Burkhard Greve

Flow cytometry in cancer stem cell analysis and separation

In recent years, a special type of cancer cell—the cancer stem cell (CSC)—has been identified and characterized for different tumors. CSCs may be responsible for the recurrence of a tumor following a primarily successful therapy and are thought to bear a high metastatic potential. For the development of efficient treatment strategies, the establishment of reliable methods for the identification and effective isolation of CSCs is imperative. Similar to their stem cell counterparts in bone marrow or small intestine, different cluster of differentiation surface antigens have been characterized, thus enabling researchers to identify them within the tumor bulk and to determine their degree of differentiation. In addition, functional properties characteristic of stem cells can be measured. Side population analysis is based on the stem cell‐specific activity of certain ATP‐binding cassette transporter proteins, which are able to transport fluorescent dyes out of the cells. Furthermore, the stem cell‐specific presence of aldehyde dehydrogenase isoform 1 can be used for CSC labeling. However, the flow cytometric analysis of these CSC functional features requires specific technical adjustments. This review focuses on the principles and strategies of the flow cytometric analysis of CSCs and provides an overview of current protocols as well as technical requirements and pitfalls. A special focus is set on side population analysis and analysis of ALDH activity. Flow cytometry‐based sorting principles and future flow cytometric applications for CSC analysis are also discussed.

A radiation-induced gene expression signature as a tool to predict acute radiotherapy-induced adverse side effects

The majority of patients tolerate radiotherapy well, but some of them suffer from severe side effects. To find genes possibly predictive for radiosensitivity, mRNA profiles were generated before and 6h after in vitro irradiation with 5Gy. We analyzed lymphocytes from four head and neck and eight breast cancer patients with strong acute radiation toxicity and from 12 matching normal reacting patients in a blind study. Expression was also measured in lymphocyte subpopulations and Epstein-Barr transformed lymphocytes. Radiation response in whole lymphocyte populations was most similar to that of B cells. In peripheral blood lymphocytes of all patients; 153 genes were identified which were statistically significantly altered by a fold change of more than 50% by irradiation. The signatures of radio-responsive genes differed tremendously between primary and transformed cells. Pathway analysis revealed genes involved in p53 signalling, cell cycle control and apoptosis in response to radiation in primary lymphocytes. In these cells, a set of 67 radiation-induced genes was identified capable of differentiating between severe radiosensitive and normal reacting patients. More than one third of such classifying genes belong to the group of apoptosis or cell cycle regulating genes. The classifying potential of the expression signature has now to be validated in further patient cohorts.

Syndecan-1 modulates β-integrin-dependent and interleukin-6-dependent functions in breast cancer cell adhesion, migration, and resistance to irradiation.

Syndecan‐1 is a cell surface heparan sulfate proteoglycan with various biological functions relevant to tumor progression and inflammation, including cell–cell adhesion, cell–matrix interaction, and cytokine signaling driving cell proliferation and motility. Syndecan‐1 is a prognostic factor in breast cancer, and has a predicitive value for neodadjuvant chemotherapy. It is still poorly understood how syndecan‐1 integrates matrix‐dependent and cytokine‐dependent signaling processes in the tumor microenvironment. Here, we evaluated the potential role of syndecan‐1 in modulating matrix‐dependent breast cancer cell migration in the presence of interleukin‐6, and its potential involvement in resistance to irradiation in vitro. MDA‐MB‐231 breast cancer cells were transiently transfected with syndecan‐1 small interfering RNA or control reagents, and this was followed by stimulation with interleukin‐6 or irradiation. Cellular responses were monitored by adhesion, migration and colony formation assays, as well as analysis of cell signaling. Syndecan‐1 depletion increased cell adhesion to fibronectin. Increased migration on fibronectin was significantly suppressed by interleukin‐6, and GRGDSP peptides inhibited both adhesion and migration. Interleukin‐6‐induced activation of focal adhesion kinase and reduction of constitutive nuclear factor kappaB signaling were decreased in syndecan‐1‐deficient cells. Focal adhesion kinase hyperactivation in syndecan‐1‐depleted cells was associated with dramatically reduced radiation sensitivity. We conclude that loss of syndecan‐1 leads to enhanced activation of β1‐integrins and focal adhesion kinase, thus increasing breast cancer cell adhesion, migration, and resistance to irradiation. Syndecan‐1 deficiency also attenuates the modulatory effect of the inflammatory microenvironment constituent interleukin‐6 on cancer cell migration.

