Contact-mediated Interactions Research Articles

Contact-mediated signaling enables disorder-driven transitions in cellular assemblies.

We show that, when cells communicate by contact-mediated interactions, heterogeneity in cell shapes and sizes leads to qualitatively distinct collective behavior in the tissue. For intercellular coupling that implements lateral inhibition, such disorder-driven transitions can substantially alter the asymptotic pattern of differentiated cells by modulating their fate choice through changes in the neighborhood geometry. In addition, when contact-induced signals influence inherent cellular oscillations, disorder leads to the emergence of functionally relevant partially-ordered dynamical states.

Flags, landscapes and signaling: contact-mediated inter-cellular interactions enable plasticity in fate determination driven by positional information

Multicellular organisms exhibit a high degree of structural organization with specific cell types always occurring in characteristic locations. The conventional framework for describing the emergence of such consistent spatial patterns is provided by Wolpert's "French flag" paradigm. According to this view, intra-cellular genetic regulatory mechanisms use positional information provided by morphogen concentration gradients to differentially express distinct fates, resulting in a characteristic pattern of differentiated cells. However, recent experiments have shown that suppression of inter-cellular interactions can alter these spatial patterns, suggesting that cell fates are not exclusively determined by the regulation of gene expression by local morphogen concentration. Using an explicit model where adjacent cells communicate by Notch signaling, we provide a mechanistic description of how contact-mediated interactions allow information from the cellular environment to be incorporated into cell fate decisions. Viewing cellular differentiation in terms of trajectories along an epigenetic landscape (as first enunciated by Waddington), our results suggest that the contours of the landscape are moulded differently in a cell position-dependent manner, not only by the global signal provided by the morphogen but also by the local environment via cell-cell interactions. We show that our results are robust with respect to different choices of coupling between the inter-cellular signaling apparatus and the intra-cellular gene regulatory dynamics. Indeed, we show that the broad features can be observed even in abstract spin models. Our work reconciles interaction-mediated self-organized pattern formation with boundary-organized mechanisms involving signals that break symmetry.

Abstract 3983: Direct contact of heterogeneous fibroblasts with cancer cells regulates the invasive phenotype of prostate cancer cells

Abstract In the tumor microenvironment of prostate cancer (PCa), cancer cells frequently invade the surrounding stroma in the way of single cell migration (SCM) or collective cell migration (CCM). This phenomenon can be responsible for the heterogeneous formation of structural atypia such as Gleason pattern. Generally, PCa cells undergo a phenotypic change which is called the epithelial-to-mesenchymal transition (EMT), e.g., downregulation of epithelial cell-cell adhesion molecules such as E-cadherin and gain of mesenchymal morphology and motility. The surrounding stroma is mainly composed of fibroblasts and extracellular matrix (ECM). We hypothesize that direct contact-mediated interactions at the interface between PCa cells and fibroblasts/ECM can trigger numerous changes in PCa cells including morphology and motility. In this study, we investigated the roles of fibroblasts in the three-dimensional (3D) morphogenesis of PCa cells on a viscous substrate. To examine the effects of fibroblasts on the 3D morphogenesis of PCa cells on Matrigel, we mixed human PCa cell line LNCaP cells with commercially available normal human prostate stromal cells (PrSC) or nine primary cultures of prostate fibroblasts (pcPrFs and NPFs) isolated from patients with PCa and bladder cancer. The 3D morphogenesis of LNCaP cells mixed with fibroblasts on Matrigel was confirmed by E-cadherin immunohistochemical staining. All ten fibroblasts alone were aggregated on Matrigel, whereas LNCaP cells alone were not. LNCaP cells mixed with six fibroblasts (PrSC, pcPrF-M5, pcPrF-M23, pcPrF-M24, pcPrF-M28, and pcPrF-M31) formed a deep crater-like hole on Matrigel. In the 3D structure, the upper and lower tiers were composed of LNCaP cells and fibroblasts, respectively. Of note, direct contact of four fibroblasts (NPF-M13, pcPrF-M7, pcPrF-M10, and pcPrF-M26) with LNCaP cells decreased the aggregation of fibroblasts on Matrigel. Five of six fibroblasts (PrSC, pcPrF-M5, pcPrF-M23, pcPrF-M28, and pcPrF-M31) induced SCM of LNCaP cells, while only one fibroblasts (pcPrF-M24) induced CCM. In the scratch assay, the cell motility of CCM-inducing fibroblasts was slower than that of SCM-inducing fibroblasts. Expression pattern of primary cilia in SCM-inducing fibroblasts was changed by mixing with LNCaP cells, whereas that in CCM-inducing fibroblasts was not. PCa is quite interesting because of the multi-focal and heterologous progression of primary tumors. The heterogeneous stromal compartment in PCa tissues contains multiple populations of fibroblasts that are associated with morphology and motility of PCa cells. Here our results showed that direct contact of heterogeneous fibroblasts with PCa cells on Matrigel regulated the invasive phenotype of PCa cells such as SCM and CCM. Impairing the malignant phenotype of fibroblasts in PCa tissues, e.g., differentiation therapy targeting tumor stroma, may abrogate PCa cell invasion. Citation Format: Kenichiro Ishii, Yasuhisa Nakagawa, Taku Shirai, Masako Ichishi, Masaya Fujiwara, Daisuke Kato, Chise Matsuda, Yoshifumi Hirokawa, Yoshiki Sugimura, Masaki Inagaki, Masatoshi Watanabe. Direct contact of heterogeneous fibroblasts with cancer cells regulates the invasive phenotype of prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3983.

