Cytotoxic lymphocytes target characteristic biophysical vulnerabilities in cancer.

Immune cells identify and destroy tumors by recognizing cellular traits indicative of oncogenic transformation. In this study, we found that myocardin-related transcription factors (MRTFs), which promote migration and metastatic invasion, also sensitize cancer cells to the immune system. Melanoma and breast cancer cells with high MRTF expression were selectively eliminated by cytotoxic lymphocytes in mouse models of metastasis. This immunosurveillance phenotype was further enhanced by treatment with immune checkpoint blockade (ICB) antibodies. We also observed that high MRTF signaling in human melanoma is associated with ICB efficacy in patients. Using biophysical and functional assays, we showed that MRTF overexpression rigidified the filamentous actin cytoskeleton and that this mechanical change rendered mouse and human cancer cells more vulnerable to cytotoxic T lymphocytes and natural killer cells. Collectively, these results suggest that immunosurveillance has a mechanical dimension, which we call mechanosurveillance, that is particularly relevant for the targeting of metastatic disease.

Caught in Action: Visualizing Dynamic Nanostructures Within Supramolecular Systems Chemistry.

Supramolecular systems chemistry has been an area of active research to develop nanomaterials with life-like functions. Progress in systems chemistry relies on our ability to probe the nanostructure formation in solution. Often visualizing the dynamics of nanostructures which transform over time is a formidable challenge. This necessitates a paradigm shift from dry sample imaging towards solution-based techniques. We review the application of state-of-the-art techniques for real-time, in situ visualization of dynamic self-assembly processes. We present how solution-based techniques namely optical super-resolution microscopy, solution-state atomic force microscopy, liquid-phase transmission electron microscopy, molecular dynamics simulations and other emerging techniques are revolutionizing our understanding of active and adaptive nanomaterials with life-like functions. This Review provides the visualization toolbox and futuristic vision to tap the potential of dynamic nanomaterials.

Supramolecular systems chemistry through advanced analytical techniques.

Supramolecular chemistry is the quintessential backbone of all biological processes. It encompasses a wide range from the metabolic network to the self-assembled cytoskeletal network. Combining the chemical diversity with the plethora of functional depth that biological systems possess is a daunting task for synthetic chemists to emulate. The only route for approaching such a challenge lies in understanding the complex and dynamic systems through advanced analytical techniques. The supramolecular complexity that can be successfully generated and analyzed is directly dependent on the analytical treatment of the system parameters. In this review, we illustrate advanced analytical techniques that have been used to investigate various supramolecular systems including complex mixtures, dynamic self-assembly, and functional nanomaterials. The underlying theme of such an overview is not only the exceeding detail with which traditional experiments can be probed but also the fact that complex experiments can now be attempted owing to the analytical techniques that can resolve an ensemble in astounding detail. Furthermore, the review critically analyzes the current state of the art analytical techniques and suggests the direction of future development. Finally, we envision that integrating multiple analytical methods into a common platform will open completely new possibilities for developing functional chemical systems.

Nanoscale Wetting of Single Viruses.

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ~1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

High-throughput protein nanopatterning.

High-throughput and large-scale patterning of enzymes with sub-10 nm resolution, the size range of individual protein molecules, is crucial for propelling advancement in a variety of areas, from the development of chip-based biomolecular nano-devices to molecular-level studies of cell biology. Despite recent developments in bio-nanofabrication technology, combining 10 nm resolution with high-throughput and large-scale patterning of enzymes is still an open challenge. Here, we demonstrate a high resolution and high-throughput patterning method to generate enzyme nanopatterns with sub-10 nm resolution by using thermochemical scanning probe lithography (tc-SPL). First, tc-SPL is used to generate amine patterns on a methacrylate copolymer film. Thermolysin enzymes functionalized with sulfonate-containing fluorescent labels (Alexa-488) are then directly immobilized onto the amine patterns through electrostatic interaction. Enzyme patterns with sub-10 nm line width are obtained as evidenced by atomic force microscopy (AFM) and fluorescence microscopy. Moreover, we demonstrate large-scale and high throughput (0.13 × 0.1 mm2 at a throughput of 5.2 × 104 µm2 h-1) patterning of enzymes incorporating 10 nm detailed pattern features. This straightforward and high-throughput method of fabricating enzyme nanopatterns will have a significant impact on future bio-nanotechnology applications and molecular-level biological studies. By scaling up using parallel probes, tc-SPL is promising for implementation to scale up the fabrication of nano-biodevices.