The adult stem cell marker Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via Notch-1 and p21WAF1/CIP1

The RNA‐binding protein Musashi‐1 has been proposed to maintain stem cell function during development and regenerative processes as a modulator of the Notch‐1 signaling pathway. Musashi‐1 expression is upregulated in endometrial carcinoma, however, its pathogenetic role in this tumor entity is unknown. Here we investigate the functional impact and mode of action of Musashi‐1 on endometrial carcinoma cell behaviour in vitro. Aldehyde dehydrogenase‐1 activity and side population (SP) measurement by Hoechst dye exclusion revealed that the Ishikawa endometrial carcinoma cell line contains a pool of putative cancer stem cells. Musashi‐1 expression is 20.8‐fold upregulated in SP+ compared to SP‐ and equally distributed between ALDH+ and ALDH‐ cell pools. siRNA‐mediated knockdown of Musashi‐1 mRNA expression lead to an altered expression of the signaling receptor Notch‐1 and its downstream targets, the transcription factor Hes‐1 and the cell cycle regulators p21WAF1/CIP1 and cyclin B1, as determined by Western blotting and quantitative real‐time PCR. Flow cytometric and ELISA analyses revealed that Musashi‐1‐mediated modulation of these factors exerted an antiproliferative effect on the cell cycle, and increased apoptosis in endometrial carcinoma cells. We conclude that Ishikawa cells contain a subpopulation of cells with stem cell‐like properties. Musashi‐1 modulates endometrial carcinoma cell cycle progression and apoptosis via the stemness‐related factors Notch‐1, Hes‐1 and p21WAF1/CIP1, thus emerging as a novel future target for endometrial carcinoma therapy.

A New No-Lyse, No-Wash Flow-Cytometric Method for the Determination of CD4 T Cells in Blood Samples

Background: Commonly used flow-cytometric methods for immunophenotyping are based on erythrocyte lysing reagents. It is known from the literature that these reagents result in a significant loss of leucocytes caused by membrane destruction. Although the dual-platform method should compensate this phenomenon, the subset-specific individual differences in sensitivity to lysing reagents lead to incorrect values. In order to overcome this problem we introduce a no-lyse, no-wash procedure in combination with absolute true volumetric counting (TVC) for the enumeration of CD4 T cells. Material and Methods: Whole-blood samples of 50 blood donors and 20 samples of patients with acquired immunodeficiency syndrome (AIDS) were treated with both a lyse, no-wash and a nolyse, no-wash procedure. Then, CD4 T cells were counted with a TVC flow cytometer (CyFlow Counter). 30 blood samples of blood donors were treated with a lyse and wash protocol and measured by the CyFlow Counter and FACS-Calibur system (reference method). A new gating strategy was used for the data analysis for both, the lyse and no-lyse method and compared to the traditional gating procedure. Results: The TVC method showed a good reproducibility for both, the lyse (CV 1.9%) and the no-lyse procedure (CV 1.5%). CD4 counts measured by the no-lyse procedure are on average 10% higher than by using the lysing protocol. The comparison of the CyFlow Counter and FACS-Calibur results showed a correlation of r = 0.961. The simplified gating strategy shows a good correlation to the traditional gating procedure (r = 0.998). Conclusion: Erythrocyte lysing procedures cause substantial cell loss with individual values for every single subclass and patient. Therefore, the use of a no-lyse procedure is recommended. The new gating strategy is fully comparable to the traditional one and simplifies enumeration of CD4 T cells. Using the no-lyse procedure in combination with the new gating strategy, it is possible to reduce the costs per sample from EUR 30.– to below EUR 2.–.

Syndecan-1 (CD138) modulates triple-negative breast cancer stem cell properties via regulation of LRP-6 and IL-6-mediated STAT3 signaling.

Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a coreceptor for growth factors and chemokines and is a molecular marker associated with epithelial-mesenchymal transition during development and carcinogenesis. Resistance of Syndecan-1-deficient mice to experimentally-induced tumorigenesis has been linked to altered Wnt-responsive precursor cell pools, suggesting a potential role of Syndecan-1 in breast cancer cell stem function. However, the precise molecular mechanism is still elusive. Here, we decipher the functional impact of Syndecan-1 knockdown using RNA interference on the breast cancer stem cell phenotype of human triple-negative MDA-MB-231 and hormone receptor-positive MCF-7 cells in vitro employing an analytical flow cytometric approach. Successful Syndecan-1 siRNA knockdown was confirmed by flow cytometry. Side population measurement by Hoechst dye exclusion and Aldehyde dehydrogenase-1 activity revealed that Syndecan-1 knockdown in MDA-MB-231 cells significantly reduced putative cancer stem cell pools by 60% and 27%, respectively, compared to controls. In MCF-7 cells, Syndecan-1 depletion reduced the side population by 40% and Aldehyde dehydrogenase-1 by 50%, repectively. In MDA-MB-231 cells, the CD44(+)CD24(-/low) phenotype decreased significantly by 6% upon siRNA-mediated Syndecan-1 depletion. Intriguingly, IL-6, its receptor sIL-6R, and the chemokine CCL20, implicated in regulating stemness-associated pathways, were downregulated by >40% in Syndecan-1-silenced MDA-MB-231 cells, which showed a dysregulated response to IL-6-induced shifts in E-cadherin and vimentin expression. Furthermore, activation of STAT-3 and NFkB transcription factors and expression of a coreceptor for Wnt signaling, LRP-6, were reduced by >45% in Syndecan-1-depleted cells compared to controls. At the functional level, Syndecan-1 siRNA reduced the formation of spheres and cysts in MCF-7 cells grown in suspension culture. Our study demonstrates the viability of flow cytometric approaches in analyzing cancer stem cell function. As Syndecan-1 modulates the cancer stem cell phenotype via regulation of the Wnt and IL-6/STAT3 signaling pathways, it emerges as a promising novel target for therapeutic approaches.

Evaluation of Different Biomarkers to Predict Individual Radiosensitivity in an Inter-Laboratory Comparison–Lessons for Future Studies

Radiotherapy is a powerful cure for several types of solid tumours, but its application is often limited because of severe side effects in individual patients. With the aim to find biomarkers capable of predicting normal tissue side reactions we analysed the radiation responses of cells from individual head and neck tumour and breast cancer patients of different clinical radiosensitivity in a multicentric study. Multiple parameters of cellular radiosensitivity were analysed in coded samples of peripheral blood lymphocytes (PBLs) and derived lymphoblastoid cell lines (LCLs) from 15 clinical radio-hypersensitive tumour patients and compared to age- and sex-matched non-radiosensitive patient controls and 15 lymphoblastoid cell lines from age- and sex- matched healthy controls of the KORA study. Experimental parameters included ionizing radiation (IR)-induced cell death (AnnexinV), induction and repair of DNA strand breaks (Comet assay), induction of yH2AX foci (as a result of DNA double strand breaks), and whole genome expression analyses. Considerable inter-individual differences in IR-induced DNA strand breaks and their repair and/or cell death could be detected in primary and immortalised cells with the applied assays. The group of clinically radiosensitive patients was not unequivocally distinguishable from normal responding patients nor were individual overreacting patients in the test system unambiguously identified by two different laboratories. Thus, the in vitro test systems investigated here seem not to be appropriate for a general prediction of clinical reactions during or after radiotherapy due to the experimental variability compared to the small effect of radiation sensitivity. Genome-wide expression analysis however revealed a set of 67 marker genes which were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation in larger cohorts in order to determine parameters potentially predictive for clinical radiosensitivity.

Stereotactic body radiotherapy for liver tumors: principles and practical guidelines of the DEGRO Working Group on Stereotactic Radiotherapy.

PurposeThis report of the Working Group on Stereotactic Radiotherapy of the German Society of Radiation Oncology (DEGRO) aims to provide a practical guideline for safe and effective stereotactic body radiotherapy (SBRT) of liver tumors.MethodsThe literature on the clinical evidence of SBRT for both primary liver tumors and liver metastases was reviewed and analyzed focusing on both physical requirements and special biological characteristics.ResultsRecommendations were developed for patient selection, imaging, planning, treatment delivery, motion management, dose reporting, and follow-up. Radiation dose constraints to critical organs at risk are provided.ConclusionSBRT is a well-established treatment option for primary and secondary liver tumors associated with low morbidity.ZusammenfassungZielDie Arbeitsgruppe Stereotaxie der Deutschen Gesellschaft für Radioonkologie (DEGRO) legt hier eine Empfehlung zur sicheren und effektiven Durchführung der SBRT von Lebertumoren vor.MethodenEine Literaturrecherche zur Untersuchung der Evidenz der SBRT sowohl für primäre Lebertumore als auch für Lebermetastasen wurde durchgeführt. Auf dieser Basis werden Empfehlungen für technisch-physikalische Voraussetzungen wie auch für die tägliche Praxis der Leber-SBRT gegeben. Weiterhin werden radiobiologische Besonderheiten dieses Verfahrens dargestellt.ErgebnissePraktische Vorgaben werden für Patientenselektion, Bildgebung, Planung, Applikation, Bewegungsmanagement, Dosisdokumentation und Follow-up gegeben. Dosisempfehlungen für die kritischen Risikoorgane werden dargestellt.SchlussfolgerungDie SBRT stellt eine etablierte Behandlungsmethode für primäre und sekundäre Lebertumore dar und ist mit niedriger Morbidität assoziiert.

