Direct Cell-cell Interactions Research Articles

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.

RBC Adhesive Capacity Is Essential for Efficient 'Immune Adherence Clearance' and Provide a Generic Target to Deplete Pathogens from Septic Patients

Sepsis is a life threatening organ dysfunction caused by a dysregulated host response to infection. It is a global health care problem and the leading cause of death in ICU patients. A rather unknown but effective mechanism to protect ourselves from such pathogen invasion is immune-adherence clearance (IAC). During IAC pathogens (including bacteria, fungi and viruses) can bind to red blood cells (RBCs) through complement opsonization and subsequent binding to complement receptor 1 (CR1, CD35) expressed on their cell surface. After binding of the pathogen to CR1, the RBCs deliver the pathogens to macrophages of the spleen and liver, where they are phagocytosed and degraded.To study the underlying mechanisms of IAC in more detail, we have developed an assay to monitor the transfer of opsonized pathogens bound to RBCs to human monocytes and/or macrophages under flow conditions, using confocal microscopy. The transfer of a variety of different pathogens, including S. aureus, E. coli and C. albicans, was studied in this assay. When looking at the transfer process in detail, it was noticed that RBC attach to the phagocyte prior to the transfer and remain attached shortly after the transfer, suggesting that RBC might be interacting with phagocytes through adhesion molecules such as integrins to establish firm binding between these two cell types. To test this hypothesis several adhesion molecules, on the phagocytes as well as on the RBC, were blocked by monoclonal antibodies and IAC was quantified. First, complement receptor 3 (CR3, amb2 CD11b/CD18 integrin) an important receptor for adhesion, migration and phagocytosis was blocked by monoclonal antibodies on the phagocytes, after which bacterial transfer was impaired. This indicated that CR3 is crucial for efficient IAC. To confirm the involvement of CR3 in IAC, we analyzed the monocytes of a known LAD-1 patient. This rare immunodeficiency is characterized by an inherited molecular defect of the β2integrin subunit (CD18) which results in impaired adhesion and migration of the patient’s leukocytes and clinically manifests in recurrent infections. Our results showed a highly reduced interaction between the phagocytes and the RBCs, resulting in a decrease in bacterial transfer of >70%.Next, we tested the effects of blocking antibodies directed against several RBC adhesion molecules on the transfer process. When blocking CD147 (Basigin, Ok blood group) and ICAM-4 (CD242, Landsteiner-Wiener), but not Glycophorin A (GPA, CD235a) on the RBC the transfer process was largely inhibited. These findings demonstrate the involvement of direct cell-cell interactions between RBC and macrophages in IAC and provide evidence that RBC adopt a "sticky" phenotype after binding a pathogen through CR1, which enables phagocytes to bind them under flow. We anticipated that blood bank filters (currently used for leukocyte depletion) might be used to bind and filter RBC-pathogen complexes from blood due to their "sticky" phenotype. This was found to be possible in a series of experiments, not only using in vitrogenerated RBC-pathogen complexes, but also using blood from septic patients. Filtration using a standard leukocyte reduction filter showed reduction of RBC-pathogen complexes close to 100%, independent of the type of pathogen. We foresee that this knowledge can be used to develop a generic approach to deplete RBCs carrying pathogens specifically from the blood stream, and thereby filter RBC-pathogen complexes from the blood of patients suffering from sepsis. DisclosuresNo relevant conflicts of interest to declare.

Breast cancer cells and bone marrow mesenchymal stromal cells: a regulated modulation of the breast tumor in the context of immune response.

The role of direct cell-cell interactions mediating selective bone metastasis by breast cancer cells (BCCs) niche is still mostly unknown. Conditioned medium and direct cell-cell contacts experiments were used to investigate the effect of bone marrow-derived mesenchymal stromal cells (MSCs), osteoprogenitor-like cells (MG-63) and osteosarcoma cells (SaOS-2) on luminal-like (MCF-7) and basal-like (MDA-MB-231) BCCs flow cytometry was used to assess the purity of isolated BCCs and osteoblasts. Expression of osteoblastic markers was investigated by semi-quantitative RT-PCR. RANKL and OPG levels were measured by ELISA. Conditioned medium from MSCs and osteoblasts induced the expression of osteoblastic markers in BCCs. While co-culture assays with SaOS-2 increased the expression of osteoblastic markers in MCF-7 cells, SaOS-2 cell conditioned medium increased the expression of RANKL, PTHrP, VEGF and NOGGIN in MCF-7 cells. Co-cultures with either MG-63 cells or MSCs induced OPG and MMP-2 in both tumor cell lines. Interestingly, conditioned medium from co-cultures of MSCs and MDA-MB-231 cells significantly decreased the proliferation of activated T lymphocytes which was reversed by addition of anti-OPG antibodies to the co-cultures. Our data suggest that MSCs strongly contribute to the adaptation and invasiveness of breast cancer cells in skeletal tissues.

