Multiplexed Detection of Molecular Interactions with DNA Origami Engineered Cells in 3D Collagen Matrices.

The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a three-dimensional (3D) microenvironment can inform detection and understanding of cell processes and the development of effective therapeutic agents. DNA origami technology allows for the design and fabrication of biocompatible and 3D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3D tissue model using a microfluidic device. We used a DNA origami cell sensing platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3D environments under controlled biomolecular transport.

DNA Origami Nanostructures Elicit Dose-Dependent Immunogenicity and Are Nontoxic up to High Doses In Vivo.

DNA origami (DO) nanotechnology enables the construction of precise nanostructures capable of functionalization with small molecule drugs, nucleic acids, and proteins, suggesting a promising platform for biomedical applications. Despite the potential for drug and vaccine delivery, the impact of DO vehicles on immunogenicity in vivo is not well understood. Here, two DO vehicles, a flat triangle and a nanorod, at varying concentrations are evaluated in vitro and with a repeated dosing regimen administered at a high dose in vivo to study early and late immunogenicity. The studies show normal CD11b+ myeloid cell populations preferentially internalize DO in vitro. DO structures distribute well systemically in vivo, elicit a modest pro-inflammatory immune response that diminishes over time and are nontoxic as shown by weight, histopathology, lack of cytokine storm, and a complete biochemistry panel at the day 10 end point. The results take critical steps to characterize the biological response to DO and suggest that DO vehicles represent a promising platform for drug delivery and vaccine development where immunogenicity should be a key consideration.

Anti-BAFF-R antibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib.

The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib has transformed chronic lymphocytic leukemia (CLL) therapy but requires continuous administration. These factors have spurred interest in combination treatments. Unlike with chemotherapy, CD20-directed antibody therapy has not improved the outcome of BTKi treatment. Whereas CD20 antigen density on CLL cells decreases during ibrutinib treatment, the B-cell activating factor (BAFF) and its receptor (BAFF-R) remain elevated. Furthermore, BAFF signaling via noncanonical NF-κB remains elevated with BTKi treatment. Blocking BAFF interaction with BAFF-R by using VAY-736, a humanized defucosylated engineered antibody directed against BAFF-R, antagonized BAFF-mediated apoptosis protection and signaling at the population and single-cell levels in CLL cells. Furthermore, VAY-736 showed superior antibody-dependent cellular cytotoxicity compared with CD20- and CD52-directed antibodies used in CLL. VAY-736 exhibited in vivo activity as a monotherapy and, when combined with ibrutinib, produced prolonged survival compared with either therapy alone. The in vivo activity of VAY-736 is dependent upon immunoreceptor tyrosine-based activation motif (ITAM)-mediated activation of effector cells as shown by using an ITAM-deficient mouse model. Collectively, our findings support targeting the BAFF signaling pathway with VAY-736 to more effectively treat CLL as a single agent and in combination with ibrutinib.

Cruciferous Vegetables, Isothiocyanates, and Bladder Cancer Prevention.

Bladder cancer is a significant health burden due to its high prevalence, risk of mortality, morbidity, and high cost of medical care. Epidemiologic evidence suggests that diets rich in cruciferous vegetables, particularly broccoli, are associated with lower bladder cancer risk. Phytochemicals in cruciferous vegetables, such as glucosinolates, which are enzymatically hydrolyzed to bioactive isothiocyanates, are possible mediators of an anticancer effect. In vitro studies have shown inhibition of bladder cancer cell lines, cell cycle arrest, and induction of apoptosis by these isothiocyanates, in particular sulforaphane and erucin. Although not yet completely understood, many mechanisms of anticancer activity at the steps of cancer initiation, promotion, and progression have been attributed to these isothiocyanates. They target multiple pathways including the adaptive stress response, phase I/II enzyme modulation, pro-growth, pro-survival, pro-inflammatory signaling, angiogenesis, and even epigenetic modulation. Multiple in vivo studies have shown the bioavailability of isothiocyanates and their antitumoral effects. Although human studies are limited, they support oral bioavailability with reasonable plasma and urine concentrations achieved. Overall, both cell and animal studies support a potential role for isothiocyanates in bladder cancer prevention and treatment. Future studies are necessary to examine clinically relevant outcomes and define guidelines on ameliorating the bladder cancer burden.

Engineering Cell Surface Function with DNA Origami.

A specific and reversible method is reported to engineer cell-membrane function by embedding DNA-origami nanodevices onto the cell surface. Robust membrane functionalization across epithelial, mesenchymal, and nonadherent immune cells is achieved with DNA nanoplatforms that enable functions including the construction of higher-order DNA assemblies at the cell surface and programed cell-cell adhesion between homotypic and heterotypic cells via sequence-specific DNA hybridization. It is anticipated that integration of DNA-origami nanodevices can transform the cell membrane into an engineered material that can mimic, manipulate, and measure biophysical and biochemical function within the plasma membrane of living cells.

The impact of cruciferous vegetable isothiocyanates on histone acetylation and histone phosphorylation in bladder cancer.

