression of the tumor´s growth. Therefore, CSCs may represent new cellular targets for anticancer therapy.

We applied the commercially available Killer Collection® compound library in high-throughput screening experiments to previously characterized CSC-enriched human primary GBM cultures. Among the 160 cellular and biochemical toxins tested, 31 candidate compounds had a significant cytotoxic or cytostatic effect – based on an initial screen using the alamarBlue® assay. Hit validation included e.g. pharmacodynamics based on alamarBlue® assay of metabolic activity, AnnexinV-analysis of apoptosis, study of growth kinetics, quantification of self-renewing, multipotent CSCs including expression profiling of stem cell markers and investigation of the mode of action. Preliminary data indicate that single dose application of the compounds also significantly reduced the metabolic activity of primary cell cultures of recurrent GBMs. Our results demonstrate that some candidate compounds indeed diminish the frequency of CSCs, while others failed to demonstrate efficiency. Ongoing experiments investigate effects of the identified candidate compound on appropriate human control cells in vitro, as well as in xenograft experiments in vivo.


Impact of long-term acidosis on the expression pattern of putative cancer stem cell biomarkers in colorectal cancer cell lines

Philipp Große-Gehling1, Constantin Mamat2, Leoni A. Kunz-Schughart1

1OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine Carl Gustav Carus, TU Dresden, Germany; 2Institute of Radiopharmacy, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany;

Background: In colorectal cancer (CRC), biomarkers such as the surface molecules CD44 and CD133 as well as the enzymatic activity of aldehyde dehydrogenase 1 (ALDH1) have been reported to be suitable for the enrichment of cancer stem cells (CSC) from both primary tumor material and cancer cell lines. The impact of microenvironmental modulations on the expression pattern of these biomarkers has not yet been explored in detail. Based on the hypothesis that the CSC population can be dynamic dependent on the tumor micromilieu, we monitored the impact of long-term acidosis on cell behavior and CSC marker expression in several CRC cell lines.

Methods: Media were established to keep the pH stably low at 6.9 and 6.4 over a period of ≥ three days by utilizing MES. HEPES-supplemented media served as osmolarity controls. HT29, HCT-116, SW620 and SW837 cells were exposed to these conditions for 5-7 days. The expression of CD44, CD133 and ALDH1 activity was analyzed by flow cytometry. Cell growth, diameter and morphology were routinely documented and complemented by a senescence test as well as cell cycle analyses.

Results & Perspective: Our investigations did not reveal significant alterations in biomarker expression or enzyme activity at pH 6.9, whereas clear modifications were observed at pH 6.4. However, although reproducible, biomarker expression changed cell line-dependently and no uniform pattern was found. Also, cell growth decelerated in a cell line-dependent manner under acidic conditions while cell diameter increased and morphology changed similarly in all investigated CRC cell lines. This led to the hypotheses that cells undergo stress-induced premature senescence at pH 6.4, but could not be confirmed in a subsequent experiment. However, cell cycle analyses revealed a significant increase of cells in the G2/M phase at pH 6.4 as compared to the respective controls. Our next goal is to study other milieu conditions related to tumor progression such as lactate accumulation with and without acidosis and modifications in oxygen supply.

This work was supported by DFG grant KU 917/7-1.


Pertuzumab is superior to Trastuzumab in rescuing the HRG-caused reversion of Lapatinib’s inhibitory effect on breast cancer cells

Simone Diermeier-Daucher, Stefanie Breindl, Stefan Buchholz, Olaf Ortmann, Gero Brockhoff

Department of Gynecology and Obstetrics, Caritas Hospital St. Josef, University of Regensburg, Germany;

Background: Monoclonal antibodies and small molecule inhibitors emerged as potent therapeutic agents in the treatment of HER2 overexpressing breast cancer. However, many patients do not adequately respond to anti-EGFR/HER2 receptor targeting. In this study we investigated receptor- and growth-stimulating effects, which potentially hamper anti-proliferative cell treatment.

Methods: BT474 and SK-BR-3 breast cancer cell lines were treated with therapeutic monoclonal antibodies Trastuzumab and Pertuzumab and with tyrosine kinase inhibitor Lapatinib alone and in different combinations. EGF or HRG were added to reveal potential growth factor-mediated compensatory effects. The treatment-specific activation status of EGFR and HER2 receptors and intracellular signaling cascades were correlated to cell cycle kinetics and apoptosis.

Results: The presence of EGF or HRG strongly impaired Lapatinib-caused growth inhibition. This compensatory effect caused by EGF, however, was reversed by additional cell treatment with either Trastuzumab or Pertuzumab. In contrast, the compensatory effect caused by HRG treatment was only reversed by Pertuzumab, but not by Trastuzumab. These data suggest that Pertuzumab might be superior to Trastuzumab in affecting a HRG-caused HER receptor interaction (activation) involved in compensation of Lapatinib-caused cell cycle exit.

Conclusions: Modular HER/ErbB receptor targeting with Lapatinib, Trastuzumab and Pertuzumab more efficiently affects receptor function than single treatment. Growth inhibition by anti-cancer drugs targeted to HER/ErbB receptors, however, can be significantly undermined in the presence of EGF and in particular by HRG treatment. This observation suggests that specific therapeutic growth factor sequestration might further enhance anti-EGFR/HER2 targeting.