T-cell clones from Th1, Th17 or Th1/17 lineages and their signature cytokines have different capacity to activate endothelial cells or synoviocytes

ObjectiveTo compare the direct effect of cytokines on synoviocytes and endothelial cells to the effects of supernatants from Th1, Th17 and Th1/17 clones and the direct cell-cell interactions with the same clones. MethodsTh17 and Th1/17 clones were obtained from the CD161+CCR6+ fraction and Th1 clones from the CD161−CCR6− fraction of human CD4+ T-cells. Endothelial cells or synoviocytes were cultured in the presence of either isolated pro-inflammatory cytokines (IL-17 and/or TNF-α) or supernatants from the T-cell clones or co-cultured with T-cell clones themselves. IL-6 and IL-8 expression and production were analyzed. ResultsIL-17 and TNF-α induced IL-6 and IL-8 expression, although IL-17 alone had a limited effect on endothelial cells compared to synoviocytes. Supernatants from activated T-helper clones also induced IL-6 and IL-8 expression but with discrepancies between endothelial cells and synoviocytes. Endothelial cells were mostly activated by Th1 clone supernatants whereas synoviocytes were activated by all T-cell subtypes. Finally, cell-cell contact experiments showed a great heterogeneity among cell clones, even from the same lineage. IL-6 expression was mostly induced by contact with Th1 clones both in endothelial and mesenchymal cells whereas IL-8 expression was induced by all T-cell clones whatever their phenotype. ConclusionWe showed that endothelial cells were much more sensitive to Th1 activation whereas synoviocytes were activated by all T-helper lineages. This work highlights the heterogeneity of interactions between T-cells and stromal cells through soluble factors or direct cell contact.

Abstract A02: The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer

Abstract To date there has been an increasing focus on the interactions between inflammatory myeloid cells and T cells in the tumor microenvironment because cytotoxic anti-tumoral T cells represent the chief effector mechanism of anti-tumoral immunity. Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in lung tumors; however, whether specialized neutrophil subpopulations capable of regulating T cell responses exist in human cancers is unknown. Our goal was to identify subsets of TANs and determine their specific roles in the regulation of T cell responses in patients with early stage lung cancer. To address this question, freshly isolated tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. An extensive phenotypic analysis of 55 early-stage human lung tumors revealed that TANs, defined as CD11b+Arg1+MPO+CD66b+CD15+ cells, contained two major sub-populations. One subset of canonical TANs expressed classic neutrophil markers. A second subset of TANs displayed a combination of neutrophil markers plus markers (CD14+HLA-DR+CCR7+CD86+) normally expressed on antigen-presenting cells (APC). This subset of TANs was found in lung tumor tissue but not in adjacent uninvolved lung. We termed this unique neutrophil population “APC-like hybrid TANs”. The frequency of these hybrid TANs varied widely within lung cancers and ranged from 0.5-25% of all TANs. Interestingly, the frequency of this hybrid population declined as tumors enlarged, and they were almost completely absent in tumors greater than 5 cm in diameter. Mechanistically, we determined that low doses of IFN-γ and GM-CSF in the tumors were required for the development of APC-like hybrid neutrophils. The high proportion of hybrid TANs (>10% of all TANs) directly correlated with the presence of IFN-γ and GM-CSF in the autologous tumor tissue. Using bone marrow-derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC-like hybrid neutrophils originate from CD11b+CD15+CD10-CD16-/low/int neutrophil progenitors in the presence of IFN-γ and GM-CSF or in tumor-conditioned media. By contrast, mature CD11b+CD15+CD10+CD16hi neutrophils did not acquire the phenotype of hybrid TANs, when cultured under these conditions. In addition, we determined that IFN-γ and GM-CSF synergistically exerted APC promoting effects on immature neutrophils via the downregulation of the transcription factor, Ikaros. However, the development of APC-like hybrid neutrophils was profoundly inhibited under hypoxic conditions. Functionally, the APC-like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) produce APC cytokines such as TNF and IL-12 after stimulation, 2) stimulate antigen non-specific autologous T cell responses induced by plate-bound anti-CD3 antibodies 3) directly stimulate antigen-specific autologous memory T cell responses to virus-derived antigens, 4) augment NY-ESO-1 specific effector T cell responses by providing a co-stimulatory signals through the OX40L, 4-1BBL CD86, CD54 molecules, and 5) cross present tumor-associated antigen as IgG-immune complex. In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early-stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC-like hybrid neutrophils. These hybrid TANs had enhanced ability to trigger and support T cell responses in direct cell-cell interactions. This property of hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction. Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Shaun O'Brien, Edmund Moon, Tom Stephen, Abhishek Rao, Alfred Garfall, Wayne Hancock, Jose Conejo-Garcia, Charuhas Deshpande, Michael Feldman, Sunil Singhal, Steven Albelda. The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A02.