Saccharomyces cerevisiae and Hanseniaspora uvarum mixed starter cultures: Influence of microbial/physical interactions on wine characteristics.

The growing trend in the wine industry is the revaluation of the role of non-Saccharomyces yeasts, promoting the use of these yeasts in association with Saccharomyces cerevisiae. Non-Saccharomyces yeasts contribute to improve wine complexity and organoleptic composition. However, the use of mixed starters needs to better understand the effect of the interaction between these species during alcoholic fermentation. The aim of this study is to evaluate the influence of mixed starter cultures, composed by combination of different S. cerevisiae and Hanseniaspora uvarum strains, on wine characteristics and to investigate the role of cell-to-cell contact on the metabolites produced during alcoholic fermentation. In the first step, three H. uvarum and two S. cerevisiae strains, previously selected, were tested during mixed fermentations in natural red grape must in order to evaluate yeast population dynamics during inoculated fermentation and influence of mixed starter cultures on wine quality. One selected mixed starter was tested in a double-compartment fermentor in order to compare mixed inoculations of S. cerevisiae/H. uvarum with and without physical separation. Our results revealed that physical contact between S. cerevisiae and H. uvarum affected the viability of H. uvarum strain, influencing also the metabolic behaviour of the strains. Although different researches are available on the role of cell-to-cell contact-mediated interactions on cell viability of the strains included in the mixed starter, to our knowledge, very few studies have evaluated the influence of cell-to-cell contact on the chemical characteristics of wine.

Self-Propelled Rods: Insights and Perspectives for Active Matter

A wide range of experimental systems including gliding, swarming and swimming bacteria, in vitro motility assays, and shaken granular media are commonly described as self-propelled rods. Large ensembles of those entities display a large variety of self-organized, collective phenomena, including the formation of moving polar clusters, polar and nematic dynamic bands, mobility-induced phase separation, topological defects, and mesoscale turbulence, among others. Here, we give a brief survey of experimental observations and review the theoretical description of self-propelled rods. Our focus is on the emergent pattern formation of ensembles of dry self-propelled rods governed by short-ranged, contact mediated interactions and their wet counterparts that are also subject to long-ranged hydrodynamic flows. Altogether, self-propelled rods provide an overarching theme covering many aspects of active matter containing well-explored limiting cases. Their collective behavior not only bridges the well-studied regimes of polar self-propelled particles and active nematics, and includes active phase separation, but also reveals a rich variety of new patterns.

A Fateful Meeting of Two Phytoplankton Species-Chemical vs. Cell-Cell-Interactions in Co-Cultures of the Green Algae Oocystis marsonii and the Cyanobacterium Microcystis aeruginosa.