Reprogrammed Schwann Cells Organize into Dynamic Tracks that Promote Pancreatic Cancer Invasion.

Nerves are a component of the tumor microenvironment contributing to cancer progression, but the role of cells from nerves in facilitating cancer invasion remains poorly understood. Here we show that Schwann cells (SC) activated by cancer cells collectively function as tumor-activated Schwann cell tracks (TAST) that promote cancer cell migration and invasion. Nonmyelinating SCs form TASTs and have cell gene expression signatures that correlate with diminished survival in patients with pancreatic ductal adenocarcinoma. In TASTs, dynamic SCs form tracks that serve as cancer pathways and apply forces on cancer cells to enhance cancer motility. These SCs are activated by c-Jun, analogous to their reprogramming during nerve repair. This study reveals a mechanism of cancer cell invasion that co-opts a wound repair process and exploits the ability of SCs to collectively organize into tracks. These findings establish a novel paradigm of how cancer cells spread and reveal therapeutic opportunities. SIGNIFICANCE: How the tumor microenvironment participates in pancreatic cancer progression is not fully understood. Here, we show that SCs are activated by cancer cells and collectively organize into tracks that dynamically enable cancer invasion in a c-Jun-dependent manner. See related commentary by Amit and Maitra, p. 2240. This article is highlighted in the In This Issue feature, p. 2221.

Time-temperature integrator based on the dewetting of polyisobutylene thin films.

This work reports the application of a patterned thin film of polyisobutylene (PIB) irradiated with an electron beam as a time-temperature integrator, i.e., a device that is able to record the thermal history of a product. The device is fabricated by irradiation with an electron beam of regions of a PIB thin film to different doses of electrons. A different dewetting behavior occurs at these regions upon thermal exposure, depending on the dose. The experimental results are quantified by means of a model of dewetting based on nucleation and growth of holes in a strong slippage regime.

Spatial mapping of the collagen distribution in human and mouse tissues by force volume atomic force microscopy.

Changes in the elastic properties of living tissues during normal development and in pathological processes are often due to modifications of the collagen component of the extracellular matrix at various length scales. Force volume AFM can precisely capture the mechanical properties of biological samples with force sensitivity and spatial resolution. The integration of AFM data with data of the molecular composition contributes to understanding the interplay between tissue biochemistry, organization and function. The detection of micrometer-size, heterogeneous domains at different elastic moduli in tissue sections by AFM has remained elusive so far, due to the lack of correlations with histological, optical and biochemical assessments. In this work, force volume AFM is used to identify collagen-enriched domains, naturally present in human and mouse tissues, by their elastic modulus. Collagen identification is obtained in a robust way and affordable timescales, through an optimal design of the sample preparation method and AFM parameters for faster scan with micrometer resolution. The choice of a separate reference sample stained for collagen allows correlating elastic modulus with collagen amount and position with high statistical significance. The proposed preparation method ensures safe handling of the tissue sections guarantees the preservation of their micromechanical characteristics over time and makes it much easier to perform correlation experiments with different biomarkers independently.

Spatial defects nanoengineering for bipolar conductivity in MoS2.