Abstract LB-288: Investigation on the interaction between human beta defensin type 3 (hBD-3) and ectodomain of EphA2 receptor using molecular dynamics simulation method

Abstract Defensins are a family of cationic cysteine-rich small molecules. In general, human beta defensins (hBD) are produced by epithelial cells, and they are important to the human innate immune system. Experimental results showed that defensins can directly bind with receptors such as chemokine receptors and act through several mechanisms that may trigger antitumor immunity or facilitate the host's immune response (or evasion) and promote tumor progression. hBD type 3 (hBD-3) is newly discovered and is unique that it has a net charge density much higher than other family members, and has the ability to undergo oligomerization. On the other hand, EPH receptors and their corresponding ligands (ephrins) are membrane-bound proteins that require direct cell-cell interactions for the activation of the receptors. Eph-ephrin system plays a critical role in tumor and vascular functions during carcinogenesis. Once EPH receptor A2 (EphA2) is ligand-independent, it can over-express itself in many types of tumors, and behave like an oncogene. Besides, many studies suggested that mutations that affect EPH receptor function play a role in cancer initiation or progression. Therefore, the EphA2-ephrin5 system can be a novel target for the development of anti-tumorigenic and anti-angiogenic therapies, and understanding its interaction with other molecules is important to demystify the cancer mechanism. Both EphA2 ectodomain (ECD) and hBD-3 exist outside of the cell membrane. In order to find out the interaction mechanism of hBD-3 with EphA2 receptor, initially blind docking using Cluspro software was performed to predict all the possible binding sites between hBD-3 oligomer and EphA2 ECD in the free and bound forms. After that, all atom molecular dynamics CHARMM simulations were performed to find out the most probable binding interfaces between hBD-3 oligomer and EphA2 ECD. It was found that hBD-3 has multiple stable binding sites on the EphA2 ECD, and its binding on the ligand-binding domain (LBD) of EphA2 could influence the binding of ephrin5 with EphA2. Those suggest the hBD-3 could play a role in cancer initiation. Citation Format: Liqun Zhang. Investigation on the interaction between human beta defensin type 3 (hBD-3) and ectodomain of EphA2 receptor using molecular dynamics simulation method. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-288.

TIM-3 expression in lymphoma cells predicts chemoresistance in patients with adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL), an aggressive type of malignant lymphoma, is highly resistant to chemotherapy. However, the detailed mechanisms of the chemoresistance of ATLL have never been elucidated. We previously demonstrated that direct cell-cell interaction between macrophages and lymphoma cells was significantly associated with lymphoma progression in patients with ATLL. The present study aimed to further analyze the effects of cell-cell interaction between macrophages and ATLL cells by means of cell culture studies and immunohistochemical analysis using human ATLL samples. It was found that direct co-culture with macrophages induced chemoresistance in the ATLL ATN-1 cell line, but not in other cell lines, including TL-Mor, ED and ATL-2S. It was also found that expression of the T cell Ig and mucin domain-containing molecule-3 (TIM-3) was induced in ATN-1 cells by their long-term co-culture with macrophages. TIM-3 gene transfection induced chemoresistance in the ATN-1 cells. Immunostaining of ATLL tissues showed TIM-3 expression in 25 out of 58 ATLL cases. Although TIM-3 expression was not associated with overall survival or T classification, it was associated with resistance to chemotherapy. TIM-3 expression is therefore considered to be a marker for predicting the efficacy of chemotherapy, and TIM-3-associated signals may be a therapeutic target for patients with ATLL.

Prostate cancer cells specifically reorganize epithelial cell-fibroblast communication through proteoglycan and junction pathways

ABSTRACTMicroenvironment and stromal fibroblasts are able to inhibit tumor cell proliferation both through secreted signaling molecules and direct cell-cell interactions but molecular mechanisms of these effects remain unclear. In this study, we investigated a role of cell-cell contact-related molecules (protein ECM components, proteoglycans (PGs) and junction-related molecules) in intercellular communications between the human TERT immortalized fibroblasts (BjTERT fibroblasts) and normal (PNT2) or cancer (LNCaP, PC3, DU145) prostate epithelial cells. It was shown that BjTERT-PNT2 cell coculture resulted in significant decrease of both BjTERT and PNT2 proliferation rates and reorganization of transcriptional activity of cell-cell contact-related genes in both cell types. Immunocytochemical staining revealed redistribution of DCN and LUM in PNT2 cells and significant increase of SDC1 at the intercellular contact zones between BjTERT and PNT2 cells, suggesting active involvement of the PGs in cell-cell contacts and contact inhibition of cell proliferation. Unlike to PNT2 cells, PC3 cells did not respond to BjTERT in terms of PGs expression, moderately increased transcriptional activity of junctions-related genes (especially tight junction) and failed to establish PC3-BjTERT contacts. At the same time, PC3 cells significantly down-regulated junctions-related genes (especially focal adhesions and adherens junctions) in BjTERT fibroblasts resulting in visible preference for homotypic PC3-PC3 over heterotypic PC3-BjTERT contacts and autonomous growth of PC3 clones. Taken together, the results demonstrate that an instructing role of fibroblasts to normal prostate epithelial cells is revoked by cancer cells through deregulation of proteoglycans and junction molecules expression and overall disorganization of fibroblast-cancer cell communication.

