Direct Cell-cell Interactions Research Articles

Functional analysis of bispecific antibody (EpCAMxCD3)‐mediated T‐lymphocyte and cancer cell interaction by single‐cell force spectroscopy

The atomic force microscopy (AFM) is a powerful tool to analyze forces generated on cellular interactions on the single-cell level. This highly sensitive device can record changes in force in the pico-Newton range, which equals single molecule bonds. Here, we have used single-cell force spectroscopy by AFM to investigate the interaction between T cells and tumor cells that is induced by the bispecific antibody HEA125xOKT3 (specificity anti-EpCAMxCD3). We show that HEA125xOKT3 induces a specific increase in adhesion force between T cells and cancer cells. The adhesive force that is generated on cell-cell contact is dependent on the applied force on initial contact and the duration of this initial contact. In summary, HEA125xOKT3 has been found to mediate contact formation by distinct processes. It induces direct cell-cell interaction, which results in the activation of T-cell signaling, facilitates the formation of supramolecular activation clusters and ultimately of an immune synapse.

Notch signaling contributes to the maintenance of both normal neural stem cells and patient-derived glioma stem cells

BackgroundCancer stem cells (CSCs) play an important role in the development and recurrence of malignant tumors including glioma. Notch signaling, an evolutionarily conserved pathway mediating direct cell-cell interaction, has been shown to regulate neural stem cells (NSCs) and glioma stem cells (GSCs) in normal neurogenesis and pathological carcinogenesis, respectively. However, how Notch signaling regulates the proliferation and differentiation of GSCs has not been well elucidated.MethodsWe isolated and cultivate human GSCs from glioma patient specimens. Then on parallel comparison with NSCs, we inhibited Notch signaling using γ-secretase inhibitors (GSI) and assessed the potential functions of Notch signaling in human GSCs.ResultsSimilar to the GSI-treated NSCs, the number of the primary and secondary tumor spheres from GSI-treated GSCs decreased significantly, suggesting that the proliferation and self-renewal ability of GSI-treated GSCs were attenuated. GSI-treated GSCs showed increased differentiation into mature neural cell types in differentiation medium, similar to GSI-treated NSCs. Next, we found that GSI-treated tumor spheres were composed of more intermediate progenitors instead of CSCs, compared with the controls. Interestingly, although inhibition of Notch signaling decreased the ratio of proliferating NSCs in long term culture, we found that the ratio of G2+M phase-GSCs were almost undisturbed on GSI treatment within 72 h.ConclusionsThese data indicate that like NSCs, Notch signaling maintains the patient-derived GSCs by promoting their self-renewal and inhibiting their differentiation, and support that Notch signal inhibitor GSI might be a prosperous candidate of the treatment targeting CSCs for gliomas, however, with GSI-resistance at the early stage of GSCs cell cycle.

Cellular Interplay between Cardiomyocytes and Nonmyocytes in Cardiac Remodeling

Cardiac hypertrophy entails complex structural remodeling involving rearrangement of muscle fibers, interstitial fibrosis, accumulation of extracellular matrix, and angiogenesis. Many of the processes underlying cardiac remodeling have features in common with chronic inflammatory processes. During these processes, nonmyocytes, such as endothelial cells, fibroblasts, and immune cells, residing in or infiltrating into the myocardial interstitium play active roles. This paper mainly addresses the functional roles of nonmyocytes during cardiac remodeling. In particular, we focus on the communication between cardiomyocytes and nonmyocytes through direct cell-cell interactions and autocrine/paracrine-mediated pathways.

