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Conference Agenda

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Session Overview
Session
Lunch / Poster Session
Time: Thursday, 13/Oct/2011: 12:30pm - 2:30pm

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”


Presentations

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.



 
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