Evident phase separation and surface segregation of hydrophobic moieties at the copolymer surface using atomic force microscopy and SFG spectroscopy.

HYPOTHESIS: Copolymers are developed to enhance the overall physical and chemical properties of polymers. The surface nature of a copolymer is relevant to creating efficient materials to improve adhesion and biocompatibility. We hypothesize that the improved adhesion, as a surface property, is due to phase separation, surface segregation, and the overall molecular organization of different polymer components at the copolymer surface. EXPERIMENTS: The surface structure of a copolymer composed of 2-hydroxyethyl methacrylate (HEMA) monomer and 2-phenoxyethyl methacrylate (PhEMA) monomer was analyzed in comparison to the polyHEMA and polyPhEMA homopolymers using atomic force microscopy (AFM) and sum frequency generation (SFG) spectroscopy. FINDINGS: The contrast in the phase images was due to the variance in the hydrophobic level provided by the hydroxyl and phenoxy modified monomers in the copolymer. The distribution of the adhesion values, supporting the presence of hydrophobic moieties, across the polymer surface defined the surface segregation of these two components. SFG spectra of the copolymer thin film showed combined spectral features of both polyHEMA and polyPhEMA thin films at the polymer surface. The tilt angles of the alpha-methyl group of homopolymers using the polarization intensity ratio analysis and the polarization mapping method were estimated to be in the range from 48° to 66°.

PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse.

BACKGROUND: The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. OBJECTIVE: We sought to determine the effect of PD-1 signaling on NK cells. METHODS: PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). RESULTS: PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. CONCLUSION: Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function.

Immunological Synapse Predicts Effectiveness of Chimeric Antigen Receptor Cells.

Chimeric antigen receptor (CAR)-modified T cell therapy has the potential to improve the overall survival of patients with malignancies by enhancing the effectiveness of CAR T cells. Precisely predicting the effectiveness of various CAR T cells represents one of today's key unsolved problems in immunotherapy. Here, we predict the effectiveness of CAR-modified cells by evaluating the quality of the CAR-mediated immunological synapse (IS) by quantitation of F-actin, clustering of tumor antigen, polarization of lytic granules (LGs), and distribution of key signaling molecules within the IS. Long-term killing capability, but not secretion of conventional cytokines or standard 4-hr cytotoxicity, correlates positively with the quality of the IS in two different CAR T cells that share identical antigen specificity. Xenograft model data confirm that the quality of the IS in vitro correlates positively with performance of CAR-modified immune cells in vivo. Therefore, we propose that the quality of the IS predicts the effectiveness of CAR-modified immune cells, which provides a novel strategy to guide CAR therapy.

Microfluidic Mapping of Cancer Cell-Protein Binding Interaction.

The interaction between tumor cells and microenvironment during metastasis is mediated by the binding of cell surface receptors, such as integrins and selectins, with protein ligands. Delineation of their binding interaction and identification of key receptors may be particularly important both in understanding extracellular matrix (ECM) remodeling and in developing potential therapeutic targets. Here we present a microfluidic chip that allows qualitative and quantitative mapping of a large population cell-protein interactions. It was found that ß1 integrin showed stronger binding interaction with collagen than with other ECM proteins. The upregulated ß1 integrin in invasive cancer cells enhanced cell-ECM interaction and may promote ECM remodeling. Cancer cells also showed strong interaction with plasma fibrinogen, the elevated level of which may help cancer cells arrest on blood vessels. We also verified that the chip may provide a platform for drug discovery by targeting integrins and cytoskeletons.

Microfluidics Cell Loading-Dock System: Ordered Cellular Array for Dynamic Lymphocyte-Communication Study.

It remains a great challenge to establish a high-throughput platform that can explore the interactions among multiple lymphocytes (>2 cells) and retrieve the interested cells for downstream analysis. This study demonstrates a microfluidics cell loading-dock system (Cell-Dock) to enclose multiple cells in 1D, 2D, and 3D chambers with high throughput and efficiency and single-cell accuracy. The loading efficiencies of 95%, 85%, and 74% for one-, three-, and five-cell systems are achieved, respectively. The Cell-Dock system provides precise and dynamic cell packing models to facilitate lymphocyte-interaction studies. The results demonstrate that individual natural killer (NK) cells may function independently rather than cooperate to lyse target cells in the defined microenvironment. Furthermore, the strong/weak NK cells are retrieved based on their on-chip cytotoxicity and mRNA sequencing is conducted to find the possible mechanisms for "serial killing," an important but unsolved issue. This study finds that the stronger NK cells overexpress multiple genes involved in cytotoxicity and adhesion molecules (including the well-known ICAM1 and seldom reported B4GALT1) might play important roles in the regulation of NK cytolysis.