Single cell 3-D platform to study ligand mobility in cell–cell contact

Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have yet to be combined and applied in a single in vitro platform to address, e.g., cell adhesion in a setting mimicking the three-dimensional environment of neighboring cells in a reductionist way. To study the effect of the lateral mobility of cell adhesive ligands in three dimensions we present and characterize a platform, which enables patterning of single cells into microwells presenting a cell membrane mimetic interface pre-patterned to its walls. Soluble E-cadherin extracellular domains coupled through an optimized streptavidin-antibody linkage to lipids in a supported lipid bilayer (SPB) were presented on the microwell walls as either laterally mobile or immobile ligands. The fluidity was controlled through a small change in temperature by choosing phospholipids for the SPB with a lipid phase transition temperature around 30 °C. The platform thus enabled the investigation of cell adhesion to either laterally immobile or mobile E-cadherin ligands presented on the same cell membrane mimetic surface. Chinese hamster ovary (CHO) cells engineered to express E-cadherin that were cultured on the platform demonstrated that enhanced cadherin lateral mobility significantly decreased the formation of actin bundles and resulted in more diffuse actin organization, while constraining the cell shape to that of the microwell. This example highlights the potential to use in vitro cell culture platforms to mimic direct cell-cell interaction in a controlled environment that nevertheless captures the dynamic nature of the native cell environment.

Dependency of CLL Cell Survival on Direct Physical Interaction with Bone Marrow Stromal Cells In Vitro Is Largely Outweighed by Secretion of Soluble Factors with Time In Culture

AbstractAbstract 1365The major pathophysiology feature of chronic lymphocytic leukemia (CLL) cells is their extended life span due to a pronounced resistance towards apoptotic stimuli in vivo. Despite this, CLL cells die within a few days when isolated from their natural microenvironment and are placed under cell culture conditions. That is why the bone marrow microenvironment has been ascribed an essential role in maintenance of the apoptotic resistance of the CLL cell. Thereby both, the physical interaction between bone marrow stromal cells and CLL cells and the secretion of soluble factors have been described to be essentially involved. We analysed the survival capacity of CLL cells in monoculture and in coculture with the bone marrow-derived stromal cell line HS5 with and without physical separation using transwells for up to 7 days by flow cytometric determination of Annexin-V/PI status. As expected, in vitro CLL cell survival was significantly reduced when physical contact between CLL cells and bone marrow stromal cells was prevented. Interestingly, this was only the case for short term cultivation for up to three days. With time under culture conditions CLL cell survival became less dependent on direct physical contact with the HS5 feederlayer, suggesting the secretion of soluble factors to compensate for the loss of pro-survival signals obtained from direct cell-cell interactions over time. This was further supported by the fact of reduced survival support for CLL cells when HS5 proliferation, hence production and secretion of soluble factors, was prevented by mitomycin treatment or formaldehyde fixation. The use of an expanded human Cytokine Antibody Array (Affymetrix), which analyses the presence of the most common 36 cytokine proteins, might offer information about the composition of soluble factors present in the supernatant which are essential for CLL cell survival in vitro. In conclusion, while direct cell-cell contact between CLL cells and bone marrow stromal cells provides an immediate protection against in vitro apoptosis of CLL cells, the secretion of soluble factors, most likely by both, CLL and bone marrow stormal cells, leads to the creation of an in vitro environment which can to a certain extent compensate for the loss of prosurvival signals obtained by direct physical interactions. Disclosures:No relevant conflicts of interest to declare.

Human adipose tissue−derived mesenchymal stem cells facilitate the immunosuppressive effect of cyclosporin A on T lymphocytes through Jagged-1−mediated inhibition of NF-κB signaling

Cyclosporine A (CsA), known as an effective immunosuppressive agent, is widely used in clinical fields. Mesenchymal stem cells may exert immunomodulatory effects on the immune system, but the exact mechanisms underlying them remain controversial. Here we investigated whether human adipose tissue-derived mesenchymal stem cells (AMSCs) facilitate in vitro the immunomodulatory effects of CsA and we explored the molecule mechanisms that may be involved. Proliferation of T lymphocytes was measured by uptake of (3)H-thymidine. Transcription and production of interleukin-2 and interferon-γ were evaluated by real-time quantitative polymerase chain reaction, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. Nuclear factor-κB (NF-κB) was assayed by Western blotting and electrophoretic mobility shift assay. Expression of Jagged-1, Jagged-2, and Delta-1 of AMSCs were surveyed by flow cytometric analysis and Western blotting. The combination of moderate-dose AMSCs and low-dose CsA was significantly more powerful than moderate-dose AMSCs or large-dose CsA alone in suppressing transcription and production of interleukin-2 and interferon-γ, activation of NF-κB, and proliferation of T lymphocytes. In addition, AMSCs expressed a high level of Jagged-1, which induced activation of Notch signaling in T lymphocytes, thus reducing NF-κB activity. Anti-Jagged-1 neutralizing antibody and N [N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester could reverse this trend. Human AMSCs facilitate the immunosuppressive effect of CsA on T lymphocytes through Jagged-1/Notch-related inhibition of NF-κB signaling. The combination of AMSCs and CsA represents a rationale therapeutic approach aimed to prevent adverse effects of CsA while maintaining its adequate immunosuppressive effect. Expression of Jagged-1 on AMSCs may provide an effective mechanism for the immunomodulatory activity of AMSCs via direct cell-cell interaction.