Status and plasticity of CD133 and CD44 expression in three colorectal cancer cell lines

Lydia Kabus, Yvette Garbe, Marit Wondrak, Leoni A. Kunz-Schughart

Tumor Pathophysiology, OncoRay – National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, TU Dresden, Germany;

Background and Aim

Several cell surface molecules are controversially discussed as markers in cancer stem cell research. Phenotype switching is one of the critical processes that affects marker expression. In addition, cytoplasmic and membrane presentation of the molecules may differ. In colorectal cancer (CRC), two of the markers of interest are CD133 and the multifunctional hyaluronan receptor CD44. The objective of the present study was to monitor protein expression profiles of colorectal cancer cells with different tumorigenic potential throughout in vitro growth and discriminate intra- and extracellular signals to unravel discrepancies between Western blot and flow cytometric antigen expression pattern.

Material and Methods

CD133, CD44 and CD24 were monitored by flow cytometry in three CRC lines (LS1034, SW620, SW1417). CD133 and CD44 expression were also evaluated by Western blot analyses using two different antibodies for each molecule of interest. The CD133/1 antibody was used with FASER amplification in viable cells, while intracellular CD133 was detected with an Alexa488-conjugated secondary antibody. CD133- and CD133+ subpopulations of LS1034 and SW1417 were sorted and subsequently reanalyzed. CD133-/CD44+, CD133+/CD44+, CD133-/CD44- and CD133+/CD44- subpopulations of SW620 cells were further reanalyzed by flow cytometry up to day 14 after sorting.

Results and Perspectives

SW620 and SW1417 show a distinct CD133+ population (>60%); a right shift of the fluorescence signal was also found in LS1034 cells, but clear CD133-positive and -negative fractions could not be discriminated. LS1034 cells are CD44-/low whereas SW620 and SW1417 cells contain high proportions of CD44+ cells in flow cytometry (>70%). Unexpectedly, CD44 expression in SW620 could not be verified in Western blot analyses. This phenomenon is under further investigation. Vice versa, CD133- sorted LS1034 and SW1417 populations showed a remarkable CD133 signal in Western blots, although a sort purity of ≥ 99% was achieved. Staining for intracellular CD133 revealed a pronounced fraction of CD133-surface-negative cells with cytoplasmic CD133 protein (LS1034: 21±3%, SW1417: 41±10%). Analyses of SW620 subfractions are ongoing, but re-expression of CD133 and CD44 on the cell surface of the respective negatively sorted populations appears to be the most prominent alteration throughout culture. Studies to monitor tumorigenic capacity and marker redistribution of subpopulations of interest in vivo are underway.

This work was supported by DFG grant KU 971/7-1.


Targeting the relapse - profiling glioblastoma residual cells

Franziska Lorbeer1, Martin Glas1, Dennis Plenker1, Daniel Trageser1, Roman Reinartz1, Anja Wieland1, Matthias Simon2, Sabine Gogolok1, Ramona Eisenreich1, Heike Hoefer1, Mihaela Keller1, Oliver Brüstle1, Björn Scheffler1

1Institute of Reconstructive Neurobiology, Germany; 2Dept. Neurosurgery, Univ. Bonn Med Ctr, 53105 Bonn;

Glioblastoma (GBM) is a highly invasive tumor of the central nervous system and accounts for one of the most aggressive types of cancer. Despite a multimodal therapeutic approach including surgical removal of the the tumor and combinational radio- and chemotherapy the median overall survival of the patients remains 14.6 months.

Relapse of the disease often occurs at the resection border. The “residual” cells left behind after surgery may be key to the rapid progression and poor prognosis of GBM. We recently demonstrated that residual GBM cells can be derived via experimental biopsy from the surgical resection margin in situ and that these cells can be purified and investigated under controlled conditions in vitro (Glas et al., Ann. Neurol. 2010). Based on their molecular and phenotypic cellular properties these cells were found to be distinct from other Glioblastoma cells.

Our work aims to validate residual cells as a unique sub-entity within GBM. In a high-throughput screening with the Killer Collection (Microsource, USA) compound library we found residual cells to show a differential behavior and an overall increased chemo-resistance. By conducting a microarray study we furthermore identified a set of 14 candidate drug targets that can be differentially exploited by pharmacodynamic in vitro experiments. The results of our study give strong evidence that residual cancer cells need to be addressed separately - and in addition to other GBM cells, in order to prevent recurrent disease. Further investigation of residual cells could reveal new avenues for the diagnosis and treatment of GBM.

 
2:30pm - 3:30pmMicro-­ and Biotechnology
Session Chair: Wolfgang Beisker
Session Chair: Lars M. Blank
Keynote Speaker: Wilhelm Huck, Radboud University, Nijmegen, Netherlands
Lecture Hall 
 

Microdroplets in Microfluidics: towards a new tool for single cell experiments

Wilhelm Huck

Radboud University, Netherlands, The;

In recent years there has been an enormous interest in exploiting droplet-based microfluidic devices for performing on-chip biochemical reactions including enzyme kinetics, protein crystallization, PCR and in vitro translation and transcription. Nanoliter droplets of water in oil emulsions can be created inside microfluidic devices and can be merged, split and sorted using electric fields, while the contents can be analyzed with sensitive optical techniques. The very high throughput of these devices, up to 10,000 per second, drives research in this area to include directed evolution on-chip and single cell gene expression experiments.

In my talk, I will present some of our recent results on in vitro translation and transcription inside droplets, parallel enzymatic reactions, emulsion separation on chip, coupling of fluorescence detection with sorting and mass spectrometric analysis of droplets contents, and progress in developing tools for single cell ‘cellulomics’.