Massive growth of single species of cyanobacteria is a common phenomenon in many eutrophicated waters worldwide. Allelopathic growth control of phytoplankton species is one suggested mechanism, but still controversially discussed. The fact that the synthesis of biological active compounds requires high energy costs and carbon investment for a single cell in contrast to high dilution rates in natural systems questions the universal validity of allelopathic mechanisms, even more as high concentrations of allelopathic substances are often needed in several experiments to cause biological effects. In this study, it was tested, if growth inhibition is induced by chemical signaling alone or via direct cell-cell interaction. As a test system, we used a co-culture of the green algae Oocystis marsonii (Trebouxiophyceae) with the cyanobacterium Microcystis aeruginosa which is known to strongly reduce the growth of the green algal competitor. In this study, direct co-culturing as well as membrane-separated growth chambers were used to test for chemical and contact-mediated interactions. In the membrane-separated chambers, both species can be co-cultivated and a membrane allows the exchange of metabolites. Growth of O. marsonii was only affected in the direct co-cultivation situation, where direct cell-to-cell contact was possible. During direct co-cultivation, deviating cellular traits, namely cell cycle pattern and large cell-aggregate formation of both species, could be detected. These data strongly support the hypothesis of a direct cell-cell-contact necessary for allelopathic growth control in this model system. Such direct contact would allow targeting allelopathic metabolites directly towards the competitor and thereby minimizing dilution effects.

Eph-ephrin signaling in nervous system development.

Ephrins and Eph receptors enable contact-mediated interactions between cells at every stage of nervous system development. In spite of their broad binding affinities, Eph proteins facilitate specificity in neuronal migration and axon targeting. This review focuses on recent studies that demonstrate how these proteins interact with each other, and with other signaling pathways, to guide specificity in a diverse set of developmental processes.

Short-range guiding can result in the formation of circular aggregates in myxobacteria populations.

Myxobacteria are social bacteria that upon starvation form multicellular fruiting bodies whose shape in different species can range from simple mounds to elaborate tree-like structures. The formation of fruiting bodies is a result of collective cell movement on a solid surface. In the course of development, groups of flexible rod-shaped cells form streams and move in circular or spiral patterns to form aggregation centers that can become sites of fruiting body formation. The mechanisms of such cell movement patterns are not well understood. It has been suggested that myxobacterial development depends on short-range contact-mediated interactions between individual cells, i.e. cell aggregation does not require long-range signaling in the population. In this study, by means of a computational mass-spring model, we investigate what types of short-range interactions between cells can result in the formation of streams and circular aggregates during myxobacterial development. We consider short-range head-to-tail guiding between individual cells, whereby movement direction of the head of one cell is affected by the nearby presence of the tail of another cell. We demonstrate that stable streams and circular aggregates can arise only when the trailing cell, in addition to being steered by the tail of the leading cell, is able to speed up to catch up with it. It is suggested that necessary head-to-tail interactions between cells can arise from physical adhesion, response to a diffusible substance or slime extruded by cells, or pulling by motility engine pili. Finally, we consider a case of long-range guiding between cells and show that circular aggregates are able to form without cells increasing speed. These findings present a possibility to discriminate between short-range and long-range guiding mechanisms in myxobacteria by experimentally measuring distribution of cell speeds in circular aggregates.

The ubiquitous neural cell adhesion molecule (N-CAM).

Adhesive interactions are important for cell trafficking, differentiation, function and tissue differentiation. Neural cell adhesion molecule (NCAM) is involved in a diverse range of contact-mediated interactions among neurons, astrocytes, oligodendrocytes, and myotubes. It is widely but transiently expressed in many tissues early in embryogenesis. Four main isoforms exist but there are many other variants resulting from alternative splicing and post-translational modifications. This review discusses the actions and association of N-CAM and variants, PSA CAM. L1CAM and receptor tyrosine kinase. Their interactions with the interstitial cells of Cajal – the pacemaker cells of the gut in the manifestation of gut motility disorders, expression in carcinomas and mesenchymal tumours are discussed.