Drugs Acting on Mast Cells Functions: A Cell Biological Perspective

Mast cells are bone marrow derived cells capable of secreting many active molecules: mediators stored in specific granules, such as histamine and heparin; small molecules produced immediately upon stimulation, such as lipid derivatives and nitric oxide; and many constitutively secreted, pleiotropic cytokines. Thanks to these secretion products and perhaps direct cell-cell interactions, mast cells play roles in inflammation and tissue repair, angiogenesis and fibrosis. Mast cells themselves respond to many mediators of their own, giving rise to autocrine loops. Successful anti-allergic therapies have typically targeted the receptors for mast cell secretory products, particularly those for histamine. Among agents directly affecting mast cells, disodium chromoglycate and glucocorticoids are known since some time, while new pharmacological approaches may stem from the recognition of an interference with mast cell growth and differentiation by cyclosporine A, monoclonal antibodies, interferons, and JAK3 inhibitors. The action of agents that affect mast cell differentiation and function is considered here from a cell and tissue biological perspective as a premise to the application of these agents to the clinics, therefore special attention has been paid to references pertaining to humans.

Regulatory B cells and their biological advances

Regulatory B cells (Bregs) belong to a B-cell subset with immunoregulatory activity, which has been evidenced in mice. The origin and phenotype of the cell subset are controversial. Bregs suppress immune responses mainly by direct cell-cell interactions, secretion of cytokines such as interleukin (IL)-10,transforming growth factor (TGF) -β, etc. It has been demonstrated that Bregs are associated with systemic lupus erythematosus as well as contact hypersensitivity (CHS),and involved in many diseases and biological processes. This review focuses on the identification, origin, function, action mechanism, relationship with diseases, prospect and challenges in researches of Bregs. Key words: B-lyphocytes,regulatory; Biology; Skin diseases

ERK‐1/2 and p38 in the regulation of hypertrophic changes of normal articular cartilage chondrocytes induced by osteoarthritic subchondral osteoblasts

Previous studies have shown the influence of subchondral bone osteoblasts (SBOs) on phenotypical changes of articular cartilage chondrocytes (ACCs) during the development of osteoarthritis (OA). The molecular mechanisms involved during this process remain elusive, in particular, the signal transduction pathways. The aim of this study was to investigate the in vitro effects of OA SBOs on the phenotypical changes in normal ACCs and to unveil the potential involvement of MAPK signaling pathways during this process. Normal and arthritic cartilage and bone samples were collected for isolation of ACCs and SBOs. Direct and indirect coculture models were applied to study chondrocyte hypertrophy under the influence of OA SBOs. MAPKs in the regulation of the cell-cell interactions were monitored by phosphorylated antibodies and relevant inhibitors. OA SBOs led to increased hypertrophic gene expression and matrix calcification in ACCs by means of both direct and indirect cell-cell interactions. In this study, we demonstrated for the first time that OA SBOs suppressed p38 phosphorylation and induced ERK-1/2 signal phosphorylation in cocultured ACCs. The ERK-1/2 pathway inhibitor PD98059 significantly attenuated the hypertrophic changes induced by conditioned medium from OA SBOs, and the p38 inhibitor SB203580 resulted in the up-regulation of hypertrophic genes in ACCs. The findings of this study suggest that the pathologic interaction of OA SBOs and ACCs is mediated via the activation of ERK-1/2 phosphorylation and deactivation of p38 phosphorylation, resulting in hypertrophic differentiation of ACCs.