Understanding the atomistic origin of defects in two-dimensional transition metal dichalcogenides, their impact on the electronic properties, and how to control them is critical for future electronics and optoelectronics. Here, we demonstrate the integration of thermochemical scanning probe lithography (tc-SPL) with a flow-through reactive gas cell to achieve nanoscale control of defects in monolayer MoS2. The tc-SPL produced defects can present either p- or n-type doping on demand, depending on the used gasses, allowing the realization of field effect transistors, and p-n junctions with precise sub-µm spatial control, and a rectification ratio of over 104. Doping and defects formation are elucidated by means of X-Ray photoelectron spectroscopy, scanning transmission electron microscopy, and density functional theory. We find that p-type doping in HCl/H2O atmosphere is related to the rearrangement of sulfur atoms, and the formation of protruding covalent S-S bonds on the surface. Alternatively, local heating MoS2 in N2 produces n-character.

Measurement of DNA morphological parameters at highly entangled regime on surfaces.

The morphology of circular DNA deposited from a solution on the mica surface is analyzed from the power spectrum density (PSD) of the atomic force microscopy (AFM) images. Sample morphology is modulated in a broad range of concentration C from isolated molecules to highly entangled networks. DNA exhibits a multiaffine behavior with two correlation length scales: the persistence length P which remains constant ( approximately 50 nm) within the C range and the intermolecular distance xi which exhibits a decay with increasing C. Applying a diffusion based model in which xi scales as xi approximately D(-0.25).C(-0.5), we extracted the DNA diffusion coefficient D approximately 2 x 10(-7) cm(2)/s. This value is consistent with a high-molecular-weight plasmid DNA supercoiled in the solution.

Sub-10 nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control.

The ability to precisely control the localization of enzymes on a surface is critical for several applications including biosensing, bionanoreactors, and single molecule studies. Despite recent advances, fabrication of enzyme patterns with resolution at the single enzyme level is limited by the lack of lithography methods that combine high resolution, compatibility with soft, polymeric structures, ease of fabrication, and high throughput. Here, a method to generate enzyme nanopatterns (using thermolysin as a model system) on a polymer surface is demonstrated using thermochemical scanning probe lithography (tc-SPL). Electrostatic immobilization of negatively charged sulfonated enzymes occurs selectively at positively charged amine nanopatterns produced by thermal deprotection of amines along the side-chain of a methacrylate-based copolymer film via tc-SPL. This process occurs simultaneously with local thermal quasi-3D topographical patterning of the same polymer, offering lateral sub-10 nm resolution, and vertical 1 nm resolution, as well as high throughput (5.2 × 104 µm2/h). The obtained single-enzyme resolution patterns are characterized by atomic force microscopy (AFM) and fluorescence microscopy. The enzyme density, the surface passivation, and the quasi-3D arbitrary geometry of these patterned pockets are directly controlled during the tc-SPL process in a single step without the need of markers or masks. Other unique features of this patterning approach include the combined single-enzyme resolution over mm2 areas and the possibility of fabricating enzymes nanogradients.

Monolayer control of discotic liquid crystal by electromigration of dewetted layers in thin film devices.

We show that ultrathin films of a semiconductive discotic liquid crystal, viz. phthalocyanines, can be organized to form a conductive channel tens of microns long between Au electrodes with thickness control over a single monolayer. Our approach exploits the electromigration of the isotropic phase formed starting from the pretransitional region of the columnar-isotropic phase transition. Dewetted isotropic material accumulates to the negative electrode by applying a longitudinal electric field of about 1 V/microm. Dewetting and electromigration expose an ultrathin film, a few monolayers thick, exhibiting columnar liquid crystal order. The layers of this ultrathin film melt progressively above T(C) and can be individually exfoliated by electromigration, starting from the ninth down to the first monolayer. The analysis of the current flowing through the junction as a function of the temperature, together with the comparative imaging of the evolution of morphology, yields a detailed picture of the changes in the dimensionality of the conductive phthalocyanine film and allows us to extract the behavior of the order parameter. The phenomenon of electromigration opens interesting questions on the technological control of individual monolayers on device patterns.

Friction and work function oscillatory behavior for an even and odd number of layers in polycrystalline MoS2.

