Cell size induced bias of current density in hypertrophic cardiomyocytes.

Alterations in ion channel expression and function known as "electrical remodeling" contribute to the development of hypertrophy and to the emergence of arrhythmias and sudden cardiac death. However, comparing current density values - an electrophysiological parameter commonly utilized to assess ion channel function - between normal and hypertrophied cells may be flawed when current amplitude does not scale with cell size. Even more, common routines to study equally sized cells or to discard measurements when large currents do not allow proper voltage-clamp control may introduce a selection bias and thereby confound direct comparison. To test a possible dependence of current density on cell size and shape, we employed whole-cell patch-clamp recording of voltage-gated sodium and calcium currents in Langendorff-isolated ventricular cardiomyocytes and Purkinje myocytes, as well as in cardiomyocytes derived from trans-aortic constriction operated mice. Here, we describe a distinct inverse relationship between voltage-gated sodium and calcium current densities and cell capacitance both in normal and hypertrophied cells. This inverse relationship was well fit by an exponential function and may be due to physiological adaptations that do not scale proportionally with cell size or may be explained by a selection bias. Our study emphasizes the need to consider cell size bias when comparing current densities in cardiomyocytes of different sizes, particularly in hypertrophic cells. Conventional comparisons based solely on mean current density may be inadequate for groups with unequal cell size or non-proportional current amplitude and cell size scaling.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

NMDAR antagonists suppress tumor progression by regulating tumor-associated macrophages.

Neurotransmitter receptors are increasingly recognized to play important roles in anti-tumor immunity. The expression of the ion channel N-methyl-D-aspartate receptor (NMDAR) on macrophages was reported, but the role of NMDAR on macrophages in the tumor microenvironment (TME) remains unknown. Here, we show that the activation of NMDAR triggered calcium influx and reactive oxygen species production, which fueled immunosuppressive activities in tumor-associated macrophages (TAMs) in the hepatocellular sarcoma and fibrosarcoma tumor settings. NMDAR antagonists, MK-801, memantine, and magnesium, effectively suppressed these processes in TAMs. Single-cell RNA sequencing analysis revealed that blocking NMDAR functionally and metabolically altered TAM phenotypes, such that they could better promote T cell- and Natural killer (NK) cell-mediated anti-tumor immunity. Treatment with NMDAR antagonists in combination with anti-PD-1 antibody led to the elimination of the majority of established preclinical liver tumors. Thus, our study uncovered an unknown role for NMDAR in regulating macrophages in the TME of hepatocellular sarcoma and provided a rationale for targeting NMDAR for tumor immunotherapy.

The type of suture material affects transverse aortic constriction-induced heart failure development in mice: a repeated measures correlation analysis.

Introduction: Transverse-aortic constriction (TAC) operation is a widely used animal model to induce hypertrophy and heart failure through left-ventricular pressure overload. In mice, the cardiac response to TAC exhibits considerable variability influenced by factors such as strain, sub-strain, age, sex and vendor. Methods: To investigate the impact of suture material (silk versus prolene) and size (6-0 versus 7-0) on the TAC-induced phenotype, we performed surgeries on male C57BL6/N mice at 9 weeks of age defining the aortic constriction by a 27G needle, thereby employing most frequently used methodological settings. The mice were randomly assigned into four separate groups, 6-0 silk, 7-0 silk, 6-0 prolene and 7-0 prolene (10 mice per group). Echocardiography was conducted before TAC and every 4 weeks thereafter to monitor the development of heart failure. Repeated measures correlation analysis was employed to compare disease progression among the different groups. Results: Our findings reveal a significant influence of the chosen suture material on TAC outcomes. Mice operated with prolene showed increased mortality, slower body weight gain, faster left-ventricular mass increase, and a faster decline in left-ventricular ejection fraction, fractional shortening and aortic pressure gradient compared to silk-operated mice. Moreover, despite non significant, using thinner suture threads (7-0) tended to result in a more severe phenotype compared to thicker threads (6-0) across all tested parameters. Discussion: Collectively, our results highlight the importance of suture material selection in determining the cardiac phenotype induced by TAC and emphasize the need to consider this factor when comparing data across different research laboratories.