SP0057 Stromal cell: Leukocyte interactions in early arthritis

Tissue fibroblasts are not passive players in the initiation and resolution of inflammation: They act as innate immune sentinels, and engage in critical soluble and contact mediated interactions with both...

2B4-mediated homotypic interactions among activating NK cells enhances activation and proliferation of NK cells via clustering and polarization of IL-2 receptors (P1101)

Abstract Clustering of activating lymphocytes has been frequently observed during early stage of immune responses, but the importance of such multi-cellular clusters has not been systematically investigated due to the lack of tools. To address it, we fabricated cell-laden microwells that allowed us to precisely control multi-cellular interactionss among activating lymphocytes. The role of contact-mediated homotypic interactions between natural killer (NK) cells during IL-2 activation was examined using the cell-laden microwells. To assess the role of NK-NK homotypic interactions on the activation of NK cells, NK cells cultured in two different types of microwells, social microwells that allow contact-mediated multicellular interactions and lonesome microwells that inhibit cell-cell contact, were compared. Activation of NK cells by IL-2 was assessed by measuring phosphorylation of STAT-5, expression of CD25 and Ki-67. Immunofluorescence microscopy was performed to assess the levels of pSTAT-5, CD25, and Ki-67, and percentages of stained cells for each marker were examined and plotted. NK cells in social microwells expressed higher levels of all three markers than those in lonesome microwells, suggesting that homotypic contact-mediated interactions can enhance the activation of NK cells. Further analysis revealed that 2B4 is a key receptor mediating contact-mediated augmentation of NK cells in social miceowells by clustering and polarizing IL-2 receptor chains.

Mesenchymal Stem Cell-Cardiomyocyte Interactions under Defined Contact Modes on Laser-Patterned Biochips

Understanding how stem cells interact with cardiomyocytes is crucial for cell-based therapies to restore the cardiomyocyte loss that occurs during myocardial infarction and other cardiac diseases. It has been thought that functional myocardial repair and regeneration could be regulated by stem cell-cardiomyocyte contact. However, because various contact modes (junction formation, cell fusion, partial cell fusion, and tunneling nanotube formation) occur randomly in a conventional coculture system, the particular regulation corresponding to a specific contact mode could not be analyzed. In this study, we used laser-patterned biochips to define cell-cell contact modes for systematic study of contact-mediated cellular interactions at the single-cell level. The results showed that the biochip design allows defined stem cell-cardiomyocyte contact-mode formation, which can be used to determine specific cellular interactions, including electrical coupling, mechanical coupling, and mitochondria transfer. The biochips will help us gain knowledge of contact-mediated interactions between stem cells and cardiomyocytes, which are fundamental for formulating a strategy to achieve stem cell-based cardiac tissue regeneration.

Novel Pharmacological Agents Differentially Modulate Cytokine Release On CLL B-Cell-Stromal Cell Co-Culture: Implications for Stromal Rescue of CLL B-Cells From Chemotherapy