Recent publications

Simultaneous Determination of Gene Expression and Enzymatic Activity in Individual Bacterial Cells in Microdroplet Compartments

J. U. Shim, L. F. Olguin, G. Whyte, D. Scott, A. Babtie, C. Abell, W. T. S. Huck, F. Hollfelder

J. Am. Chem. Soc. 2009, 131, 15251-15256.

Coupling Microdroplet Microreactors with Mass Spectrometry: Reading the Contents of Single Droplets Online

L. M. Fidalgo, G. Whyte, B.T. Ruotolo J. L. P. Benesch, F. Stengel, C. Abell, C. V. Robinson, W. T. S. Huck

Angew. Chem. Int. 2009, 48, 3665-3668

From Microdroplets to Microfluidics: Selective Emulsion Separation in Microfluidic Devices

L. M. Fidalgo, G. Whyte, D. Bratton, C. F. Kaminski, C. Abell, W. T. S. Huck

Angew. Chem. Int. 2008, 47, 2042-2045


Microtechnologies: New Dimensions for Cytometry

Jonathan West

ISAS, Germany;

Single cell analysis has been underpinned by the microscope and the flow cell cytometer (FCC). Microscopy images stationary cells to reveal spatial information with sub-cellular resolution, whereas FCC is a high throughput continuous flow process for describing the population by capturing single cell events. The two technologies have continued to evolve to the present day state-of-the-art. In parallel with these developments planar array and fluidic microtechnologies have emerged as complementary tools which can bring new qualities and quantities of information to modern cytometry. In my presentation I will illustrate the reach of microtechnologies by using examples from research in my laboratory which spans pharmaceutical and neurotoxicology screening as well as basic research concerning communication processes at the single cell level.

Patterning the adhesion microenvironment can be used for spatially standardizing the culture microenvironment [1] for reproducible and statistically powerful cell analytics. In the first case, uniform tumour spheroids were mass produced (>10000) [2] and the array dimensions were varied to modulate growth, metabolic and pathophysiological characteristics and the associated chemosensitivity traits [3]. In a second case, the neurite outgrowth assay, a classical morphological end-point measure for neurotoxicology was refined in the form of the network formation assay (NFA). Here, the array format eliminates length measurements and also assesses connectivity, a higher order predictor of functional impact than outgrowth alone [4]. The NFA was used for the rapid and reproducible assessment of the inhibitory and degenerative effects of neurotoxins, and the effects of neuroprotective agents [4,5].

Single cell arrays can also be prepared using deterministic microfluidic approaches. Using a novel valving concept where the cells naturally transform from a suspension to an adhesive state, we used this array platform for the high throughput pairing of single cells in readiness for investigating juxtacrine signalling [6]. Using similar fluidic principles a microfluidic circuit was developed for establishing compartmentalized neuronal co-cultures with one-to-one connectivity [7]. These are being used by our collaborators to investigate disease propagation following fluidically targeted chemical or pathogenic insults. Lastly, microfluidics presents the opportunity to define cellular reaction sequences with unprecedented temporal resolution. To this end we have developed a continuous flow single cell manipulation platform which resembles traditional FCC but instead is used to stimulate, incubate and capture ligand–receptor signal transduction events with millisecond resolution [8]. This opens the possibility of capturing the timing and sequence of molecular transitions occurring at the communication front between the cell and its microenvironment.

References

1. J.P. Frimat et al, Anal. Bioanal. Chem., 2009, 395(3), 601–609.

2. F. Hirschhaeuser et al, J. Biotechnol., 2010, 148, 3–15.

3. H. Hardelauf et al, Lab Chip, 2011, 11(3), 419–428.

4. J.P. Frimat et al, Lab Chip, 2010, 10, 701–709.

5. H. Hardelauf et al, Lab Chip, 2011, 11(16), 2763 – 2771.

6. J.P. Frimat et al, Lab Chip, 2011, 11(2), 231–237.

7. H. Hardelauf et al, 6th IEEE MMB Conference, Lucerne, Switzerland, 2011.

8. Y.Y. Chiang et al, 6th IEEE MMB Conference, Lucerne, Switzerland, 2011.


Sub-μm Fluidic Structures for Single Cell Trapping and Analysis of Prokaryotic Production Strains

Dietrich Kohlheyer

Forschungszentrum Juelich GmbH, Germany;

This abstract describes a microfluidic chip to hydrodynamically capture single bacteria cells for long-term growth and production studies. The trapping and cultivation of single bacteria within a laminar flow, as well as fluorescence based lysine detection was successfully performed. The presented system is specially designed for parallel single cell experiments to determine growth rates and production heterogeneity. In-house developed industrial productions strains mainly based on E. coli and C. glutamicum will be used in further experiments. An important finding within this field was shown by Wang et al. in 2010. They developed a micro system having parallel growth channels to study growth behavior of E. coli [1]. The microfluidic system shown here, allows the application of chemical gradients for screening and heterogeneity studies. Furthermore, trapping of bacteria in one focal plane enables fluorescence based measurements of bacteria arrays in quasi high-throughput. Fully motorized live cell imaging microscopy and well controllable cell environment allows for long-term investigation in a time lapsed manner [2].

Using soft-lithographic methods, a disposable poly(dimethylsiloxane) (PDMS) microfluidic chip was fabricated, containing several arrays of traps for single bacteria capturing.

This work lays the foundation for future system biological and metabolic investigations such as cell heterogeneity studies or mutant screening of high content producing cells within our group and institute. It is expected to present latest results on single cell analysis within microfluidic devices.

1. Ping Wang, Lydia Robert, James Pelletier, Wei Lien Dang, Francois Taddei, Andrew Wright and Suckjoon Jun, Robust Growth of Escherichia coli. Current Biology, 2010. 20(12): p. 1099-1103.