Abstract 1421: Inhibition of melanoma growth by activation of Notch pathway in stromal fibroblasts

Abstract Background. Tumor microenvironment influences tumor initiation and progression. Fibroblasts are major components of tumor stroma and critically involved in regulating tumor growth and angiogenesis through secretion of soluble factors, synthesis of extracellular matrix and direct cell-cell interaction. The Notch pathway regulates a variety of cellular activities. However, it is unknown whether and how manipulation of Notch signaling in stromal fibroblasts affects melanoma growth. Objectives. This study aimed to investigate the effects of Notch-activated stromal fibroblasts on melanoma growth and elucidate the underlying mechanism. Methods. Enforced activation of Notch pathway was achieved by transducing fibroblasts with lentiviral vector encoding activated Notch1 (NIC). The effect of Notch1-activated stromal fibroblasts on melanoma growth was tested by in vitro co-culture and in vivo co-injection approaches. The soluble factor which produced by Notch1-activated fibroblasts was identified by PCR Array and validated by Western blot analysis. Function of the candidate factor in mediating the inhibitory effect of Notch1-activated stromal fibroblasts on melanoma growth was examined through siRNA-mediated blockade approach and tested by in vitro cell co-culture and in vivo tumor xenografting animal models. Results. Notch1-activated fibroblasts significantly inhibited melanoma growth by inducing tumor cell apoptosis and suppressing tumor angiogenesis. The inhibitory effect of NIC-engineered fibroblasts was mediated by up-regulating WISP-1 expression. Elevated WISP-1 suppressed melanoma growth while blockade of WISP-1 liberated the inhibitory effect. Conclusions. Activation of Notch pathway in stromal fibroblasts can suppress melanoma growth, partially through WISP-1. Our study provides a novel approach to target tumor microenvironment through modulating stromal fibroblasts by manipulation of Notch signaling, and highlights WISP-1 as a potential therapeutic target in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1421.

Mesenchymal Stem Cells Contribute to Tumor Cell Proliferation by Direct Cell-Cell Contact Interactions

ABSTRACTBone marrow (BM)-derived mesenchymal stem cells (MSCs) have been implicated in tumor progression, making MSCs important targets for anti-cancer strategies. In this study, we show that MSCs promote tumor growth in vivo in a lymphoma xenograft model. We show that MSCs provide direct cell–cell contact interactions and, to a lesser extend, soluble factors that promote tumor cell proliferation and survival in vitro. PTK787/ZK 222584 reduces tumor growth-promoting effects of MSCs both in vitro and in vivo. Our results address the importance of targeting the MSCs for future anti-cancer strategies.

Sperm Proteomics Reveals Intensified Selection on Mouse Sperm Membrane and Acrosome Genes

Spermatozoa are a focal point for the impact of sexual selection due to sperm competition and sperm-female interactions in a wide range of sexually reproducing organisms. In-depth molecular investigation of the ramifications of these selective regimes has been limited due to a lack of information concerning the molecular composition of sperm. In this study, we utilize three previously published proteomic data sets in conjunction with our whole mouse sperm proteomic analysis to delineate cellular regions of sperm most impacted by positive selection. Interspecific analysis reveals robust evolutionary acceleration of sperm cell membrane genes (which include genes encoding acrosomal and sperm cell surface proteins) relative to other sperm genes, and evidence for positive selection in approximately 22% of sperm cell membrane components was obtained using maximum likelihood models. The selective forces driving the accelerated evolution of these membrane proteins may occur at a number of locations during sperm development, maturation, and transit through the female reproductive tract where the sperm cell membrane and eventually the acrosome are exposed to the extracellular milieu and available for direct cell-cell interactions.

Intramyocardial Fibroblast Myocyte Communication

Cardiac fibroblasts are emerging as key components of normal cardiac function, as well as the response to stressors and injury. These most numerous cells of the heart interact with myocytes via paracrine mechanisms, alterations in extracellular matrix homeostasis, and direct cell-cell interactions. It is possible that they are a contributor to the inability of adult myocytes to proliferate and may influence cardiac progenitor biology. Furthering our understanding of how cardiac fibroblasts and myocytes interact may provide an avenue to novel treatments for heart failure prevention. This review discusses the most recent concepts in cardiac fibroblast-myocyte communication and areas of potential future research.