AbstractAbstract 3927 PURPOSE:CLL B-cells interact with their microenvironment in a variety of ways that influence leukemia cell survival. Contact mediated interactions are the most widely studied. More recently, bidirectional cytokine mediated interactions have been found to modulate spontaneous and drug induced CLL B-cell death. Specifically, upregulation of CCL2, CCL3, and CCL4 are found when CLL B-cells are co-cultured with various accessory cells. We set out to more comprehensively profile the effect of CLL-stromal cell co-culture on immune cytokine regulation and to evaluate the effect of novel therapeutic agents in clinical testing for patients with CLL on these interactions. METHODS:Freshly isolated CLL B-cells (n=24) were cultured in AIM V media for 24 hours alone or in direct contact with HS-5 stromal cells. HS-5 cells were cultured alone in AIM V to determine basal level of immune cytokine secretion. Immune cytokine levels in the media under these 3 conditions (CLL alone; HS5 alone; co-culture) were assessed using multiplex cytokine arrays (Invitrogen). The protective effects of the 6 most highly upregulated cytokines on spontaneous apoptosis of CLL B-cells after 7 day culture were then examined by culturing leukemic cells with and without recombinant cytokines. The effect of sub-lethal doses of the PI3K inhibitor CAL-101, the AKT inhibitor MK2206 and the Bruton's Tyrosine Kinase inhibitor PCI-32765 were examined. To compare the effect of these drugs to a more conventional chemotherapeutic agent, we evaluated fludarabine (F-Ara-A) under the same conditions. To explore the ability of these novel agents to overcome stromal rescue, CLL B-cells pre-exposed to bendamustine (BEN) were cultured in media alone, or in direct contact with stroma with and without sub-lethal doses of CAL-101, MK2206, or PCI-32756. Leukemic cell viability was assessed by flow cytometry using annexin/PI staining. RESULTS:Six cytokines increased ≥4 fold (e.g. >400%, all p<0.0001) on coculture relative to the expected additive value of individual cultures. Consistent with previous reports, (Schulz 2011; Burger 2009) CCL3 and CCL4 were found to have the greatest upregulation (both >20 fold the additive value). The other highly upregulated cytokines detected have not been previously reported to be influenced by coculture: IL12 (15 fold additive value), IL1β (11 fold additive value), TNF-α (11 fold additive value), and IL-1RA (4 fold additive value). Although transwell experiments (n=6) suggested upregulation of these cytokines was largely contact dependent, substantial upregulation of several cytokines still occurred without direct contact. Each of the 6 most upregulated cytokines reduced spontaneous apoptosis when CLL B-cells were cultured for 7 days in media plus recombinant cytokine.When CLL B-cells and HS-5 cells were co-cultured with CAL-101, MK-2206, or PCI-32765, the effects on cytokine secreted upregulation were cytokine and drug specific (e.g. one drug affected individual cytokines differently; different drugs affected different cytokines). For example, PCI-3276 and CAL-101 had similar affects on the upregulation of CCL3 and CCL4 on co-culture but opposite effects on TNF-α and IL-1β. In contrast, F-Ara-A had minimal effect on upregulation of any of these 6 cytokines.Finally, we evaluated the ability of these novel agents to overcome stromal rescue of CLL B-cells treated with conventional chemotherapeutic agents. At sublethal doses CAL-101, MK2206, PCI-32765 each significantly overcame stromal rescue of BEN exposed CLL B-cells. (Fig.1) CONCLUSIONS:Direct physical interaction of CLL B-cells with bone marrow stromal cells resulted in profound upregulation of multiple immune cytokines. The 6 most upregulated cytokines identified in these experiments all reduced spontaneous apoptosis of CLL B-cells on extended culture. Sublethal doses of the PI3K inhibitor CAL-101, the AKT inhibitor MK2206, and the Bruton's Tyrosine Kinase inhibitor PCI-32765 had distinct effects on stromal cell mediated cytokine upregulation. Importantly, each of these 3 drugs also overcame stromal mediated rescue of BEN exposed CLL B-cells. These in vitro findings support the ongoing clinical trials of BEN in combination with CAL-101 (ASH 2011), PCI-32765 (ASCO 2012) or MK2206 (Ding et. al., unpublished observation). [Display omitted] Disclosures:Kay:Celgene: Research Funding. Shanafelt:Celgene: Research Funding; Hospira: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Polyphenon E International: Research Funding.

IFN type I and type II independent enhancement of B cell TLR7 expression by natural killer cells

The PRR TLR7 plays a key role in the activation of autoantigen-reactive B cells. This response is increased markedly by IFN-α, produced by accessory cells, as a result of the up-regulation of TLR7. We report herein an alternative pathway by which TLR7 expression can be augmented. This finding was derived from continuation of ongoing studies to uncover interactions between NK and B cells. Here, we have compared gene expression profiles by microarray analysis of B cells before and after their interaction with purified NK cells. The most outstanding alteration of genes transcribed in B cells is a significant increase in the expression of many members of the ISG family, among which is TLR7. Further analysis revealed that the enhancement of TLR7 on B cells is not mediated via type I or type II IFN but by another cytokine, IL-28, a type III IFN, which acts in concert with contact-mediated interactions with NK cells. This increased expression allows B cells to respond more readily upon stimulation by its ligand and may increase in vivo responses to other TLR7 ligands, such as autoantigens, prior to or jointly with stimulation by other cytokines.