2. Alexander Grünberger, Christopher Probst, Stephan Binder, Lothar Eggeling, Wolfgang Wiechert and Dietrich Kohlheyer, Single cell trapping and analysis of prokaryotic production strains in sub-μm fluidic structures, Proceedings of Micro TAS Conference 2011 (accepted for publication), October 2-6 2011, Seattle, USA


The Envirostat Chip - Contactless Isolation and Controlled Cultivation of Single Bacteria

Frederik Sven Ole Fritzsch1, Katrin Rosenthal1, Anna Kampert1, Christian Dusny1, Hendrik Kortmann1, Oliver Frick2, Lars Mathias Blank2, Andreas Schmid1,2

1Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., D-44227 Dortmund, Germany; 2Laboratory of Chemical Biotechnology, TU Dortmund University, D-44227 Dortmund, Germany;

Cell to cell differences (cell heterogeneity) within a population can be analyzed by flow cytometry. Its opto based snapshots are however restricted to one point in time and hence do not allow time resolved investigations of single cells. Time resolved analysis of single cells during growth in a controlled environment enables deciphering of cellular mechanisms, such as cause and effect in regulation cascades. The analytical investigation of such mechanisms on the level of a single cell is challenging.

Rarely considered during single cell analysis, contact-free cultivation is desirable to avoid surface induced unknown changes in cell phenotype. Our recently developed Envirostat technology allows contactless and environmental controlled single cell cultivation employing negative dielectrophoresis (nDEP) for cell trapping in a flow of growth medium. The technology enables systematic perturbation experiments as carried out in many systems biology studies, but on the single cell level. Hence the experimentator is not relying on cell averages. The technology was used to analyze growth and protein secretion of single yeast. Until now, it was not possible to use contactless nDEP cultivation for the majority of bacteria because of their small cell size.

We will present a nDEP microfluidic chip that overcomes this limitation with a sophisticated microelectrode and microchannel design, developed and characterized using computational modeling. Microelectrode geometries optimized for bacteria cell dimensions and designed microfluidics enables: first, single bacteria isolation from other cells, secondly, a fluid removal of dead cells and residues of media used for population cultivation and third a precise environmental controlled nDEP trapping of a chosen cell in a microchamber for single cell cultivation. The contactless cultivated single bacterium is continuously in a flow of medium which can be used for environmental controlled perturbation experiments. Subsequent sampling of secreted molecules can be taken by an on-tube-seal microfluidic connection and for example be analyzed by mass spectroscopy. Finally, the analyzed single cell or daughter cell can be transferred in a cultivation well to retrieve a population. Importantly, the manufactured chip is biocompatible and enables isolation and cultivation of single bacteria in an environment defined by the experimentator and therefore overcomes major limitations in microfluidic based single bacteria analysis.

 
3:30pm - 5:00pmKlaus-Goerttler Session
Session Chair: Hendrik Kortmann
Session Chair: Lars M. Blank

Keynote Speaker: Andreas Manz, Korea Institute of Science and Technolgy, Saarbruecken, Germany


Lecture Hall 
 

Microfluidics & Miniaturization - the Teenage Years are Over

Andreas Manz

Korea Institute of Science and Technolgy (KIST);

.After a brief overview of historic micro- and nanotechnology for “lab on chip” devices, I will focus on 2 examples of near market applications:

Bacteria causing infections can be identified by detecting specific DNA sequences. This method is widely used and has a high degree of specificity. However, for this purpose, the micro organisms will have to be captured, and it is not clear whether they are dead or alive. Metabolite studies have the advantage that remote evidence for live cells can be identified.

I will present portable equipment for volatile metabolite quantification, based on multi-capillary GC and ion-mobility detection [1]. The raw data is mathematically treated to receive best discrimination of patient groups. Applications for diagnostics of lung infections and lung cancer will be shown. In addition, a chip-based hand held real-time PCR instrument will be presented. A "virtual reactor" approach, i.e., a free droplet in oil, is used for thermal cycling and subsequent melting temperature profiling of the PCR product [2].

Furthermore, there will be a few other examples of recent microfluidic work.

[1] Perl, T., Vautz, W., Nolte, J., Baumbach, J.I. & Quintel, M. Ion mobility spectrometry of human pathologic bacteria - metabolic profiling by volatile organic compounds. Infection 37, 24-24 (2009).

[2] Pavel Neuzil, Chunyan Zhang, Juergen Pipper, Sharon Oh, Lang Zhuo, Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes. Nucleic Acids Research, 2006, Vol. 34, doi:10.1093/nar/gkl416


Microfluidic single-cell analysis: NF-kappaB signaling as a case study

Savas Tay

ETH Zürich;

Microfluidics is a powerful technology that is impacting biomedical sciences in a profound way. I will talk about microfluidic single cell analysis in the context of NF-kappaB signaling dynamics. NF-kappaB is one of the better studied innate immune pathways, albeit mostly using traditional, population averaged assays. Although the individual components of NF-kappaB are (relatively) well understood, how these components act as a system at the cellular level has remained an open question. Using an automated high-throughput microfluidic cell culture system and single-cell microscopy, we measured and quantified NF-kappaB transcription factor activity in thousands of live cells under TNF-alpha doses covering four orders of magnitude (Nature 466, 267, 2010). In contrast to population assays, NF-kappaB activation is found to be a switch-like process at the single cell level, with fewer cells responding at lower doses. The activated cells up-regulate early genes independent of the TNF-alpha concentration in line with the digital activation of NF-kappaB, while only high dose stimulation results in the expression of late-term genes. Using our comprehensive data, we developed a mathematical model that reproduces both the digital and analog dynamics as well as the gene expression profiles at all measured conditions, constituting a broadly applicable model of NF-kappaB signaling. These results highlight the value of high-throughput quantitative measurements at the single-cell level in understanding how biological systems operate. I will also briefly talk about other aspects of NF-kappaB signaling under LPS mediated TLR4 activation, and collective migration characteristics of cells in 2-D microfluidic environments.