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

In situ generation of H2O2 using CaO2 as peroxide storage depot for haloperoxidase mimicry with surface-tailored Bi-doped mesoporous CeO2 nanozymes.

Designing the size, morphology and interfacial charge of catalyst particles at the nanometer scale can enhance their performance. We demonstrate this with nanoceria which is a functional mimic of haloperoxidases, a group of enzymes that halogenates organic substrates in the presence of hydrogen peroxide. These reactions in aqueous solution require the presence of H2O2. We demonstrate in situ generation of H2O2 from a CaO2 reservoir in polyether sulfone (PES) and poly(vinylidene fluoride) (PVDF) polymer beads, which circumvents the external addition of H2O2 and expands the scope of applications for haloperoxidase reactions. The catalytic activity of nanoceria was enhanced significantly by Bi3+ substitution. Bi-doped mesoporous ceria nanoparticles with tunable surface properties were prepared by changing the reaction time. Increasing reaction time increases the surface area SBET of the mesoporous Bi0.2Ce0.8O1.9 nanoparticles and the Ce3+/Ce4+ ratio, which is associated with the ζ-potential. In this way, the catalytic activity of nanoceria could be tuned in a straightforward manner. H2O2 required for the reaction was released steadily over a long period of time from a CaO2 storage depot incorporated in polyether sulfone (PES) and poly(vinylidene fluoride) (PVDF) beads together with Bi0.2Ce0.8O1.9 particles, which may be used as precision fillers and templates for biological applications. The spheres are prepared as a dry powder with no surface functionalization or coatings. They are inert, chemically stable, and safe for handling. The feasibility of this approach was demonstrated using a haloperoxidase assay.

Communication Breakdown: Into the Molecular Mechanism of Biofilm Inhibition by CeO2 Nanocrystal Enzyme Mimics and How It Can Be Exploited.

Bacterial biofilm formation is a huge problem in industry and medicine. Therefore, the discovery of anti-biofilm agents may hold great promise. Biofilm formation is usually a consequence of bacterial cell-cell communication, a process called quorum sensing (QS). CeO2 nanocrystals (NCs) have been established as haloperoxidase (HPO) mimics and ecologically beneficial biofilm inhibitors. They were suggested to interfere with QS, a mechanism termed quorum quenching (QQ), but their molecular mechanism remained elusive. We show that CeO2 NCs are effective QQ agents, inactivating QS signals by bromination. Catalytic bromination of 3-oxo-C12-AHL a QS signaling compound used by Pseudomonas aeruginosa, was detected in the presence of CeO2 NCs, bromide ions, and hydrogen peroxide. Brominated acyl-homoserine lactones (AHLs) no longer act as QS signals but were not detected in the bacterial cultures. Externally added brominated AHLs also disappeared in P. aeruginosa cultures within minutes of their addition, indicating that they are rapidly degraded by the bacteria. Moreover, we detected the catalytic bromination of 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO), a multifunctional non-AHL QS signal from P. aeruginosa with antibacterial and algicidal properties controlling the expression of many virulence genes. Brominated HQNO was not degraded by the bacteria in vivo. The repression of the Pseudomonas quinolone signal (PQS) production and biofilm formation in P. aeruginosa through the catalytic formation of Br-HQNO on surfaces with coatings containing CeO2 enzyme mimics validates the non-toxic strategy for the development of anti-infectives.

Tuning ceria catalysts in aqueous media at the nanoscale: how do surface charge and surface defects determine peroxidase- and haloperoxidase-like reactivity.