A large effort is underway to investigate the properties of two-dimensional (2D) materials for their potential to become building blocks in a variety of integrated nanodevices. In particular, the ability to understand the relationship between friction, adhesion, electric charges and defects in 2D materials is of key importance for their assembly and use in nano-electro-mechanical and energy harvesting systems. Here, we report on a new oscillatory behavior of nanoscopic friction in continuous polycrystalline MoS2 films for an odd and even number of atomic layers, where odd layers show higher friction and lower work function. Friction force microscopy combined with Kelvin probe force microscopy and X-ray photoelectron spectroscopy demonstrates that an enhanced adsorption of charges and OH molecules is at the origin of the observed increase in friction for 1 and 3 polycrystalline MoS2 layers. In polycrystalline films with an odd number of layers, each crystalline nano-grain carries a dipole due to the MoS2 piezoelectricity, therefore charged molecules adsorb at the grain boundaries all over the surface of the continuous MoS2 film. Their displacement during the sliding of a nano-size tip gives rise to the observed enhanced dissipation and larger nanoscale friction for odd layer-numbers. Similarly, charged adsorbed molecules are responsible for the work function decrease in odd layer-number.

Key factors of scanning a plant virus with AFM in air and aqueous solution.

For tobacco mosaic virus (TMV) as a model virus, this article shows typical issues of scanning soft biological matter by atomic force microscopy (AFM). TMV adsorbed on chemically different flat surfaces, gold, mica, and APDMES-functionalized silicon, is studied in air and aqueous environment. In air, the TMV particles arrangement shows some variety, depending on the substrate. The height of TMV is reduced to 13.7, 15.8, and 15.6 nm, for gold, APDMES, and mica, respectively while the width is about ∼30 nm due to the influence of the tip radius. In aqueous solution, the surface charges of the virus and the solid support play an important role in the virus adsorption process. While deposition on negatively charged mica is favored only at low pH values, it is shown that positively charged APDMES functionalized silicon can be a suitable substrate to work with at neutral pHs. The effects of cantilever oscillation's free amplitude (A0 ) and the amplitude set-point (A) are also assessed here. While high A0 prompt reversible deformation of TMV in measurements performed in air, irreversible damage of the virus in liquid conditions (water) is observed using stiff cantilevers (0.35 N m-1 ) and high A0 (81 nm), leading to a 6 nm reduction in the height of TMV after the first scan. Finally, low values of the amplitude set-point (A/A0 = 0.3), which means applying higher forces to the sample, also brings the damage of TMV virus assemblies, reducing its monolayer roughness to 0.3 nm. Microsc. Res. Tech. 80:18-29, 2017. © 2016 Wiley Periodicals, Inc.

Quantification of interacting cognate odorants with olfactory receptors in nanovesicles.

This study aims to improve our understanding of the interaction between olfactory receptors and odorants to develop highly selective biosensing devices. Natural nanovesicles (NVs) from Saccharomyces cerevisiae, ~100 nm in diameter, carrying either the human OR17-40 or the chimpanzee OR7D4 olfactory receptor (OR) tagged with the c-myc epitope at their N-terminus, are presented as model systems to quantify the interaction between odorant and olfactory receptors. The level of expression of olfactory receptors was determined at individual NVs using a novel competitive ELISA immunoassay comparing the values obtained against those from techniques involving the solubilization of cell membrane proteins and the identification of c-myc-carrying receptors. Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips indicate that cognate odorants bind to their Ors, thereby quantifying the approximate number of odorants that interact with a given olfactory receptor. The selectivity of OR17-40-carrying NVs towards helional and OR7D4-carrying NVs towards androstenone has been proven in cross-check experiments with non-specific odorant molecules (heptanal and pentadecalactone, respectively) and in control receptors.

Multifrequency Force Microscopy of Helical Protein Assembly on a Virus.