The Envirostat: A Lab-on-a-Chip Reactor Concept for Single Cell Biotechnology

Hendrik Kortmann

Novartis Vaccines, Germany;

Cells, equal or not equal? That is the question pursued in this presented thesis. A question not easily answerable by population data reporting averaged read outs of millions of cells. Here spatiotemporal single cell analysis under fully controlled environmental conditions was the aim, approached by the design, construction and use of a lab-on-a-chip reactor concept, the Envirostat.

To accomplish single cell cultivation under environmental controlled and constant conditions, the commercially lab on a chip was adapted. A new interface was constructed for fluidic and electric coupling of the fragile lab-on-a-chip system. Cultivation temperature during cell trapping was controlled by combination of the negative dielectrophoresis trapping immanent Joule heating and an integrated temperature-device. The Envirostat concept of constant environmental conditions during cultivation of a single yeast cell was simulated by computation fluid dynamics. Cultivations starting from single yeast cells in the Envirosta demonstrate its usability as bioreactor.

Furthermore, the combination of the Envirostat with confocal microscopy enabled real time protein secretion rate monitoring of trapped fission yeast cells. The results indicated that few (four) eGFP secreting cells can be reliably differentiated from non-secreting cells with the promising prospect to gain detailed time resolved information of protein secretion kinetics of an individual cell.

Summing up, in the presented thesis the Envirostat for the analysis of few down to a single cell was developed, characterized, and its usability shown.

 
5:30pm - 7:00pmMeeting of DGfZ members
 
7:00pm - 8:00pmBreak and Departure for the Dinner
 
8:00pm - 11:59pmConference Dinner
Brauhaus Machold
A very popular and spacious pub built into the rooms of a former brewery protected under the cultural heritage code. Upscale place for young and old and everyone in between. Dress Code: whatever you like

 
Date: Friday, 14/Oct/2011
9:00am - 10:15amInnate Immunity
Session Chair: Ulrich Sack
Session Chair: Frank Schildberg

Keynote Speaker: Stefan Bauer, Universität Marburg, Institut für Immunologie, Marburg, Germany


Lecture Hall 
 

Toll-like receptors and the recognition of nucleic acids

Stefan Bauer

Philipps-Universität Marburg, Germany;

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are indispensable components in living organisms. DNA stores genetic information while RNA serves as genome for some viruses is a prerequisite for protein translation and has important regulatory functions.

Evidence accumulated over the past few decades that nucleic acid when released from pathogens or host cells can activate the innate immune system and lead to enhanced anti-tumour activity and type I interferon production. Details on nucleic acid recognizing receptors such as Toll-like receptors will be discussed.


Exploring innate immune signaling via genome-wide shRNA screens

Andrea Ablasser

University of Bonn, Germany;

The innate immune system is the first line of defense against invading pathogens. Members of the Toll-like receptor (TLR) family act as primary sensors that detect a wide range of microbial components and elicit innate immune responses. All TLR signaling pathways culminate in the activation of the transcription factor nuclear factor-κB (NF-κB), which controls the expression of an array of inflammatory genes. In addition, each TLR elicits specific antimicrobial defenses owing to differential usage of intracellular adaptor proteins or due to activation of cell-specific signaling cascades. Given the extraordinary complexity of immune regulation via TLRs, this can be best investigated with systematic, genome-wide loss of function approaches. Here we describe a comprehensive strategy for large-scale pooled short hairpin RNA (shRNA) screening in macrophages that combines FACS sorting for genome-wide negative selection and next-generation sequencing for deconvolution of candidate genes. We applied this methodology to explore the TLR4 signalling cascade and were able to identify known regulators as well as unknown candidates. We are convinced that a more detailed understanding of the molecular networks underlying TLR signal transduction will reveal new drug targets and strategies to fine tune TLR-mediated immune responses.


NK Cells in Hodgkin Lymphoma are impaired but can be activated

Katrin S. Reiners, Jörg Kessler, Maike Sauer, Andreas Engert, Elke Pogge von Strandmann

University Hospital Cologne, Germany, Department of Internal Medicine I, Laboratory for Immunotherapy;

Introduction: NK cells represent the key component of the innate immune system to recognize and eliminate cancer cells. Defects in NK cell function including impaired cytotoxicity/cytokine secretion, aberrant receptor expression profile, NK cell number and NK cell anergy are reported in non Hodgkin lymphoma and correlate with a bad prognosis. So far, nothing is known about the phenotype of peripheral NK cells and serum levels of ligands for NK cell receptors in Hodgkin Lymphoma (HL) patients. Here, cytotoxicity, expression pattern of activating NK cell receptors and the serum levels of several ligands for the key cytotoxic receptors NKG2D and NKp30 are determined.

Methods: The cytotoxicity of NK cells isolated from HL patients was analysed by europium release assay using the HL cell line L428 as target cells. The serum level of the NKp30-ligand BAT3 and ligands for NKG2D (MICA, MICB and ULBP1,2,3) was estimated in sera of 117 HL patients and 40 healthy donors by ELISA. The expression pattern of NKp30, NKp44, NKp46, CD16 and the activation markers CD25, CD69 and CD71 was determined by 4-colour FACS analysis of peripheral blood lymphocytes.

Results: The cytotoxicity assays reveal a significantly reduced killing efficacy of NK cells from HL patients against the Hodgkin cell line L428 in comparison to NK cells from healthy donors. Correlating with the impaired NK cell function, we observed that the serum level for BAT3 and MICA was significantly elevated in HL patients, whereas other ligands (MICB and ULBP1,2,3) remained unchanged. NKG2D showed a significantly decreased expression on NK cells of HL patients. No significant difference was observed for all other receptors and activation markers tested.