Cruciferous vegetable intake is associated with reduced risk of bladder cancer, yet mechanisms remain unclear. Cruciferous vegetable isothiocyanates (ITCs), namely sulforaphane (SFN) and erucin (ECN), significantly inhibit histone deacetylase (HDAC) activity in human bladder cancer cells representing superficial to invasive biology (59-83% inhibition with 20µM, 48h treatment), and in bladder cancer xenografts (59±3% ECN inhibition). Individual HDACs inhibited by SFN and ECN include HDACs 1, 2, 4 and 6. Interestingly, global acetylation status of histones H3 or H4 remain unaltered. The interplay between HDAC inhibition and modest modulation of AcH3 and AcH4 status is partially explained by decreased histone acetyl transferase activity (48.8±5.3%). In contrast, a significant decrease in phosphorylation status of all isoforms of histone H1 was observed, concomitant with increased phosphatase PP1ß and PP2A activity. Together, these findings suggest that ITCs modulate histone status via HDAC inhibition and phosphatase enhancement. This allows for reduced levels of histone H1 phosphorylation, a marker correlated with human bladder cancer progression. Therefore, ITC-mediated inhibition of histone H1 phosphorylation presents a novel direction of research in elucidating epidemiological relationships and supports future food-based prevention strategies. SIGNIFICANCE: Collectively, our findings suggest that the cruciferous vegetable isothiocyanates: sulforaphane (SFN) and erucin (ECN), impact histones status in bladder cancer cells by modulating specific HDACs and HATs, and enhancing phosphatase activity, resulting in reduction of histone H1 phosphorylation. These findings are significant due to the fact that our previous work positively correlated histone H1 phosphorylation with bladder cancer carcinogenesis and progression. Therefore, we propose that SFN and ECN may inhibit bladder carcinogenesis via epigenetic modulation of gene expression associated with histone H1 phosphorylation. These efforts may elucidate biomarkers useful in epidemiologic studies related to cruciferous vegetable intake and cancer risk or provide intermediate biomarkers for food-based clinical intervention studies in high-risk cohorts.

Probing Nucleosome Stability with a DNA Origami Nanocaliper.

The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structural changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances, including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6, 26, and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remain fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding but also significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.

DNA Origami: Folded DNA-Nanodevices That Can Direct and Interpret Cell Behavior.

DNA origami is a DNA-based nanotechnology that utilizes programmed combinations of short complementary oligonucleotides to fold a large single strand of DNA into precise 2D and 3D shapes. The exquisite nanoscale shape control of this inherently biocompatible material is combined with the potential to spatially address the origami structures with diverse cargoes including drugs, antibodies, nucleic acid sequences, small molecules, and inorganic particles. This programmable flexibility enables the fabrication of precise nanoscale devices that have already shown great potential for biomedical applications such as: drug delivery, biosensing, and synthetic nanopore formation. Here, the advances in the DNA-origami field since its inception several years ago are reviewed with a focus on how these DNA-nanodevices can be designed to interact with cells to direct or probe their behavior.

Daunorubicin-Loaded DNA Origami Nanostructures Circumvent Drug-Resistance Mechanisms in a Leukemia Model.

Many cancers show primary or acquired drug resistance due to the overexpression of efflux pumps. A novel mechanism to circumvent this is to integrate drugs, such as anthracycline antibiotics, with nanoparticle delivery vehicles that can bypass intrinsic tumor drug-resistance mechanisms. DNA nanoparticles serve as an efficient binding platform for intercalating drugs (e.g., anthracyclines doxorubicin and daunorubicin, which are widely used to treat acute leukemias) and enable precise structure design and chemical modifications, for example, for incorporating targeting capabilities. Here, DNA nanostructures are utilized to circumvent daunorubicin drug resistance at clinically relevant doses in a leukemia cell line model. The fabrication of a rod-like DNA origami drug carrier is reported that can be controllably loaded with daunorubicin. It is further directly verified that nanostructure-mediated daunorubicin delivery leads to increased drug entry and retention in cells relative to free daunorubicin at equal concentrations, which yields significantly enhanced drug efficacy. Our results indicate that DNA origami nanostructures can circumvent efflux-pump-mediated drug resistance in leukemia cells at clinically relevant drug concentrations and provide a robust DNA nanostructure design that could be implemented in a wide range of cellular applications due to its remarkably fast self-assembly (≈5 min) and excellent stability in cell culture conditions.

Prohibitins and the cytoplasmic domain of CD86 cooperate to mediate CD86 signaling in B lymphocytes.

CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that promote NF-κB activation to increase Oct-2 and mature IgG1 mRNA and protein expression, as well as the rate of IgG1 transcription, without affecting class switch recombination. One of the most proximal signaling intermediates identified is phospholipase Cγ2, a protein reported to bind tyrosine residues, which are absent in the cytoplasmic domain of CD86. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins prohibitin (Phb)1 and Phb2 bind to CD86. The basal expression of Phb1/2 and association with CD86 was low in resting B cells, whereas the level of expression and association increased primarily after priming with CD40. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by short hairpin RNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine protein kinase C phosphorylation sites, which did not affect Phb1/2 binding to CD86. Using this approach, we also show that Phb1/2 and the CD86 cytoplasmic domain are required for the CD86-induced phosphorylation of IκBα, which we previously reported leads to NF-κB p50/p65 activation, whereas only Phb1/2 was required for the CD86-induced phosphorylation of phospholipase Cγ2 and protein kinase Cα/ß(II), which we have previously reported leads to NF-κB (p65) phosphorylation and subsequent nuclear translocation. Taken together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell through differential phosphorylation of distal signaling intermediates required to increase IgG1.