High-resolution microscopy techniques have been extensively used to investigate the structure of soft, biological matter at the nanoscale, from very thin membranes to small objects, like viruses. Electron microscopy techniques allow for obtaining extraordinary resolution by averaging signals from multiple identical structures. In contrast, atomic force microscopy (AFM) collects data from single entities. Here, it is possible to finely modulate the interaction with the samples, in order to be sensitive to their top surface, avoiding mechanical deformations. However, most biological surfaces are highly curved, such as fibers or tubes, and ultimate details of their surface are in the vicinity of steep height variations. This limits lateral resolution, even when sharp probes are used. We overcome this problem by using multifrequency force microscopy on a textbook example, the Tobacco Mosaic Virus (TMV). We achieved unprecedented resolution in local maps of amplitude and phase shift of the second excited mode, recorded together with sample topography. Our data, which combine multifrequency imaging and Fourier analysis, confirm the structure deduced from averaging techniques (XRD, cryoEM) for surface features of single virus particles, down to the helical pitch of the coat protein subunits, 2.3 nm. Remarkably, multifrequency AFM images do not require any image postprocessing.

Capillary and van der Waals interactions on CaF2 crystals from amplitude modulation AFM force reconstruction profiles under ambient conditions.

There has been much interest in the past two decades to produce experimental force profiles characteristic of the interaction between nanoscale objects or a nanoscale object and a plane. Arguably, the advent of the atomic force microscope AFM was instrumental in driving such efforts because, in principle, force profiles could be recovered directly. Nevertheless, it has taken years before techniques have developed enough as to recover the attractive part of the force with relatively low noise and without missing information on critical ranges, particularly under ambient conditions where capillary interactions are believed to dominate. Thus a systematic study of the different profiles that may arise in such situations is still lacking. Here we employ the surfaces of CaF2, on which nanoscale water films form, to report on the range and force profiles that might originate by dynamic capillary interactions occurring between an AFM tip and nanoscale water patches. Three types of force profiles were observed under ambient conditions. One in which the force decay resembles the well-known inverse-square law typical of van der Waals interactions during the first 0.5-1 nm of decay, a second one in which the force decays almost linearly, in relatively good agreement with capillary force predicted by the constant chemical potential approximation, and a third one in which the attractive force is almost constant, i.e., forms a plateau, up to 3-4 nm above the surface when the formation of a capillary neck dominates the tip-sample interaction.

Force measurements on natural membrane nanovesicles reveal a composition-independent, high Young's modulus.

Mechanical properties of nano-sized vesicles made up of natural membranes are crucial to the development of stable, biocompatible nanocontainers with enhanced functional, recognition and sensing capabilities. Here we measure and compare the mechanical properties of plasma and inner membrane nanovesicles ∼80 nm in diameter obtained from disrupted yeast Saccharomyces cerevisiae cells. We provide evidence of a highly deformable behaviour for these vesicles, able to support repeated wall-to-wall compressions without irreversible deformations, accompanied by a noticeably high Young's modulus (∼300 MPa) compared to that obtained for reconstituted artificial liposomes of similar size and approaching that of some virus particles. Surprisingly enough, the results are approximately similar for plasma and inner membrane nanovesicles, in spite of their different lipid compositions, especially on what concerns the ergosterol content. These results point towards an important structural role of membrane proteins in the mechanical response of natural membrane vesicles and open the perspective to their potential use as robust nanocontainers for bioapplications.

Synchronized optical and electrical characterization of discotic liquid crystals thin films.

We describe a setup suitable for simultaneously measuring optical and electrical properties of a liquid crystal mesophase upon temperature variation, and the difference in the order parameters between the bulk and the interface with the substrate. It integrates high-resolution polarized light optical microscopy, temperature regulation, and electrical measurements in a controlled atmosphere with a software kernel that controls the instruments and synchronizes the data streams. A user-friendly interface allows us to program multistep experiments controlling all the instruments and data acquisition by a specifically designed scheduler. We tested our system on a thin film of alkoxy-substituted phthalocyanines deposited on a test pattern with interdigitated electrodes. We studied the optical and electrical behavior in the proximity of the bulk phase transition to isotropic liquid, identifying a few ordered monolayers anchored to the substrate above the transition temperature.