Mesenchymal stem cells: Molecular characteristics and clinical applications

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.

Hippo pathway-independent nuclear export of the transcriptional activator YAP regulates proliferation and migration in HCC cells

Aims: The Hippo pathway is a central regulator of organ size in different organisms. Hippo signalling is regulated by direct cell-cell interaction and contact inhibition, leading to phosphorylation and cytoplasmic retention of the transcriptional co-activator YAP (yes-associated protein). Because most human cancer cells become refractory to contact inhibition, it was suggested that this pathway and especially the nuclear accumulation of YAP is involved in liver cancer development.

Direct cell–cell contact between periodontal ligament fibroblasts and osteoclast precursors synergistically increases the expression of genes related to osteoclastogenesis

The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell-cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell-cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co-culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule-1 (ICAM-1) and of osteoclastogenesis-related genes (RANKL, RANK, TNF-alpha, and IL-1beta) was highly up-regulated in the co-cultures compared to mono-cultures and the 5-10-fold up-regulation reflected a synergistic increase due to direct cell-cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)(2)VitD(3) and dexamethasone. In case of indirect cell-cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast-like cells that were formed in co-culture with periodontal ligament fibroblasts was significantly augmented compared to mono-cultures. Our data indicate that cell-cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts.

NK Cell Enhancement of Antigen Presentation by B Lymphocytes

Ag presentation to CD4 T cells can be mediated by a number of cell types depending on the anatomical site in which Ag is first encountered. For blood borne Ags, cells localized in situ in the spleen should be major players. There is now much evidence that B cell Ag presentation may be particularly important in the priming of memory T cells. The majority of NK cells are also localized the spleen. Inasmuch as we have previously shown that NK cells can modulate various aspects of B cell differentiation, we entertained the possibility that NK cells can also influence Ag presentation by B cells. By specific depletion of NK cells before immunization, we show herein that NK cells play an important role in modulating the ability of B cells to process and present Ag to T cells. These effects are particularly important in the generation of memory T cells. The findings are further substantiated by in vitro experiments showing that the enhancement does not require IFN-gamma but is mediated by direct cell-cell interaction. These results show, for the first time, that the rapid activation of a component of the innate response can even exert effects on the Ag-specific memory response.

Molecular and Immunological Effects of Thalidomide in Chronic Lymphocytic Leukemia.