Designing the shape and size of catalyst particles, and their interfacial charge, at the nanometer scale can radically change their performance. We demonstrate this with ceria nanoparticles. In aqueous media, nanoceria is a functional mimic of haloperoxidases, a group of enzymes that oxidize organic substrates, or of peroxidases that can degrade reactive oxygen species (ROS) such as H2O2 by oxidizing an organic substrate. We show that the chemical activity of CeO2-x nanoparticles in haloperoxidase- and peroxidase-like reactions scales with their active surface area, their surface charge, given by the ζ-potential, and their surface defects (via the Ce3+/Ce4+ ratio). Haloperoxidase-like reactions are controlled through the ζ-potential as they involve the adsorption of charged halide anions to the CeO2 surface, whereas peroxidase-like reactions without charged substrates are controlled through the specific surface area SBET. Mesoporous CeO2-x particles, with large surface areas, were prepared via template-free hydrothermal reactions and characterized by small-angle X-ray scattering. Surface area, ζ-potential and the Ce3+/Ce4+ ratio are controlled in a simple and predictable manner by the synthesis time of the hydrothermal reaction as demonstrated by X-ray photoelectron spectroscopy, sorption and ζ-potential measurements. The surface area increased with synthesis time, whilst the Ce3+/Ce4+ ratio scales inversely with decreasing ζ-potential. In this way the catalytic activity of mesoporous CeO2-x particles could be tailored selectively for haloperoxidase- and peroxidase-like reactions. The ease of tuning the surface properties of mesoporous CeO2x particles by varying the synthesis time makes the synthesis a powerful general tool for the preparation of nanocatalysts according to individual needs.

Pembrolizumab plus docetaxel for the treatment of recurrent/metastatic head and neck cancer: A prospective phase I/II study.

BACKGROUND: Taxane-based checkpoint inhibitor combination therapy might improve the outcome in recurrent/metastatic (R/M) head and neck cancer (HNSCC) patients. Thus, we investigated the efficacy and safety of docetaxel (DTX) plus pembrolizumab (P) in a prospective phase I/II trial. METHODS: Platinum-resistant R/M HNSCC patients received DTX 75 mg/m^2 plus P 200 mg for up to six cycles followed by P maintenance therapy. The primary endpoint was overall response rate (ORR) and safety. Secondary endpoints comprised disease control rate (DCR), overall survival (OS) and progression free survival (PFS). RESULTS: Twenty-two patients were enrolled. Nine patients (40.9%) had a primary tumor in the oropharynx, 8 (36.4%) in the oral cavity, 3 (13.6%) in the hypopharynx and 2 (9.1%) in the larynx. The ORR was 22.7% (95% CI 10.1%-43.4%) and one (4.5%) complete response was achieved. The DCR was 54.6% (95% 34.7%-73.1%). The median PFS was 5.8 months (95% CI 2.7-11.6) and the median OS 21.3 months (95% CI 6.3-31.1). The 1-year PFS and OS rates were 27.3% and 68.2%, respectively. While the most frequent adverse event (AE) was myelosuppression, which was reported in all 22 patients, 3 (13.6%) patients experienced grade 3 febrile neutropenia. The most common immune-related AEs were grade skin rash (40.9%) and hypothyroidism (40.9%). One patient (4.5%) experienced grade 5 immune thrombocytopenia. CONCLUSION: DXT in combination with P shows promising activity accompanied with a manageable side effect profile in pre-treated R/M HNSCC patients.

Transparent polycarbonate coated with CeO2 nanozymes repel Pseudomonas aeruginosa PA14 biofilms.

Highly transparent CeO2/polycarbonate surfaces were fabricated that prevent adhesion, proliferation, and the spread of bacteria. CeO2 nanoparticles with diameters of 10-15 nm and lengths of 100-200 nm for this application were prepared by oxidizing aqueous dispersions of Ce(OH)3 with H2O2 in the presence of nitrilotriacetic acid (NTA) as the capping agent. The surface-functionalized water-dispersible CeO2 nanorods showed high catalytic activity in the halogenation reactions, which makes them highly efficient functional mimics of haloperoxidases. These enzymes are used in nature to prevent the formation of biofilms through the halogenation of signaling compounds that interfere with bacterial cell-cell communication ("quorum sensing"). Bacteria-repellent CeO2/polycarbonate plates were prepared by dip-coating plasma-treated polycarbonate plates in aqueous CeO2 particle dispersions. The quasi-enzymatic activity of the CeO2 coating was demonstrated using phenol red enzyme assays. The monolayer coating of CeO2 nanorods (1.6 µg cm-2) and the bacteria repellent properties were demonstrated by atomic force microscopy, biofilm assays, and fluorescence measurements. The engineered polymer surfaces have the ability to repel biofilms as green antimicrobials on plastics, where H2O2 is present in humid environments such as automotive parts, greenhouses, or plastic containers for rainwater.

Selective Synthesis of Monodisperse CoO Nanooctahedra as Catalysts for Electrochemical Water Oxidation.