Conclusion: Our results suggest that soluble BAT3 and MICA, ligands for NKp30 and NKG2D, contribute to the NK cell inhibition in HL patients. Since soluble ligands for NK cell receptors are known to inhibit NK cell-cytotoxicity, the release of these ligands might represent an immune escape mechanism of HL tumors to avoid detection and killing by the innate immune system. To overcome NK cell inhibition in HL patients we design, express and purify bispecific proteins (immunoligands) that target NKG2D and a HL-specific tumorantigen. Work to activate HL-derived NK cells ex vivo will be discussed.

 
10:15am - 10:30amCoffee break
 
10:30am - 11:30amSpecial Lecture: Meet the Expert
Session Chair: Hyun-Dong Chang

Keynote Speaker: Garry Nolan, Dept of Microbiology & Immunology, Stanford University, CA, USA


Lecture Hall 
 

Systems Immunology at the Single Cell Level

Garry Nolan

Stanford University, United States of America;

Single cell analysis of functional cell states via flow cytometry has been an essential tool for dissecting the functional complexity of hematopoiesis. We have focused upon the development of intracellular assays of signaling systems can be correlated to functional subsets of cells in complex populations based on active kinase states or other signaling junctions. Such correlations could be important to document and order the immune system and its disorders into understandable progressions.

Using a next-generation single-cell ‘mass cytometry’ platform we quantify surface and cytokine or drug responsive indices of kinase targets with 34 parameters analysis (e.g. 31 antibodies, viability, DNA content and relative cell size). In healthy human blood and bone marrow, we identified cell subsets spanning the hematopoietic hierarchy using a panel of up to 31 surface markers and used an automated algorithm to delineate all cell subpopulations into a progressive heirarchy. Additionally, 18 simultaneous functional markers and a battery of ex vivo stimuli and inhibitors were used to interrogate signaling. This novel algorithm for unsupervised high-dimensional clustering of single-cell data revealed a continuum of phenotypically contiguous subpopulations. This continuum was demarcated by surface marker expression, but paralleled by gradual changes in intracellular signaling architecture.

The application of these multidimensional approaches to human acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) samples, as well as differentiating ES cells will be presented. The simultaneous measurement of surface marker, cell cycle, apoptosis, and intracellular signaling parameters has revealed substantial “order” within the presumed heterogeneity in the populations of these progressions of cell states. This information is used to provide a high-content view of differential in vitro drug activity across these distinct blast subpopulations for cancer—again indicating there exists remarkable “structure” within the tumor that is suggestive of either an adulterated differentiation cascade or a pseudo-hierarchy of differentiation. For differentiating ES cells we find signatures of pluripotent marker states that predict dead ends for differentiation or indicate a progression towards a defined lineage. Collectively, these results have exposed unappreciated layers of human hematopoietic organization, and provide an opportunity to re-evaluate diseases and pharmacological therapeutics as specific perturbations to this inherent order.

 
11:30am - 12:30pmLunch
 
12:30pm - 2:00pmThe Pathogen and the Host
Session Chair: Dirk Bumann
Session Chair: Frank Schmidt

Keynote Speaker: Ulrich Schaible, Forschungszentrum Borstel, Borstel, Germany


Lecture Hall 
 

Analysing life forms of pulmonary bacterial pathogens

Ulrich E. Schaible

Forschungszentrum Borstel, Germany;

The lung is target organ of a number of bacterial pathogens including the notorious tubercle bacillus (Mycobacterium tuberculosis, Mtb) as well as various agents causing pneumonia including the emerging Gram-negative opportunist Stenotrophomonas maltophilia (Sm). Mtb is able to live and proliferate inside host macrophages and other phagocytes by manipulating its host cell's intracellular vesicle transport system.

In order to dissect the virulence properties of intracellular tubercle bacilli, we used different separation methods to purify intracellular mycobacteria for proteomics, transcriptomics and lipidomics. To mimick the mycobacteria-host interphase, we employ magnetic beads coupled to Mtb structures such as glycolipids to study host-pathogen interaction in a simplified model. Obligate extracellular bacterial pathogens of the lung such as Sm often can generate biofilms, which may interfere with phagocytosis by innate host defense cells and enhance drug resistance. In order to reveal the differential living conditions in Sm biofilms, we compared biofilm-forming vs. planctonic GFP expressing Sm by proteomics demonstrating a biofilm-specific proteome.

The characterization of distinct life forms, which arise during a life cycle of pathogenic bacteria, such as intra- vs. extracellular or biofilm vs. planctonic, is facilitated by specific separation methods.


In vivo proteome analysis of internalized Staphylococcus aureus by human bronchial epithelial cells

Henrike Pförtner1, Marc Burian1, Petra Hildebrandt2, Vishnu Dhople1, Jan Liese3, Frank Schmidt2, Uwe Völker1

1Department of Functional Genomics, Ernst-Moritz-Arndt-University of Greifswald, Germany; 2ZIK-FunGen Junior Group Applied Proteomics, Ernst-Moritz-Arndt-University of Greifswald, Germany; 3Medical Microbiology and Hygiene, Eberhard Karls University of Tübingen, Germany;

Staphylococcus aureus , the cause of a wide spectrum of severe community-acquired and nosocomial infections is traditionally not considered to be an intracellular pathogen. However, there is evidence that S. aureus can be internalized and persist in non-professional phagocytic cells in vitro . During the internalization process, S. aureus has to adapt to the new intracellular environment to survive or even persist within the host. Due to low numbers of bacteria available from an internalization experiment, the investigation of the bacterial adaptation to the host environment is still a challenge and therefore little is known about these adaptive changes on the proteome level.