Chitosan-hyaluronan based 3D co-culture platform for studying the crosstalk of lung cancer cells and mesenchymal stem cells

The controversial roles of mesenchymal stem cells (MSCs) in lung cancer development are not yet resolved because of the lack of an extracellular environment that mimics the tumor microenvironment. Three-dimensional (3D) culture system is an emerging research tool for biomedical applications such as drug screening. In this study, MSCs and human non-small cell lung carcinoma cells (A549) were co-cultured on a thin biomaterial-based substratum (hyaluronan-grafted chitosan, CS-HA; ∼2μm), and they were self-organized into the 3D tumor co-spheroids with core-shell structure. The gene expression levels of tumorigenicity markers in cancer cells associated with cancer stemness, epithelial-mesenchymal transition (EMT) property, and cell mobility were up-regulated for more than twofold in the MSC-tumor co-spheroids, through the promoted expression of certain tumor enhancers and the direct cell-cell interaction. To verify the different extents of tumorigenicity, A549 cells or those co-cultured with MSCs were transplanted into zebrafish embryos for evaluation in vivo. The tumorigenicity obtained from the zebrafish xenotransplantation model was consistent with that observed in vitro. These evidences suggest that the CS-HA substrate-based 3D co-culture platform for cancer cells and MSCs may be a convenient tool for studying the cell-cell interaction in a tumor-like microenvironment and potentially for cancer drug testing. Statement of SignificanceMesenchymal stem cells (MSCs) have been found in several types of tumor tissues. However, the controversial roles of MSCs in cancer development are still unsolved. Chitosan and hyaluronan are commonly used materials in the biomedical field. In the current study, we co-cultured lung cancer cells and MSCs on the planar hyaluronan-grafted chitosan (CS-HA) hybrid substrates, and discovered that lung cancer cells and MSCs were rapidly self-assembled into 3D tumor spheroids with core-shell structure on the substrates after only two days in culture. Therefore, CS-HA based 3D co-culture platform can be applied to exploration of the relationship between cancer cells and MSCs and other cancer-related medical applications such as drug screening.

Neutrophilia and lymphopenia as prognostic factors in non-small cell lung cancer.

e20587Abstract Background: Neutrophil count is associated with prognosis in some cancers. Direct cell-cell interactions between neutrophils and tumor cells enhance tumor growth in Non-small cell lu...

Effects of substrate stiffness and cell-cell contact on mesenchymal stem cell differentiation

The mechanical properties of the microenvironment and direct contact-mediated cell-cell interactions are two variables known to be important in the determination of stem cell differentiation fate, but little is known about the interplay of these cues. Here, we use a micropatterning approach on polyacrylamide gels of tunable stiffnesses to study how homotypic cell-cell contacts and mechanical stiffness affect different stages of osteogenesis of mesenchymal stem cells (MSCs). Nuclear localization of transcription factors associated with osteogenesis depended on substrate stiffness and was independent of the degree of cell-cell contact. However, expression of alkaline phosphatase, an early protein marker for osteogenesis, increased only in cells with both direct contact with neighboring cells and adhesion to stiffer substrates. Finally, mature osteogenesis, as assessed by calcium deposition, was low in micropatterned cells, even on stiff substrates and in multicellular clusters. These results indicate that substrate stiffness and the presence of neighboring cells regulate osteogenesis in MSCs.

Relationship of blood monocytes with chronic lymphocytic leukemia aggressiveness and outcomes: a multi-institutional study.