Recently, several novel mechanisms of tumor progression were characterized in chronic lymphocytic leukemia (CLL), stressing an important role of tumor microenvironment. Direct cell-cell interactions as well as soluble factors might support tumor growth. Pro-survival signals might be supported by the tumor necrosis factor (TNF) or some distinctive members of TNF family as well as angiogenic factors. Thalidomide has been shown to be a promising immunomodulatory drug (IMiD) that targets not only leukemic cells but also the tumor microenvironment and by inhibiting angiogenesis. However, so far little is known about the molecular effects of IMiDs in-vivo. Therefore, we investigated cellular and molecular changes induced by a 7 day thalidomide mono-treatment of CLL patients. In forty cases TNF expression levels were assessed using a high sensitivity ELISA test, and T-cell subpopulations were analyzed by flow cytometry. To evaluate the influence of thalidomide on gene expression (GEP), 20 paired pre-treatment and thalidomide-treated samples were analyzed using Affymetrix U133plus2.0 microarrays. Thalidomide therapy was effective in decreasing the number of CLL cells (CD5+CD19+) from 51.75 G/L to 31.7 G/L after treatment. The number of CD3 lymphocytes showed no significant change during thalidomide therapy. No effect on CD4+ as well as CD8+ T cells was observed. Interestingly, we found significant decrease in the number of CD4+CD25hiFOXP3+ T regulatory cells (Tregs) after thalidomide (p=0.05). Thalidomide therapy did not reduce the number of other T-cell subpopulations reported to possess regulatory properties such as CD8+CD28−, CD4+GITR+, CD4+CD62L+ and CD3+TCRγδ+. The changes in Tregs during thalidomide therapy differed in cases who responded to thalidomide with a WBC reduction compared to non-responders. In cases with a WBC reduction there was a greater fold of Tregs reduction. With regard to TNF, we observed no significant changes in the TNF plasma levels after thalidomide treatment. However, the expression of TNF R1 was significantly higher in patients without WBC reduction following thalidomide (p=0.03). Gene expression profiling revealed a thalidomide-induced signature comprising 123 differentially expressed genes (paired t-test with random variance model, p<0.001). Upon thalidomide treatment we observed an up-regulation of genes known to be involved in mediating thalidomide response, such as FAS and CDKN1A. In addition, we detected novel candidate genes, such as STAT1 and IKZF1. By gene set enrichment analysis investigating 271 BioCarta pathways we found 22 pathways to be significantly deregulated (random variance model, p<0.005). These included pathways involved in apoptosis, angiogenesis, cytokine, PDGF and p38 MAPK signaling. Cases with a WBC reduction following thalidomide administration were characterized by a lower expression of pro-survival cytokines such as IL8 and lower levels of TGFB1, whereas genes involved in apoptosis like e.g. CASP1 were more highly expressed than in non-responders. On the other hand, non-responders showed higher ZAP70 expression as determined by microarray analysis and higher expression levels of anti-apoptotic genes, such as TRAF1, and genes involved in angiogenesis, such as ECGF1. However, only thalidomide responders showed also a correlation with CD40L signaling. We were also able to define a thalidomide response signature. For example, pretreatment samples derived from cases responding to thalidomide with a WBC reduction differed from non-responders with regard to the expression of JUN and CASP9. In conclusion, our study provides novel biological insights into the molecular effects of thalidomide, which might act (i) by enhancing apoptosis of CLL cells and (ii) by reducing Tregs, thereby enabling T-cell dependent tumor rejection.

Chronic Lymphocytic Leukemia (CLL) Cells Require Microenvironmental Stimuli to Resist Apoptosis through Activation of STAT3 Mediated by VEGF.

B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, but incompetent B-cells due to a decrease of apoptosis rather than an increase in proliferation. Vascular endothelial growth factor (VEGF) has been suggested to play an important role in this so called apoptotic block. However, so far little is understood whether VEGF is acting mainly as a microenvironmental stimulus and/or whether CLL cells themselves contribute to the enhanced apoptotic resistance by maintaining an autocrine VEGF loop. Moreover, it is unknown by which mechanisms VEGF prevents apoptosis and whether this can be circumvented by inhibition of VEGF signaling. By quantitative real time PCR we found no significant difference in mRNA VEGF levels in B-cells from CLL patients and healthy donors after isolation from blood. In contrast, ELISA revealed clearly increased levels of secreted VEGF in plasma of CLL patients and in the supernatant under culture conditions compared to healthy individuals. In addition, we found the VEGF receptor 2 (VEGFR2), which is existent in CLL and healthy B-cells, in a phosphorylated, hence activated state, to a significantly higher extent in CLL cells as assessed by intracellular phospho flow cytometry. In conclusion, despite its expression in healthy B-cells VEGF does not seem to be secreted and therefore, no VEGF receptor phosphorylation takes place. Whereas CLL cells exhibit a long life span in vivo, they die rapidly in vitro, suggesting major survival factors being existent in the CLL cells microenvironment. We found levels of secreted VEGF in supernatant decreasing with time in culture, going along with decreasing levels of phosphorylated VEGFR2 and increasing cell death as assessed by Annexin V-FITC/PI staining. This further supports the role of VEGF in CLL cell survival. Coculturing primary CLL cells with the bone marrow stromal derived cell line HS5 dramatically increased VEGF transcription and secretion and improved cell survival. Hence, VEGF expression in CLL cells is not only mediated by autocrine, but also paracrine stimuli involving bone marrow stromal. Knocking down VEGF in HS5 cells and subsequent coculture with CLL cells might prove the major role of VEGF in this survival supporting coculture setting. Besides coculturing also supplement of culture medium with recombinant human VEGF (rhVEGF) increased survival, but to a lesser extent than coculture, indicating a direct cell-cell interaction as advantageous. Furthermore, we found a downregulation of anti apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia 1 (MCL1) and BclXL upon VEGF stimulation. Also cyclinD1 was upregulated as seen by immunoblotting. We further tried to discover the underlying mechanism of how VEGF mediates its pro survival effect and found STAT3 to become phosphorylated on tyrosine 705 upon VEGF stimulation. In CLL STAT3 is known to be constitutively phosphorylated on serine 727. This phosphorylation is not sufficient to induce target gene expression though. We could show that Y705 phosphorylation of STAT3 is responsible for upregulation of anti apoptotic BCLXL and cyclinD1. A PCR array detecting mRNA levels of 84 transcription factors in untreated and VEGF stimulated CLL cells shall provide more information about mechanistical details how VEGF mediates it pro survival effect. Since VEGF seems to be a major player in CLL cell survival it might be a suitable target to overcome the apoptotic block. In first experiments we found an induction of apoptosis after neutralization of VEGF or inhibition of the VEGF receptor. This additionally highlights the severe importance of VEGF in the apoptotic block in CLL cells. Therefore, VEGF might serve as an excellent therapeutic target in CLL.