Cell-laden microwells for the study of multicellularity in lymphocyte fate decisions

Cell–cell cooperativity in populations of motile and transiently interacting immune cells has been difficult to assess in the absence of tools to control proximity and communication. Here, we describe the generation of cell-laden microwells that can precisely control contact-mediated interactions and multicellular ‘quorum’ decisions in lymphocytes. Different types of fate decisions for activating T cells can be shown to variously obey ‘binary’ or ‘density’ outcomes, correlated with cell–cell contact, using this new platform.

N-Cadherin-Mediated Microenvironmental Interactions Protect CML Stem Cells from Imatinib Mediated Apoptosis.

The therapeutic success of Imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by incomplete elimination of malignant hematopoietic stem cells (HSC). The bone marrow microenvironment supports the self-renewal, proliferation and survival of HSC. We investigated whether microenvironmental interactions protected CML HSPC from elimination by IM. CML CD34+CD38− primitive and CD34+CD38+ committed progenitors were cultured for 96 hours with IM (5μM) and with or without irradiated hTERT-immortalized primary human marrow stromal cells. Stroma coculture effectively protected CML progenitors from IM-induced apoptosis (20±6% apoptosis of CD38− cells without stroma and 4±1% apoptosis with stroma, p=0.04, n=4; 13±4% apoptosis of CD38+ cells without stroma and 7±1% apoptosis with stroma, p=0.04, n=4). Stroma coculture did not affect IM-mediated inhibition of CML progenitor proliferation. CML CD34+ cells cultured with and without stroma were injected into irradiated NOD-SCID-IL2Rγ- chain deleted (NSG) mice. Prior coculture with stroma prior to injection resulted in significantly increased engraftment of CML cells in the BM and spleen of NSG mice both at 4 weeks (short-term repopulation) and 10 weeks (long-term repopulation) after transplantation, both with and without IM treatment (without IM, 53±9% human cells in BM after 4 weeks with stroma and 27±5% without stroma, p=0.02, n=4; with IM, 13±2% with stroma and 5±2% without stroma, p=0.01, n=4). Q-PCR analysis showed that stroma coculture was associated with increased engraftment of BCR-ABL+ cells both with and without IM treatment. Culture of CML CD34+ cells in Transwell inserts (0.45μm pores) over stromal layers resulted in partial reversal of protective effect of stroma, suggesting that direct stromal contact contributed to protection of CML progenitors from IM-induced apoptosis (13±2% apoptosis with stroma, 20±5% with Transwell, and 30±3% without stroma). N-cadherin, a cell-cell adhesion protein expressed on HSPC and stromal cells, may contribute to drug resistance in Ph+ lymphoblastic leukemia. Blocking antibodies (Ab) to N-cadherin partially reversed the protective effect of stroma on CML cells treated with IM (33±2% apoptosis with anti-N-cadherin Ab, 26±4% without Ab, p=0.01, n=3). Similarly an N-cadherin-blocking cyclical peptide enhanced apoptosis of IM-treated CML progenitors cultured on stroma (31±4% apoptosis with N-cadherin peptide, 26±3% with control peptide, p=0.03, n=3). CML progenitors cultured on N-cadherin coated plates showed modestly reduced apoptosis compared with controls indicating that N-Cadherin may also directly transduce anti-apoptotic signals (24±4% apoptosis with N-cadherin, 28±3% with BSA, p=0.001, n=4). A minority of CML progenitors expressed N-cadherin on the cell surface. Expression was higher in CD38− cells (17%) compared with CD38+ cells (6%). The percentage of cell surface N-cadherin +ve cells increased after IM treatment in the presence of stroma but not after culture with stroma or IM alone (no stroma no IM 6.5%, stroma alone 6.8%, IM alone 9.9%, stroma and IM both17.8%). We show that purified surface N-cadherin +ve CML CD34+ cells are capable of engrafting NSG mice (0.9±0.4% human cells for N-cadherin +ve and 0.6±0.2% for N-cadherin -ve cells, p=0.12, n=4). Interestingly, surface N-cadherin -ve cells also expressed N-cadherin RNA on Q-PCR, and antibody labeling after fixation and permeablization indicated that a large proportion of these cells expressed intracellular N-cadherin protein, suggesting that they could potentially express surface N-cadherin. In conclusion, our studies indicate that the marrow microenvironment enhances CML HSPC preservation and protects CML HSC from pro-apoptotic effects of IM treatment through direct cell-cell contact-mediated interactions mediated at least in part through N-cadherin. Measures aimed at interfering with N-cadherin mediated microenvironmental interactions could enhance eradication of residual malignant HSC in CML patients receiving IM treatment.