Monocyte-derived cells, constituents of the cancer microenvironment, support chronic lymphocytic leukemia (CLL) cell survival in vitro via direct cell-cell interaction and secreted factors. We hypothesized that circulating absolute monocyte count (AMC) reflects the monocyte-derived cells in the microenvironment, and that higher AMC is associated with increased CLL cell survival in vivo and thus inferior CLL patient outcomes. We assessed the extent to which AMC at diagnosis of CLL is correlated with clinical outcomes, and whether this information adds to currently used prognostic markers. We evaluated AMC, clinically used prognostic markers, and time to event data from 1,168 CLL patients followed at the Mayo Clinic, the Duke University Medical Center, and the Durham VA Medical Center. Elevated AMC was significantly associated with inferior clinical outcomes, including time to first therapy (TTT) and overall survival (OS). AMC combined with established clinical and molecular prognostic markers significantly improved risk-stratification of CLL patients for TTT. As an elevated AMC at diagnosis is associated with accelerated disease progression, and monocyte-derived cells in the CLL microenvironment promote CLL cell survival and proliferation, these findings suggest that monocytes and monocyte-derived cells are rational therapeutic targets in CLL. Am. J. Hematol. 91:687-691, 2016. © 2016 Wiley Periodicals, Inc.

Fusion cytokine IL-2-GMCSF enhances anticancer immune responses through promoting cell-cell interactions.

Background Potent antitumor responses can be induced through cytokine immunotherapy. Interleukin (IL)-2 and granulocyte–macrophage colony-stimulating factor (GM-CSF) are among the most effective cytokines to induce tumor-specific systemic immune responses and can act synergistically. To overcome the limitations of combined use of these two cytokines, we have constructed an IL2-GMCSF fusion protein and characterized its antitumor effects in this study.MethodsThe expression of IL-2 receptor and GM-CSF receptor of cell lines were detected with quantitative real-time PCR. On this basis, the bioactivities of IL2-GMCSF, especially effects on DC2.4 cells were assayed. Another function of IL2-GMCSF—bridge two types of cells—was assessed by cell contact counting and cytotoxicity assays. The anti-tumor activity in vivo of IL2-GMCSF was evaluated in the melanoma model. The statistical significance among treatment groups were determined by One-Way ANOVA.ResultsThe fusion protein IL2-GMCSF maintained the activities of IL-2 and GM-CSF, and could significantly promote DC2.4 cell activities, including phagocytosis, proliferation and cytokine secretion. In addition to the inherent cytokine activity, IL2-GMCSF bridges direct cell–cell interactions and enhances splenocyte killing efficacy against multiple tumor cell lines in vitro. Co-injection of IL2-GMCSF and inactivated B16F10 mouse melanoma cells induced complete immunoprotective responses in about 30 % of mice.ConclusionThese results suggested that IL2-GMCSF can efficiently regulate immune responses against tumors. Furthermore, as the bridging effect relies on both IL-2R and GM-CSFR and promotes interactions between immune and tumor cells, IL2-GMCSF may be utilized as a useful tool for dissecting specific immune responses for future clinical applications.

Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P < 0.01) and increased local ECM remodeling quantified by confocal microscopy. Under coculture conditions that allow indirect cellular interaction via paracrine factors but prevent direct cell-cell contact, monocytes had minimal effects on myofibroblast activity (17.9 ± 11.1% vs. 6.4 ± 7.0% increase, respectively; P < 0.01). When cells were cultured under direct contact conditions, multiplex analysis of the coculture media revealed an increase in the paracrine factors TGF-β1 and matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P < 0.001). TGF-β blockade abolished the monocyte-induced increase in cardiac myofibroblast activity. These data suggest that direct cell-cell interaction between monocytes and cardiac myofibroblasts stimulates TGF-β-mediated myofibroblast activity and increases remodeling of local matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1. These data implicate inflammation as a potential driver of cardiac fibrosis.

Biomimetic channel modeling local vascular dynamics of pro-inflammatory endothelial changes.

Endothelial cells form the inner lining of blood vessels and are exposed to various factors like hemodynamic conditions (shear stress, laminar, and turbulent flow), biochemical signals (cytokines), and communication with other cell types (smooth muscle cells, monocytes, platelets, etc.). Blood vessel functions are regulated by interactions among these factors. The occurrence of a pathological condition would lead to localized upregulation of cell adhesion molecules on the endothelial lining of the blood vessel. This process is promoted by circulating cytokines such as tumor necrosis factor-alpha, which leads to expression of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell surface among other molecules. ICAM-1 is critical in regulating endothelial cell layer dynamic integrity and cytoskeletal remodeling and also mediates direct cell-cell interactions as part of inflammatory responses and wound healing. In this study, we developed a biomimetic blood vessel model by culturing confluent, flow aligned, endothelial cells in a microfluidic platform, and performed real time in situ characterization of flow mediated localized pro-inflammatory endothelial activation. The model mimics the physiological phenomenon of cytokine activation of endothelium from the tissue side and studies the heterogeneity in localized surface ICAM-1 expression and F-actin arrangement. Fluorescent antibody coated particles were used as imaging probes for identifying endothelial cell surface ICAM-1 expression. The binding properties of particles were evaluated under flow for two different particle sizes and antibody coating densities. This allowed the investigation of spatial resolution and accessibility of ICAM-1 molecules expressed on the endothelial cells, along with their sensitivity in receptor-ligand recognition and binding. This work has developed an in vitro blood vessel model that can integrate various heterogeneous factors to effectively mimic a complex endothelial microenvironment and can be potentially applied for relevant blood vessel mechanobiology studies.