Microfluidic cytometric analysis of cancer cell transportability and invasiveness.

The extensive phenotypic and functional heterogeneity of cancer cells plays an important role in tumor progression and therapeutic resistance. Characterizing this heterogeneity and identifying invasive phenotype may provide possibility to improve chemotherapy treatment. By mimicking cancer cell perfusion through circulatory system in metastasis, we develop a unique microfluidic cytometry (MC) platform to separate cancer cells at high throughput, and further derive a physical parameter 'transportability' to characterize the ability to pass through micro-constrictions. The transportability is determined by cell stiffness and cell-surface frictional property, and can be used to probe tumor heterogeneity, discriminate more invasive phenotypes and correlate with biomarker expressions in breast cancer cells. Decreased cell stiffness and cell-surface frictional force leads to an increase in transportability and may be a feature of invasive cancer cells by promoting cell perfusion through narrow spaces in circulatory system. The MC-Chip provides a promising microfluidic platform for studying cell mechanics and transportability could be used as a novel marker for probing tumor heterogeneity and determining invasive phenotypes.

Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics.

The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. In this study, we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single-cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically "stacked" cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1, and the cytotoxic synapse at the single-cell level. In addition to the technique innovation, we have demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, programed death protein-1 microclusters at the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines.

Quantitative orientation analysis by sum frequency generation in the presence of near-resonant background signal: acetonitrile on rutile TiO2 (110).

Sum frequency generation (SFG) spectroscopic techniques are used to investigate the molecular orientation of adsorbed acetonitrile on rutile TiO2 (110) at the solid-vapor interface. Generally, most molecular orientation analyses using SFG have been performed on dielectric substrates, to avoid the spectral interference between resonant and the near-resonant background signal. Although rutile crystal can be treated as a dielectric substrate, its electronic state contributes to the intensity and interferes with the resonant signal when the SFG frequency is close to its band gap energy. In addition, the rutile crystal is a uniaxial birefringent material, and the (110) surface is anisotropic, which further complicates the spectral analysis. In this study, various SFG measurement techniques were applied, and quantitative analytical methods were established to interpret the surface orientation of an adsorbed molecule. SFG vibrational spectra of acetonitrile on rutile TiO2 (110) surface have been measured using distinct polarization combinations, polarization mapping, and null angle method. By varying the polarization combinations of SFG, the magnitude and shape of the spectra undergo substantial change, which originate from the interference between the near-resonant signal from the rutile substrate and the resonance signal from the acetonitrile. Theory, simulation, and analytical methods for obtaining quantitative orientation information of a molecule on an anisotropic semiconductor substrate in the presence of a near-resonant signal are presented.

Photoregulated fluorescence switching in axially coordinated tin(IV) porphyrinic dithienylethene.

Photochromic fluorophore Sn(TTP)(DTE)2 , in which two phenolic derivatives of 1,2-dithienylethene are axially coordinated to (5,10,15,20-tetratolylporphyrinato)tin(IV) in trans position, has been synthesized and fully characterized by various spectroscopic methods. We have also investigated the photoregulated fluorescence switching behavior of Sn(TTP)(DTE)2 . The fluorescence of the porphyrin macrocycle in Sn(TTP)(DTE) 2 greatly depends on the state of the 1,2-dithienyletene photochromic switch. In the open state (Sn(TTP)(o-DTE)2), the porphyrin exhibits high fluorescence intensity at 609 and 664 nm when excited at 410 nm. When the photocyclization reaction was carried out by irradiating Sn(TTP)(o-DTE)2 with the UV light (approximately 365 nm), the fluorescence intensity of the porphyrin macrocycle decreased. Back irradiation with visible light at wavelengths greater than 500 nm regenerated Sn(TTP)(o-DTE)2 and almost restored the original fluorescence spectrum. The fluorescence intensity of the porphyrin fluorophore is efficiently regulated by photochromic switching between Sn(TTP)(o-DTE)2 and Sn(TTP)(c-DTE)2 in several cycles, clearly demonstrating that the Sn(TTP)(DTE)2 can act as a system for reversible data processing using fluorescence as the detection method.