Thermal decomposition is a promising route for the synthesis of metal oxide nanoparticles because size and morphology can be tuned by minute control of the reaction variables. We synthesized CoO nanooctahedra with diameters of ∼48 nm and a narrow size distribution. Full control over nanoparticle size and morphology could be obtained by controlling the reaction time, surfactant ratio, and reactant concentrations. We show that the particle size does not increase monotonically with time or surfactant concentration but passes through minima or maxima. We unravel the critical role of the surfactants in nucleation and growth and rationalize the observed experimental trends in accordance with simulation experiments. The as-synthesized CoO nanooctahedra exhibit superior electrocatalytic activity with long-term stability during oxygen evolution. The morphology of the CoO particles controls the electrocatalytic reaction through the distinct surface sites involved in the oxygen evolution reaction.

Nanocomposite antimicrobials prevent bacterial growth through the enzyme-like activity of Bi-doped cerium dioxide (Ce1-xBixO2-δ).

Preventing bacterial adhesion on materials surfaces is an important problem in marine, industrial, medical and environmental fields and a topic of major medical and societal importance. A defense strategy of marine organisms against bacterial colonization relies on the biohalogenation of signaling compounds that interfere with bacterial communication. These reactions are catalyzed by haloperoxidases, a class of metal-dependent enzymes, whose activity can be emulated by ceria nanoparticles. The enzyme-like activity of ceria was enhanced by a factor of 3 through bismuth substitution (Ce1-xBixO2-δ). The solubility of Bi3+ in CeO2 is confined to the range 0 < x < 0.25 under quasi-hydrothermal conditions. The Bi3+ cations are located close to the nanoparticle surface because their ionic radii are larger than those of the tetravalent Ce4+ ions. The synthesis of Ce1-xBixO2-δ (0 < x < 0.25) nanoparticles was upscaled to yields of ∼50 g. The halogenation activity of Ce1-xBixO2-δ was demonstrated with phenol red assays. The maximum activity for x ≈ 0.2 is related to the interplay of the ζ-potential of surface-engineered Ce1-xBixO2-δ nanoparticles and their BET surface area. Ce0.80Bi0.20O1.9 nanoparticles with optimized activity were incorporated in polyethersulfone beads, which are typical constituents of water filter membrane supports. Although Ce1-xBixO2-δ nanoparticles are not bactericidal on their own, naked Ce1-xBixO2-δ nanoparticles and polyethersulfone/Ce1-xBixO2-δ nanocomposites showed a strongly reduced bacterial coverage. We attribute the decreased adhesion of the Gram-negative soil bacterium Pseudomonas aeruginosa and of Phaeobacter gallaeciensis, a primary bacterial colonizer in marine biofilms, to the formation of halogenated signaling compounds. No biocides are needed, H2O2 (formed in daylight) and halide are the only substrates required. The haloperoxidase-like activity of Ce1-xBixO2-δ may be a promising starting point for the development of environmentally friendly, "green" nanocomposites, when the use of conventional biocides is prohibited.

Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function.

T cells engineered to express chimeric antigen receptors (CAR-T cells) have shown impressive clinical efficacy in the treatment of B cell malignancies. However, the development of CAR-T cell therapies for solid tumors is hampered by the lack of truly tumor-specific antigens and poor control over T cell activity. Here we present an avidity-controlled CAR (AvidCAR) platform with inducible and logic control functions. The key is the combination of (i) an improved CAR design which enables controlled CAR dimerization and (ii) a significant reduction of antigen-binding affinities to introduce dependence on bivalent interaction, i.e. avidity. The potential and versatility of the AvidCAR platform is exemplified by designing ON-switch CARs, which can be regulated with a clinically applied drug, and AND-gate CARs specifically recognizing combinations of two antigens. Thus, we expect that AvidCARs will be a highly valuable platform for the development of controllable CAR therapies with improved tumor specificity.

Loss of NKG2D in murine NK cells leads to increased perforin production upon long-term stimulation with IL-2.