A Boolean Function for Neural Induction Reveals a Critical Role of Direct Intercellular Interactions in Patterning the Ectoderm of the Ascidian Embryo

A complex system of multiple signaling molecules often produce differential gene expression patterns in animal embryos. In the ascidian embryo, four signaling ligands, Ephrin-A.d (Efna.d), Fgf9/16/20, Admp, and Gdf1/3-r, coordinately induce Otx expression in the neural lineage at the 32-cell stage. However, it has not been determined whether differential inputs of all of these signaling pathways are really necessary. It is possible that differential activation of one of these signaling pathways is sufficient and the remaining signaling pathways are activated in all cells at similar levels. To address this question, we developed a parameter-free method for determining a Boolean function for Otx expression in the present study. We treated activities of signaling pathways as Boolean values, and we also took all possible patterns of signaling gradients into consideration. We successfully determined a Boolean function that explains Otx expression in the animal hemisphere of wild-type and morphant embryos at the 32-cell stage. This Boolean function was not inconsistent with three sensing patterns, which represented whether or not individual cells received sufficient amounts of the signaling molecules. These sensing patterns all indicated that differential expression of Otx in the neural lineage is primarily determined by Efna.d, but not by differential inputs of Fgf9/16/20, Admp, and Gdf1/3-r signaling. To confirm this hypothesis experimentally, we simultaneously knocked-down Admp, Gdf1/3-r, and Fgf9/16/20, and treated this triple morphant with recombinant bFGF and BMP4 proteins, which mimic Fgf9/16/20 and Admp/Gdf1/3-r activity, respectively. Although no differential inputs of Admp, Gdf1/3-r and Fgf9/16/20 signaling were expected under this experimental condition, Otx was expressed specifically in the neural lineage. Thus, direct cell–cell interactions through Efna.d play a critical role in patterning the ectoderm of the early ascidian embryo.

Targeting Rac-Gtpases Reverses Stroma-Induced Resistance to Notch and mTOR-Inhibition in Acute T-Cell Leukemia

Molecular hallmarks of T-ALL are the aberrant activation of NOTCH signaling and high activity of the PI3K-AKT-mTOR pathway. Upregulated mTOR and NOTCH have been linked to the resistance of T-ALL to chemotherapy as well as to high frequencies of leukemia-initiating cells. Hence, the mTOR and the NOTCH pathways are promising therapeutic targets in T-ALL. However, clinical success of the mTOR inhibitor Rapamycin in acute leukemia has been disappointing. Similar results have been observed in mouse models of T-ALL treated with Notch inhibitors. To investigate the impact of mTOR and Notch inhibition in a genetically complex T-ALL, we developed an aggressive murine T-ALL model, driven by tyrosine kinase signaling, loss of Pten, Cux1-haploinsufficiency and constitutive Notch signaling. In vitro, T-ALL blasts were highly sensitive to inhibition of AKT, mTOR and Notch signaling. We transplanted the leukemias into secondary recipients and initiated treatment with Rapamycin after the onset of leukemia. Rapamycin significantly prolonged survival of the animals (placebo: 27 days, Rapamycin 49 days, p<0.001). Eventually, all Rapamycin treated animals succumbed to the T-ALL that extensively infiltrated the bone marrow and solid organs despite continuous drug administration. When Rapamycin-resistant blasts were explanted and cultured in petri dishes they again became susceptible to Rapamycin, demonstrating a context-dependent resistance rather than outgrowth of intrinsically resistant clones. Gene set enrichment analysis revealed that Rapamycin-resistant T-ALL in vivo upregulated genetic networks associated with cell-cell interactions. Stromal cell support from OP9-cells as well as from mesenchymal stem cells recapitulated the in vivo effect and induced resistance to mTOR and Notch-inhibition in T-ALL blasts. Coating the tissue culture wells with Collagen, Fibronectin, Retronectin or Matrigel, did not elicit resistance. By using trans-well assays we show that the stroma-induced resistance was dependent on direct cell-cell interactions. Immunoblots and PhosFlow probing the mTORC1/C2 and Notch pathway demonstrated an identical drug effect on their intracellular targets in resistant T-ALL blasts cultured on stroma cells and susceptible cells in suspension. Since the number of molecules potentially involved in cell-to-cell contacts is vast, we focused on central nodes that organize this process in order to find a potentially druggable target that is critically involved in stroma-induced resistance. Transcriptome profiling pointed towards upregulation of Rac-associated pathways. We determined the activation of Rac1 by PAK-pull down assays in T-ALL blasts grown in suspension or on stromal cells. We observed an increase (FC=1.96 ± 0.58, p=0.04) in activated Rac1 in the T-ALL blasts in contact with a stromal layer. To determine whether Rac activation plays a role in stroma-induced resistance, we devised a strategy to abrogate Rac signaling in T-ALL blasts, but not in the stromal cells, since inhibition of Rac in stromal cells by the Rac-inhibitor NSC23766 led to the their detachment. Furthermore, Rac1,2 and 3 can be functionally redundant, making knock down experiments using shRNAs challenging. The Clostridium difficile serotype F strain 1470 produces toxin B isoform (TcdBF), that selectively glucosylates and inactivates Rac(1,2,3). We pretreated T-ALL blasts with TcdBF and observed a dose-dependent functional inhibition of Rac GTPases monitored by dephosphorylation of the Rac effector kinase pS144/141-PAK-1/2. T-ALL blasts were then incubated for 5 hours with increasing toxin doses, washed 3 times and incubated in toxin-free medium. Eighteen hours after the end of the exposure to the toxins, Rac was still inhibited. Strikingly, in the TcdBF-pretreated T-ALL, the stroma-induced resistance effect was abrogated and clusters of apoptotic cells were clearly visible (>2 fold reduction of the input, p=0.002). In contrast, the carrier-treated T-ALL exhibited resistance to the inhibitors on stroma (>10 fold expansion of the input, p<0.0003). Altogether, we identify the Rac-GTPases as a nexus of stroma-induced drug resistance and show that inhibition of Rac and mTOR is synthetically lethal to T-ALL blasts T-ALL blasts that are in contact with stromal cells, paving the way to augment the effectiveness of small molecule inhibitors in acute leukemia. DisclosuresNo relevant conflicts of interest to declare.