Protection of CML Progenitors from Bcr-Abl Tyrosine Kinase Inhibitor Mediated Apoptosis by the Bone Marrow Stromal Microenvironment.

The therapeutic success of imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by persistence of malignant hematopoietic stem and progenitor cells (HSPC). The bone marrow microenvironment regulates the self-renewal, proliferation and differentiation of HSPC. We investigated the role of microenvironmental interactions in resistance of CML HSPC to elimination by BCR-ABL tyrosine kinase inhibitors (TKI). CML CD34+CD38− primitive progenitor cells and CD34+CD38+ committed progenitor cells were cultured for 96 hours with IM (5μM), nilotinib (5μM) and dasatinib(150nM), in medium supplemented with low concentrations of growth factors, with and without irradiated primary human marrow stromal cells (immortalized by ectopic telomerase expression) followed by an assessment of apoptosis and proliferation. Culture with stroma did not result in significant alteration of apoptosis in the absence of TKI treatment (3.1±0.7% apoptosis for primitive progenitors with stroma and 2.7±0.9% without stroma, 3.7±0.2% for committed progenitors with stroma and 4.7±2.1% without stroma). Coculture with stroma completely protected CML primitive and committed progenitors from TKI-induced apoptosis. CML CD34+CD38− cells demonstrated 20±6% apoptosis following culture with IM in the absence of stroma, but only 3.8±1% apoptosis in the presence of stroma (p=0.04, n=4). Similarly, apoptosis with nilotinib decreased from 12.5±1.8% without stroma to 2.9±0.3% with stroma (p=0.033), and apoptosis with dasatinib decreased from 7.1±0.04% without stroma to 2.7±0.2% with stroma (p=0.001). Apoptosis of CML CD34+CD38+ cells also significantly decreased following TKI treatment with 12.9±4.0%, 10.6±3.2%, 8.4±2.3% apoptosis observed after IM, nilotinib and dasatinib treatment respectively without stroma and 7.1±1.2%, 4.8±1.0%, 3.7±0.4% with stroma, (p=0.04, p=0.03 and p=0.02 respectively, n=4). Culture with stroma resulted in mild reduction in CML progenitor proliferation in the absence of TKI treatment, but TKI treatment resulted in similar degrees of inhibition of proliferation regardless of the presence of stroma. Culture of CML CD34+ cells in a Transwell insert with 0.45μm pores, allowing free diffusion of stromal factors but preventing direct contact with stroma, was associated with reduction in the protective effect of stroma coculture (32.2% apoptosis without stroma, 14.7% with stroma, and 24.6% with Transwell insert). Addition of blocking antibodies to a4 integrin and N-cadherin did not affect survival of CML CD34+ cells in the absence of IM, but resulted in enhanced apoptosis of CML CD34+ cells cocultured with stroma after addition of IM (20.4% apoptosis without antibody, 28.9% with anti-N-cadherin, and 29.8% with anti-integrin antibody). We conclude that the bone marrow stromal microenvironment protects CML primitive and committed progenitors from pro-apoptotic effects of BCR-ABL TKI treatment. Direct contact-mediated interactions, likely through VLA-4 and N-Cadherin, play an important role in protecting CML CD34+ cells from TKI-mediated apoptosis. These observations indicate that measures aimed at interfering with the protective effects of stroma could be of benefit for the eradication of residual malignant progenitors in CML patients receiving BCR-ABL TKI treatment.