NK cells are innate lymphocytes responsible for lysis of pathogen-infected and transformed cells. One of the major activating receptors required for target cell recognition is the NK group 2D (NKG2D) receptor. Numerous reports show the necessity of NKG2D for effective tumor immune surveillance. Further studies identified NKG2D as a key element allowing tumor immune escape. We here use a mouse model with restricted deletion of NKG2D in mature NKp46+ cells (NKG2DΔNK ). NKG2DΔNK NK cells develop normally, have an unaltered IFN-γ production but kill tumor cell lines expressing NKG2D ligands (NKG2DLs) less efficiently. However, upon long-term stimulation with IL-2, NKG2D-deficient NK cells show increased levels of the lytic molecule perforin. Thus, our findings demonstrate a dual function of NKG2D for NK cell cytotoxicity; while NKG2D is a crucial trigger for cytotoxicity of tumor cells expressing activating ligands it is also capable to limit perforin production in IL-2 activated NK cells.

JAK/STAT Cytokine Signaling at the Crossroad of NK Cell Development and Maturation.

Natural Killer (NK) cells are cytotoxic lymphocytes of the innate immune system and play a critical role in anti-viral and anti-tumor responses. NK cells develop in the bone marrow from hematopoietic stem cells (HSCs) that differentiate through common lymphoid progenitors (CLPs) to NK lineage-restricted progenitors (NKPs). The orchestrated action of multiple cytokines is crucial for NK cell development and maturation. Many of these cytokines such as IL-2, IL-7, IL-12, IL-15, IL-21, IL-27, and interferons (IFNs) signal via the Janus Kinase / Signal Transducer and Activator of Transcription (JAK/STAT) pathway. We here review the current knowledge about these cytokines and the downstream signaling involved in the development and maturation of conventional NK cells and their close relatives, innate lymphoid cells type 1 (ILC1). We further discuss the role of suppressor of cytokine signaling (SOCS) proteins in NK cells and highlight their potential for therapeutic application.

Aggressive B-cell lymphomas in patients with myelofibrosis receiving JAK1/2 inhibitor therapy.

Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1-/- mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.

Dysregulated IL-18 Is a Key Driver of Immunosuppression and a Possible Therapeutic Target in the Multiple Myeloma Microenvironment.

Tumor-promoting inflammation and avoiding immune destruction are hallmarks of cancer. Here, we demonstrate that the pro-inflammatory cytokine interleukin (IL)-18 is critically involved in these hallmarks in multiple myeloma (MM). Mice deficient for IL-18 were remarkably protected from Vk∗MYC MM progression in a CD8+ T cell-dependent manner. The MM-niche-derived IL-18 drove generation of myeloid-derived suppressor cells (MDSCs), leading to accelerated disease progression. A global transcriptome analysis of the immune microenvironment in 73 MM patients strongly supported the negative impact of IL-18-driven MDSCs on T cell responses. Strikingly, high levels of bone marrow plasma IL-18 were associated with poor overall survival in MM patients. Furthermore, our preclinical studies suggested that IL-18 could be a potential therapeutic target in MM.

NK Cell-Specific CDK8 Deletion Enhances Antitumor Responses.

Cyclin-dependent kinase 8 (CDK8) is a member of the transcription-regulating CDK family. CDK8 activates or represses transcription by associating with the mediator complex or by regulating transcription factors. Oncogenic activity of CDK8 has been demonstrated in several cancer types. Targeting CDK8 represents a potential therapeutic strategy. Because knockdown of CDK8 in a natural killer (NK) cell line enhances cytotoxicity and NK cells provide the first line of immune defense against transformed cells, we asked whether inhibiting CDK8 would improve NK-cell antitumor responses. In this study, we investigated the role of CDK8 in NK-cell function in vivo using mice with conditional ablation of CDK8 in NKp46+ cells (Cdk8fl/flNcr1Cre). Regardless of CDK8 expression, NK cells develop and mature normally in bone marrow and spleen. However, CDK8 deletion increased expression of the lytic molecule perforin, which correlated with enhanced NK-cell cytotoxicity in vitro This translates into improved NK cell-mediated tumor surveillance in vivo in three independent models: B16F10 melanoma, v-abl+ lymphoma, and a slowly developing oncogene-driven leukemia. Our results thereby define a suppressive effect of CDK8 on NK-cell activity. Therapies that target CDK8 in cancer patients may enhance NK-cell responses against tumor cells. Cancer Immunol Res; 6(4); 458-66. ©2018 AACR.