Stereotactic body radiotherapy for liver tumors

PurposeThis report of the Working Group on Stereotactic Radiotherapy of the German Society of Radiation Oncology (DEGRO) aims to provide a practical guideline for safe and effective stereotactic body radiotherapy (SBRT) of liver tumors.MethodsThe literature on the clinical evidence of SBRT for both primary liver tumors and liver metastases was reviewed and analyzed focusing on both physical requirements and special biological characteristics.ResultsRecommendations were developed for patient selection, imaging, planning, treatment delivery, motion management, dose reporting, and follow-up. Radiation dose constraints to critical organs at risk are provided.ConclusionSBRT is a well-established treatment option for primary and secondary liver tumors associated with low morbidity.ZusammenfassungZielDie Arbeitsgruppe Stereotaxie der Deutschen Gesellschaft für Radioonkologie (DEGRO) legt hier eine Empfehlung zur sicheren und effektiven Durchführung der SBRT von Lebertumoren vor.MethodenEine Literaturrecherche zur Untersuchung der Evidenz der SBRT sowohl für primäre Lebertumore als auch für Lebermetastasen wurde durchgeführt. Auf dieser Basis werden Empfehlungen für technisch-physikalische Voraussetzungen wie auch für die tägliche Praxis der Leber-SBRT gegeben. Weiterhin werden radiobiologische Besonderheiten dieses Verfahrens dargestellt.ErgebnissePraktische Vorgaben werden für Patientenselektion, Bildgebung, Planung, Applikation, Bewegungsmanagement, Dosisdokumentation und Follow-up gegeben. Dosisempfehlungen für die kritischen Risikoorgane werden dargestellt.SchlussfolgerungDie SBRT stellt eine etablierte Behandlungsmethode für primäre und sekundäre Lebertumore dar und ist mit niedriger Morbidität assoziiert.

Association of telomerase activity with radio- and chemosensitivity of neuroblastomas

BackgroundTelomerase activity compensates shortening of telomeres during cell division and enables cancer cells to escape senescent processes. It is also supposed, that telomerase is associated with radio- and chemoresistance. In the here described study we systematically investigated the influence of telomerase activity (TA) and telomere length on the outcome of radio- and chemotherapy in neuroblastoma.MethodsWe studied the effects on dominant negative (DN) mutant, wild type (WT) of the telomerase catalytic unit (hTERT) using neuroblastoma cell lines. The cells were irradiated with 60Co and treated with doxorubicin, etoposide, cisplatin and ifosfamide, respectively. Viability was determined by MTS/MTT-test and the GI50 was calculated. Telomere length was measured by southernblot analysis and TA by Trap-Assay.ResultsCompared to the hTERT expressing cells the dominant negative cells showed increased radiosensitivity with decreased telomere length. Independent of telomere length, telomerase negative cells are significantly more sensitive to irradiation. The effect of TA knock-down or overexpression on chemosensitivity were dependent on TA, the anticancer drug, and the chemosensitivity of the maternal cell line.ConclusionsOur results supported the concept of telomerase inhibition as an antiproliferative treatment approach in neuroblastomas. Telomerase inhibition increases the outcome of radiotherapy while in combination with chemotherapy the outcome depends on drug- and cell line and can be additive/synergistic or antagonistic. High telomerase activity is one distinct cancer stem cell feature and the here described cellular constructs in combination with stem cell markers like CD133, Aldehyddehydrogenase-1 (ALDH-1) or Side population (SP) may help to investigate the impact of telomerase activity on cancer stem cell survival under therapy.