Viability in Late Stages of Ex Vivo Erythropoiesis Is Enhanced by Increased Cell Density

Erythropoiesis is one of the body's most productive cell production processes yielding 2×1011 new red cells from hematopoietic stem cells (HSCs) of the bone marrow every day. Intensive research has focused on mimicking this process ex vivo through application of various growth factor combinations or co-culture with stromal cells. To develop a scalable and reproducible system for large scale production of red blood cells we have investigated in vitro erythropoiesis of peripheral blood derived CD34+ cells with primary focus on the impact of the microenvironment on the process. The influence of cultivation conditions on expansion of erythroid progenitor cells and their terminal differentiation to mature red blood cells were studied in stroma-free liquid culture supplemented with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO). Peripheral blood derived CD34+ cells were expanded by more than 105 fold over a 3 week period. This degree of expansion has only been achieved previously for CD34+ cells derived from more potent stem cell sources such as cord blood, bone marrow and G-CSF mobilized peripheral blood (Giarratana et al, Nat Biotechnol 2005). The natural environment of human erythropoiesis, the bone marrow, is a very crowded milieu where hematopoietic precursors and other cells are packed in close proximity. Cell crowdedness was found to have significant influences on ex vivo erythropoiesis. Cell density per surface area rather than cell concentration per media volume determined cell expansion during exponential growth where more crowded cells showed reduced overall expansion. In cultures inoculated at 4×105 cells/ml (2.1×105 cells/cm2) increasing cell density per area (i.e. decreasing surface area to volume ratio) 4fold (to 8.4×105 cells/cm2) resulted in 35±12% reduction of total expansion (p<0.05, unpaired Student's t-test). While 4fold increase of cell density in cultures seeded at 1×106 cells/ml (from 5.3×105 cells/cm2 to 2.1×106 cells/cm2) reduced overall expansion by 51±9% (p<0.01). In late stage erythropoiesis, however, when cells had become arrested in G1 and no longer proliferated, cell density was seen to enhance cell viability. Dilution series of late stage erythroblasts showed that although cell viability gradually decreased over a 14 day cultivation period the decreasing rate was lower in cells cultivated at higher density as shown in the Figure. Enhanced viability in crowded culture conditions could reflect the cells' dependency on direct cell-cell interactions as found in the marrow environment. Cultures grown to high cell densities of 2–3×106 cells/cm2 showed higher maturation efficiency than previously obtained in this cultivation set-up with more than 80% of cells being CD71-/GpA+. Enucleation yields of up to 45% were achieved indicating a significant amount of terminal maturation to red blood cells. Efficient maturation and particularly enucleation have in many cases been found to be dependent on or improved by interactions with feeder cells or macrophages (Fujimi et al, Int J Hematol 2008). Keeping erythroid cells at high densities during late stages of erythropoiesis possibly helps to mimic their in vivo environment, thus allowing for better survival and efficient terminal maturation without the need for co-culture with other cells. [Display omitted]