Wharton's Jelly- MSC As Effective Tool for Expansion of Purified Human Cord Blood CD34+ Hematopoietic Stem Cells

Umbilical cord is an extra-embryonic-annex rich of both hematopoietic stem and progenitor cells (HSPC) and mesenchymal stem cells (MSC) and it is easily accessible. The HSPC derived from umbilical cord blood (UCB) are promising as graft for allogeneic bone marrow (BM) transplantation and as source of target cells for autologous HSPC gene correction. UCB-HSPC have several advantages compared to adult ones: a less risk of graft-versus-host disease, a higher frequency of progenitors with a greater clonogenic potential and more susceptibility to be transduced by lentiviral vectors. Nonetheless, the HSPC yield from single cord blood unit is not sufficient for these clinical approaches in adults. Therefore, ex-vivo expansion of HSPC in media supplemented by cytokines and/or in vitro culture systems with feeder layers, is a valid approach to exceed this limit. MSC are a component of BM-microenvironment that play a key role in supporting of hematopoiesis by ability to secrete soluble factors and probably by the direct cell-cell interaction too.In this work, we investigated the ability of umbilical cord extracellular matrix-MSC (Wharton's Jelly-MSC) to support the ex-vivo expansion of UBC- purified CD34+ cells. In particular, we evaluated the fold increase, and the frequency of CD34+ cell and CD34+subtypes during expansion at the following culture conditions: by direct contact with WJ-MSC layer, by exposure to the soluble factors secreted by WJ-MSC layer in transwell system. The fold expansion was compared with the CD34+ cells expanded in a customized serum-free medium.CD34+ cells were isolated by immuneselection from 8 fresh UCB. The WJ-MSC were isolated from UC cut-pieces by non-enzymatic procedure but thanks to their capacity to migrate to plastic substrate. At the confluence of 60-70% the WJ-MSC were treated with mytomicin-C to arrest the cell cycle. After 48h, the immune-selected CD34+ cells were seeded in WJ-MSC at the density of 5-10 x104 in 12 well plates by direct or indirect contact (by transwell system). CD34+cells were grown in absence of feeder layers at the same conditions. Early hematopoietic cytokines (Flt-3, TPO, SCF) were supplemented in all three conditions and freshly replaced every two days of culture.Numbers and frequency of CD34+cells were evaluated according to ISHAGE method and CD34+ subtyping was performed by four color method to investigated the co-expression of the primitive surface antigens (CD38, CD133, CD90).The frequency of CD34+ cells at day 5 of culture decreased only 10% and was about 50% after 8 days of culture in conditions. The expansion of CD34 + cells at direct contact with WJ-MSC was superior (5.5 fold increase) compared to that of the other two conditions (3 fold on average).At day 8of culture, the CD34+ cells expanded 12 fold at direct contact with feeder layer, about 7 fold in a transwell system and 6 fold in basic medium. No substantial differences in the grade of expansion was revealed in heterologous vs homologous co-cultures of HSPC/WJ-MSC.Noteworthy is that in the contact system in addition to the fluctuating CD34+ cells harvested from the medium (floating CD34+ cells), we found approximately 50% of the total CD34+ cells be adherent to WJ-MSC layer, these cells were released only after enzymatic proteolytic treatment.Subtyping the CD34+cell population growing in contact to the WJ-MSC or in the conditioned medium we found that the CD34+/CD133+cell population was maintained high (72% ±12 over the total CD34+ cells) as in unmanipulated CB-HSC. The CD34+CD38- cells decreased by 2,5 fold in both systems, as early as day 5 of culture. However, in the contact system this population was 3 times more represented in the attached CD34+ cell fraction. The CD34+/CD90+ subtype was also expanded (more than 8 fold) particularly in the attached fraction, as early as 5 days of culture and was maintained to the endWJ-MSC supported ex-vivo HSPC expansion with superior effect in a cell contact system. Two phenotypically different populations of HSPC developed in this system with an increased frequency of CD34+ cells that co-expressed markers typical of more early progenitors in the attached CD34+ cell fraction. We are assessing the significance of these differences by performing molecular and functional studies of WJ-MSC-supported HSPC.This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 DisclosuresNo relevant conflicts of interest to declare.