We were recently able to analyze internalized S. aureus RN1HG wild type cells with our newly developed workflow, which combines a classical infection assay with pulse-chase labeling, high capacity cell sorting, and gel-free proteomics.

Before internalization, S. aureus RN1HG cells were fully labeled with heavy amino acids using the SILAC method and then co-cultivated with S9 human bronchial epithelial cells allowing internalization. Due to the continuous GFP expression of S. aureus , bacteria could be enriched after lysis of the S9 cells by FACS on a filter membrane. The Staphylococci on the membrane were subjected to tryptict digestion and generated peptides were measured by nanoLC-ESI-MS/MS. Using a sorting time of 45 min, 300 to 700 S. aureus proteins from only 2-5 ∙ 10^6 cells could be identified and quantified over a period of 6.5 hours post-internalization.

In a further experiment we compared the adaptive changes of RN1HG and its isogenic ∆agr mutant. The most promising approach to compare these adaptive and competitive changes would be simultaneous co-infection of the wild type and the mutant. The striking advantage of a co-infection assay is that both strains internalize and adapt to the host under exactly the same conditions.

For distinct enrichment of RN1HG and the ∆agr mutant, the mutant had to be labeled with a fluorescent marker allowing the separation from GFP of the wild type. After validation, the fluorescent marker gpCerulean showed a good intensity and a clear distinction to GFP in FACSAria. Therefore, we are now able to sort co-internalized wild type and ∆agr mutant parallel in distinct wells of a 96-well plate.

In a first proteomics experiment, we have enriched 10^3 - 10^4 co-internalized Staphylococci via FACSAria and identified about 200 - 300 proteins. The pros and cons of the approach and observed adaptations in the protein profiles will be discussed in the presentation.


Flow Cytometry as Efficient Tool in Filarial Research

Sebastian Ziewer1, Coralie Martin2, Achim Hörauf1, Sabine Specht1

1Universitiy Hosital Bonn, Germany; 2Muséum National d’Histoire Naturelle, France;

Lymphatic filariasis (LF) and onchocerciasis are parasitic nematode infections that are responsible for a major disease burden in the African continent. Disease symptoms are induced by the immune reactions of the host, with lymphoedema and hydrocoele in LF, and dermatitis and ocular inflammation in onchocerciasis. The infection is acquired during a blood meal of an insect vector thereby transmitting infective filarial stages onto the human host, where they develop into adults and produce millions of new larvae, the microfilariae (Mf). Current disease elimination strategies of the WHO are based on mass drug administration (MDA) of microfilaricides.

However, success of MDA is compromised by logistical aspects such as long-time treatment, as well as by adverse events to therapy and suboptimal responses to the drugs. Alternative approaches are urgently needed and a vaccine targeting the blood-circulating Mf stage would be a pivotal step in disease control in order to stop transmission. Here we show a successful immunization protocol against the MF stage in the murine model for filariasis.

Flowcytometry has become one of the most powerful tools in immunological research and consequently has also been used to study LF, e.g. for staining immune cells from the site of infection in the murine model of LF. Here we show that flowcytometry can be used to count and analyze the multi-cellular 80-100 µm tall filarial Mf stage. Flowcytometric analysis of the Mf stage may become a powerful tool to investigate the mechanisms that form the basis for the Mf-specific responses of the successful immunization.

In addition we show a flowcytometry-based characterization of immune cells from the site of infection in the murine model of LF. Given that the cellular content from the site of infection is composed of several different cell types, we finally want to illustrate some of the “pitfalls” that have to be considered in the interpretation of that kind of data.


Interaction of platelets with bacteria visualized by correlated light and electron microscopy and tomography

Josef Neumüller1, Claudia Meißlitzer-Ruppitsch1, Renate Renz2, Christof Jungbauer2, Adolf Ellinger1, Margit Pavelka1

1Medical University of Vienna, Center of Anatomy and Cell Biology, Dept. for Cell Biology and Ultrastructure Research, Austria; 2Blood donation Center of the Austrian Red Cross for Vienna, Lower Austria and Burgenland, Austria;

Platelets (PLT) obtained by thrombapheresis were spiked with bacteria to see how they interact with each other in respect to PLT activation, sequestration or phagocytosis. Since bacteria can be covered by the surface connected open canalicular system (OCS) of PLT we used ruthenium red (RR) as a tracer for the OCS which enabled discriminating completely engulfed bacteria from those that are only partially covered by the OCS. In addition, the reorganization of the cytoskeleton including the circumferential microtubular coil and the actin microfilaments was investigated.

PLT were spiked with commercially available preparations of Staphylococcus aureus and Escherichia coli (pHrodoTM, Invitrogen) in a ratio of approximately 1:10. After 30 minutes of coincubation, PLT were allowed to adhere to glass coverslips and spiked with bacteria or incubated in PBS; both treatments lasted for 30 min. Alternatively, spiking with bacteria was carried out in suspension or when PLT in suspension interacted with bacteria immobilized on a glass surface. The samples were fixed 15 min. with 1% formaldehyde for light microscopy or with 2.5% glutaraldehyde (60 min.) and 1% OsO4 (2 h.) for electron microscopy. OCS tracing was carried out with 2% OsO4 and 3% RR in 0.1M cacodylate buffer, for 90 min. Electron tomography (ET) was carried on 300 nm semithin sections by acquisition of tilting series of ±65° with an increment of 1° and reconstruction using the weighted back projection method. F-actin and α-tubulin staining was performed with phalloidin-TRITC and an Alexa Fluor 488 anti-tubulin-α MoAb respectively.