Enhanced cell-to-cell contacts between activated microglia and pyramidal cell dendrites following kainic acid-induced neurotoxicity in the hippocampus

Microglia participate in immune responses in the brain. However, little is known about the contact-mediated interaction between microglia and neurons. We report here that the cell-to-cell contacts between microglial processes and dendrites of hippocampal CA1 neurons were dramatically increased in density and area following local injection of kainic acid (KA). A similar KA-induced increase in the degree of intercellular contacts was observed in mice lacking telencephalin (TLCN), a neuronal dendritic adhesion molecule of ICAM family. The results suggest that adhesive contacts independent of TLCN and contact-mediated interactions between microglia and dendrites were promoted by excitotoxic brain injury.

An early CD4+ T cell-dependent immunoglobulin A response to influenza infection in the absence of key cognate T-B interactions.

Contact-mediated interactions between CD4+ T cells and B cells are considered crucial for T cell–dependent B cell responses. To investigate the ability of activated CD4+ T cells to drive in vivo B cell responses in the absence of key cognate T–B interactions, we constructed radiation bone marrow chimeras in which CD4+ T cells would be activated by wild-type (WT) dendritic cells, but would interact with B cells that lacked expression of either major histocompatibility complex class II (MHC II) or CD40. B cell responses were assessed after influenza virus infection of the respiratory tract, which elicits a vigorous, CD4+ T cell–dependent antibody response in WT mice. The influenza-specific antibody response was strongly reduced in MHC II knockout and CD40 knockout mice. MHC II–deficient and CD40-deficient B cells in the chimera environment also produced little virus-specific immunoglobulin (Ig)M and IgG, but generated a strong virus-specific IgA response with virus-neutralizing activity. The IgA response was entirely influenza specific, in contrast to the IgG2a response, which had a substantial nonvirus-specific component. Our study demonstrates a CD4+ T cell–dependent, antiviral IgA response that is generated in the absence of B cell signaling via MHC II or CD40, and is restricted exclusively to virus-specific B cells.

Expression of a novel factor, short-type PB-cadherin, in Sertoli cells and spermatogenic stem cells of the neonatal rat testis.

In the rodent testis, contact-mediated interactions between gonocytes, or neonatal stem cells, and Sertoli cells are critical for development. Previously, we showed that the neural cell adhesion molecule (NCAM) serves as a Sertoli cell-gonocyte attachment factor in neonates. Its expression decreases dramatically by 1 week of age and eventually disappears in vivo, and appears to be down-regulated by thyroid hormone (tri-iodothyronine (T(3))). In this study, we used a cDNA microarray to screen for additional adhesion factors which might be important in testes of developing rats and detected expression of a novel factor, short-type PB-cadherin (STPB-C). Next, RT-PCR was used to generate cDNA for STPB-C from total RNA isolated from co-cultures, cDNA was cloned into pPCR-Script Amp SK(+) cloning vector, and plasmid DNA was isolated and sequenced to confirm the fidelity of the STPB-C cDNA portion of the plasmid. In situ hybridization analyses of testicular sections indicated that STPB-C expression in neonates is localized in the cytoplasm of many, but not all, gonocytes and in the cytoplasm of most of the surrounding Sertoli cells. Parallel hybridizations carried out on co-cultures also demonstrated a strong cytoplasmic signal in some gonocytes and in the great majority of the Sertoli cells of the underlying monolayer. With Northern analyses we found that STPB-C is expressed in vivo at high levels between days 1 and 5, with a subsequent large drop by day 10 and thereafter, suggesting that its expression may be associated with Sertoli or germ cell differentiation. Subsequent analyses of co-cultures exposed under a variety of conditions to T(3) suggest that, unlike NCAM, STPB-C is not regulated by this hormone. Next, we studied production of STPB-C protein by using an antiserum recognizing a peptide sequence unique to this factor in Western blotting and in immunolocalization. Signal was detected both intracellularly and at cell surfaces in most Sertoli cells and many gonocytes, although many of the latter cell type were also found to be negative for the protein, suggesting a potential role for STPB-C in survival and further development of some of these germ cells from which all subsequent spermatogenic cells originate.