High-Throughput Single-Cell Kinetics of Virus Infections in the Presence of Defective Interfering Particles

Defective interfering particles (DIPs) are virus mutants that lack essential genes for growth. In coinfections with helper virus, the diversion of viral proteins to the replication and packaging of DIP genomes can interfere with virus production. Mounting cases of DIPs and DIP-like genomes in clinical and natural isolates, as well as growing interest in DIP-based therapies, underscore a need to better elucidate how DIPs work. DIP activity is primarily measured by its inhibition of virus infection yield, an endpoint that masks the dynamic and potentially diverse individual cell behaviors. Using vesicular stomatitis virus (VSV) as a model, we coinfected BHK cells with VSV DIPs and recombinant helper virus carrying a gene encoding a red fluorescent protein (RFP) whose expression correlates with the timing and level of virus release. For single cells within a monolayer, 10 DIPs per cell suppressed the reporter expression in only 1.2% of the cells. In most cells, it slowed and reduced viral gene expression, manifested as a shift in mean latent time from 4 to 6 h and reduced virus yields by 10-fold. For single cells isolated in microwells, DIP effects were more pronounced, reducing virus yields by 100-fold and extending latent times to 12 h, including individual instances above 20 h. Together, these results suggest that direct or indirect cell-cell interactions prevent most coinfected cells from being completely suppressed by DIPs. Finally, a gamma distribution model captures well how the infection kinetics quantitatively depends on the DIP dose. Such models will be useful for advancing a predictive biology of DIP-associated virus growth and infection spread. During the last century, basic studies in virology have focused on developing a molecular mechanistic understanding of how infectious viruses reproduce in their living host cells. However, over the last 10 years, the advent of deep sequencing and other powerful technologies has revealed in natural and patient infections that viruses do not act alone. Instead, viruses are often accompanied by defective virus-like particles that carry large deletions in their genomes and fail to replicate on their own. Coinfections of viable and defective viruses behave in unpredictable ways, but they often interfere with normal virus growth, potentially enabling infections to evade host immune surveillance. In the current study, controlled levels of defective viruses are coinfected with viable viruses that have been engineered to express a fluorescent reporter protein during infection. Unique profiles of reporter expression acquired from thousands of coinfected cells reveal how interference acts at multiple stages of infection.

Transcriptomics Identify CD9 as a Marker of Murine IL-10-Competent Regulatory B Cells.

Regulatory B cells (Breg) have immune suppressive functions in various autoimmune/inflammation models and diseases and are found to be enriched in diverse B cell subsets. The lack of a unique marker or set of markers efficiently identifying Breg cells impedes detailed investigation into their origin, development, and immunological roles. Here, we perform transcriptome analysis of IL-10-expressing B cells to identify key regulators for Breg biogenesis and function and identify CD9, a tetraspanin-family transmembrane protein, as a key surface marker for most mouse IL-10(+) B cells and their progenitors. CD9 plays a role in the suppressive function of IL-10(+) B cells in ex vivo T cell proliferation assays through a mechanism that is dependent upon B/T cell interactions. CD9(+) B cells also demonstrate inhibition of Th1-mediated contact hypersensitivity in an in vivo model system. Taken together, our findings implicate CD9 in the immunosuppressive activity of regulatory B cells.