PLT, adherent to glass surfaces, formed a granulomer and a hyalomer. In TEM, slightly adherent PLT showed the circumferential microtubular coil with adjacent lacunae of the dense tubular system (DTS). In firmly adherent PLT, this ring was centralized, lining the granulomer containing organelles. This centralization of the microtubular coil could be also shown by fluorescence microscopy..

Both species of bacteria formed cell contacts with PLT. Activated PLT extended filopodia forming aggregates by free cell contacts sequestering bacteria. Conspicuous PLT aggregates occurred also due to interaction with immobilized bacteria. RR decorated only particular OCS compartments while other remained separated from the PLT surface which could be convincingly underlined by ET. Bacteria in the PLT suspension induced a significant PLT activation demonstrated by centralization of the microtubular coil and by accumulation of F-actin around the granulomer. The delivery of α granules could also be verified at the EM level.

Further investigations should clarify, whether PLT are able to kill bacteria using their lysosomal enzymes or are only able to transport them to sites of degradation or to deposit them to particular sites of the body, where bacteria-induced inflammation could be induced. Possible consequences for transfusion medicine and human pathology will be discussed.

 
2:00pm - 2:30pmCoffee break
 
2:30pm - 3:30pmCell Signalling
Session Chair: Hyun-Dong Chang
Lecture Hall 
 

Quantitative single cell analysis of endogenous transcription factor expression levels reveals NFATc2 and c-fos as limiting factors for IL-2 production

Tobias Scheel1, Hanna Bendfeldt1, Manuela Benary2, Hanspeter Herzel2, Ria Baumgrass1

1Deutsches Rheuma-Forschungszentrum Berlin, Germany; 2Institute for Theoretical Biology, Humboldt University zu Berlin, Germany;

The regulation of Interleukin-2 (IL-2) expression in human Th cells is of great importance for the balance of the immune system: effective immune responses vs. central tolerance. Therefore, precise fine-tuning of IL-2 expression is crucial for adjusting the immune response.

So far it is unknown, whether IL-2 producing and nonproducing Th cells are different in the expression levels and activatability of the main TCR-dependent transcription factors. Using transcription factor analysis on single cell level by flow cytometry and mathematical analysis we studied the transcription factor networks regulating IL-2 production in human memory Th cells. We showed that physiological differences in the expression level of NFATc2 and c-fos, but not of c-jun and NF-κBp65, are limiting for the decision whether IL-2 is expressed or not. Particularly, stochastic variation in the expression of c-fos leads to substantial diversity of IL-2 expression in approximately 40% of the memory Th cells. The remaining cells exhibit a high probability of IL-2 expression, thereby ensuring robustness in IL-2 response within the population. Manipulation of c-fos de novo synthesis by the small molecular inhibitor U0126 confirms the importance of a certain endogenous c-fos expression level for IL-2 production.

These findings reveal how T cells benefit from regulated variation in transcription factor expression to achieve variability of cytokine expression in a controlled manner.

The work was supported by the German Federal Ministry of Education and Research (BMBF; Forsys-Partner) and by the Deutsche Forschungsgemeinschaft (DFG; SFB-TR52).


Incubation of human mobilized peripheral and cord blood and bone marrow cells with anti-hVEGFR2 and anti-hS1P3 alters gene expression of pluripotent cell populations.

Anush Vemir Karapetyan, Ahmed Abdel-Latif

University of Kentucky, United States of America;

Hypothesis: antibodies to certain receptors may cause conformational changes in receptor and activate downstream signal transduction cascade.

Vascular endothelial growth factor receptors (VEGFR) belong to receptor tyrosine kinases.

Binding of vascular endothelial growth factor ( VEGF) to extracellular portion of receptor causes conformational changes in intracellular part of receptor. This event leads to activation of kinase activity of receptor with subsequent signal transduction eventually resulting in alteration of genome response. There are 3 types of VEGF receptors with different affinities to various splices of VEGF molecule. In our experiment we were looking on VEGFR2 expression on lineage negative progenitor cells. As lineage positive we classify cells expressing lymphoid and myeloid surface markers.

Sphingosine 1-phosphate (S1P) receptors belong to G-coupled proteins, also responding and transducing extracellular signals through conformational changes caused by extracellular ligand-receptor interaction. There are 5 known S1P receptors designated as S1P1, 2,3,4,5

By using flow cytometry we examined the expression of VEGFR2, CD105, CD117, CXCR4 in lineage negative CD34+ populations after incubation of white blood cells of mPB, CB and BM with antibodies to human VEGFR2. The population of lineage-CD34+VEGFR2+ cells significantly rises after two days of incubation and the expression of CD105 and CD117 changes on some lineage negative subpopulations.

We also examined the phosphorylation of Akt -kinase after incubation with anti-hS1P3.

The positive change in phosphorylation level of Akt after 10 min of incubation mPB with anti-hS1P3 compared to control demonstrates the activation of S1P3 receptors by its antibody.

By using RT-qPCR we also examined the alterations in gene expression of CB cells after incubation with anti-hVEGFR2 and anti-hS1P3 antibodies for 1, 2 and 3 days.

The expression levels of mRNA of homeodomain proteins Nkx2.5 and NANOG and transcription factors GATA4 and oct 4 were several times altered.

As is known GATA4 and Nkx2.5 are expressed in precardiac cells and are considered to direct the differentiation of progenitor cells to cardiac cells.

Instead NANOG and oct4 are major factors expressed in pluripotent cells and responsible for maintaining pluripotency.

Thus VEGFR2 and S1P3 receptors are involved in differentiation of pluripotent progenitor cells and their activity may be regulated by the antibodies to these receptors.

 
3:30pm - 4:30pmFarewell Coffee
 

 
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