Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that ...measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .
Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. ...In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring β-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.
•N-linked glycosylation is required for physical contact between PD-L1 and PD-1•EGF/EGFR stimulates PD-L1 glycosylation via B3GNT3 glycosyltransferase•Glycosylated-PD-L1 antibody induces PD-L1 internalization•Glycosylated-PD-L1-ADC possesses potent toxicity as well as bystander effects
Li et al. show that glycosylation of PD-L1 is essential for PD-L1/PD-1 interaction and immunosuppression in triple-negative breast cancer (TNBC). They generate a glycosylation-specific antibody that induces PD-L1 internalization and an antibody-drug conjugate with potent anti-tumor activities in TNBC models.
Current lithography techniques, which employ photon, electron, or ion beams to induce chemical or physical reactions for micro/nano-fabrication, have remained challenging in patterning chemically ...synthesized colloidal particles, which are emerging as building blocks for functional devices. Herein, we develop a new technique - bubble-pen lithography (BPL) - to pattern colloidal particles on substrates using optically controlled microbubbles. Briefly, a single laser beam generates a microbubble at the interface of colloidal suspension and a plasmonic substrate via plasmon-enhanced photothermal effects. The microbubble captures and immobilizes the colloidal particles on the substrate through coordinated actions of Marangoni convection, surface tension, gas pressure, and substrate adhesion. Through directing the laser beam to move the microbubble, we create arbitrary single-particle patterns and particle assemblies with different resolutions and architectures. Furthermore, we have applied BPL to pattern CdSe/ZnS quantum dots on plasmonic substrates and polystyrene (PS) microparticles on two-dimensional (2D) atomic-layer materials. With the low-power operation, arbitrary patterning and applicability to general colloidal particles, BPL will find a wide range of applications in microelectronics, nanophotonics, and nanomedicine.
Semiconductor quantum dots (QDs) feature excellent properties, such as high quantum efficiency, tunable emission frequency, and good fluorescence stability. Incorporation of QDs into new devices ...relies upon high-resolution and high-throughput patterning techniques. Herein, we report a new printing technique known as bubble printing (BP), which exploits a light-generated microbubble at the interface of colloidal QD solution and a substrate to directly write QDs into arbitrary patterns. With the uniform plasmonic hot spot distribution for high bubble stability and the optimum light-scanning parameters, we have achieved full-color QD printing with submicron resolution (650 nm), high throughput (scanning rate of ∼10–2 m/s), and high adhesion of the QDs to the substrates. The printing parameters can be optimized to further control the fluorescence properties of the patterned QDs, such as emission wavelength and lifetime. The patterning of QDs on flexible substrates further demonstrates the wide applicability of this new technique. Thus, BP technique addresses the barrier of achieving a widely applicable, high-throughput and user-friendly patterning technique in the submicrometer regime, along with simultaneous fluorescence modification capability.
Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of ...intracellular processes using single-particle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups—immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.
We report molecular-fluorescence enhancement via the blue-shifted plasmon-induced resonance energy transfer (PIRET) from single Au nanorods (AuNRs) to merocyanine (MC) dye molecules. The blue-shifted ...PIRET occurs when there is a proper spectral overlap between the scattering of AuNRs and the absorption of MC molecules. Along with the quenching of scattering from AuNRs, the blue-shifted PIRET enhances the fluorescence of nearby molecules. On the basis of the fluorescence enhancement, we conclude that AuNRs can be used as donors with clear advantages to excite the fluorescence of molecules as acceptors in AuNR–molecule hybrids. On the one hand, compared to conventional molecular donors in Förster resonance energy transfer (FRET), AuNRs have much larger absorption cross sections at the plasmon resonance frequencies. On the other hand, energy-transfer efficiency of PIRET decreases at a lower rate than that of FRET when the donor–acceptor distance is increased. Besides, the blue-shifted PIRET allows excitation with incident light of lower energy than the acceptor’s absorption, which is difficult to achieve in FRET because of the Stokes shift. With the capability of enhancing molecular fluorescence with excitation light of low intensity and long wavelength, the blue-shifted PIRET will expand the applications of nanoparticle–molecule hybrids in biosensing and bioimaging by increasing signal-to-noise ratio and by reducing photodamage to biological cells and organelles at the targeted areas.
Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including ...cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ∼15 μm. Here we present a three-dimensional tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 μm. The system has a response time of 1 ms with a temporal resolution down to 50 μs in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolour imaging. Here we demonstrate three-dimensional tracking of epidermal growth factor receptor complexes at a depth of ∼100 μm in tumour spheroids.
Here, we present a three-dimensional two-color dual-particle tracking (3D-2C-DPT) technique that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution ...down to 5 ms. The dual-targets can be tracked with separation distances from 33 to 250 nm with tracking precisions of ∼15 nm (for static targets) and ∼35 nm (for freely diffusing targets). Since each target is individually localized, a wealth of data can be extracted, such as the relative 3D position, the 2D rotation, and the separation distance between the two targets. Using this technique, we turn a double-stranded DNA (dsDNA)-linked dumbbell-like dimer into a nanoscopic optical ruler to quantify the bending dynamics of nicked or gapped dsDNA molecules in free solution by manipulating the design of dsDNA linkers (1-nick, 3-nt, 6-nt, or 9-nt single-strand gap), and the results show the increase of k on (linear to bent) from 3.2 to 10.7 s–1. The 3D-2C-DPT is then applied to observe translational and rotational motions of the landing of an antibody-conjugated nanoparticle on the plasma membrane of living cells, revealing the reduction of rotations possibly due to interactions with membrane receptors. This study demonstrates that this 3D-2C-DPT technique is a new tool to shed light on the conformational changes of biomolecules and the intermolecular interactions on plasma membrane.
Semiconductor quantum dots (QDs) are attractive for a wide range of applications such as displays, light-emitting devices, and sensors due to their properties such as tunable fluorescence wavelength, ...high brightness, and narrow bandwidth. Most of the applications require precise patterning of QDs with targeted properties on solid-state substrates. Herein, we have developed a haptic-interfaced bubble printing (HIBP) technique to enable high-resolution (510 nm) high-throughput (>10
μm s
) patterning of QDs with strong emission tunability and to significantly enhance the accessibility of the technique
a smartphone device. The scalability and versatility of the HIBP are demonstrated in our arbitrary patterning of QDs on plasmonic substrates. A detailed study of plasmonic and photothermal interactions is performed
programmed stage movements to realize tunability of the emission wavelength and lifetime. Finally, the influence of the hand movement on the properties of the printed QDs in terms of emission wavelength tuning from yellow to blue is established. This work provides a single-step macroscale platform to manipulate nanoscale properties at high resolution and high throughput.
Derailed transmembrane receptor trafficking could be a hallmark of tumorigenesis and increased tumor invasiveness, but receptor dynamics have not been used to differentiate metastatic cancer cells ...from less invasive ones. Using single-particle tracking techniques, we developed a phenotyping asssay named Transmembrane Receptor Dynamics (TReD), studied the dynamics of epidermal growth factor receptor (EGFR) in seven breast epithelial cell lines and developed a phenotyping assay named Transmembrane Receptor Dynamics (TReD). Here we show a clear evidence that increased EGFR diffusivity and enlarged EGFR confinement size in the plasma membrane (PM) are correlated with the enhanced metastatic potential in these cell lines. By comparing the TReD results with the gene expression profiles, we found a clear negative correlation between the EGFR diffusivities and the breast cancer luminal differentiation scores (r = -0.75). Upon the induction of epithelial-mesenchymal transition (EMT), EGFR diffusivity significantly increased for the non-tumorigenic MCF10A (99%) and the non-invasive MCF7 (56%) cells, but not for the highly metastatic MDA-MB-231 cell. We believe that the reorganization of actin filaments during EMT modified the PM structures, causing the receptor dynamics to change. TReD can thus serve as a new biophysical marker to probe the metastatic potential of cancer cells and even to monitor the transition of metastasis.
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