Macrophage-Targeted Therapy Unlocks Antitumoral Cross-talk between IFNγ-Secreting Lymphocytes and IL12-Producing Dendritic Cells.

Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches. We showed that tumor control was not caused by CSF1R+ cell depletion; instead, CSF1R targeting reshaped the CSF1R+ cell landscape, which unlocked cross-talk between antitumoral CSF1R- cells. These cells included IFNγ-producing natural killer and T cells, and an IL12-producing dendritic cell subset, denoted as DC3, which were all necessary for CSF1R inhibitor-mediated lung tumor control. These data indicate that CSF1R targeting can activate a cardinal cross-talk between cells that are not macrophages and that are essential to mediate the effects of T cell-targeted immunotherapies and promote antitumor immunity.See related Spotlight by Burrello and de Visser, p. 4.

Single Extracellular Vesicle Protein Analysis Using Immuno-Droplet Digital Polymerase Chain Reaction Amplification.

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno-droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody-DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read-out by droplet imaging. In these proof-of-principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.

Expanding the Scope of Antibody Rebridging with New Pyridazinedione-TCO Constructs.

Disulfide rebridging methods have recently emerged as a route to hinge region-specific antibody modification, and there exist numerous examples of successful rebridging chemistry applied to clinically relevant human IgG1 antibodies. Here, dibromopyridazinedione disulfide rebridging is adapted to fast trans-cyclooctene/tetrazine (TCO/Tz) bioorthogonal ligations and extended beyond therapeutic human IgG1 antibodies for the first time to include mouse and rat monoclonal antibodies integral to multiplexed analytical diagnostics. In spite of a common architecture, only a subset of antibody host species and IgG isotype subclasses can be rebridged, highlighting the intricate relationship between hinge region sequence, structure, biological activity, and the conjugation chemistry of IgG antibodies.

Glass Chemistry to Analyze Human Cells under Adverse Conditions.

Emerging point-of-care diagnostic tests capable of analyzing whole mammalian cells often rely on the attachment of harvested cells to glass surfaces for subsequent molecular characterization. We set out to develop and optimize a kit for the diagnosis of lymphoma in low- and middle-income countries where access to advanced healthcare testing is often absent or prone to error. Here, we optimized a process for the lyophilization of neutravidin-coated glass and cocktails of antibodies relevant to lymphoma diagnosis to establish long-term stability of reagents required for point-of-care cell capture technology. Lyophilized glass slides showed no decline in their performance compared to freshly prepared neutravidin glass and preserved capture efficiency for 5 weeks under easily attainable storage conditions. We demonstrate the successful performance of the low-cost, lyophilized kit in a cell capture assay to enable true point-of-care analyses under adverse conditions. We anticipate that the strategy can be expanded to other cancer cell types or cell-derived vesicle analysis.

Mapping Forbidden Emission to Structure in Self-Assembled Organic Nanoparticles.

The interplay between micromorphology and electronic properties is an important theme in organic electronic materials. Here, we show that a spirofluorene-functionalized boron-dipyrromethene (BODIPY) with an alkyl norbornyl tail self-assembles into nanoparticles with qualitatively different properties as compared to the polymerized species. Further, the nanoparticles exhibit a host of unique emissive properties, including photobrightening, a blue satellite peak, and spectral diffusion. Extensive photophysical characterization, including single-particle imaging and spectroscopy, and time-resolved fluorescence, coupled with electronic structure calculations based on an experimentally determined crystal structure, allow a mechanism to be developed. Specifically, BODIPY chromophores are observed to form quasi-two-dimensional layers, where stacking of unit cells adds either J-aggregate character or H-aggregate character depending on the direction of the stacking. Particularly strongly H-coupled domains show the rare process of emission from an upper exciton state, in violation of Kasha's rule, and result in the blue satellite peak. The spatial heterogeneity of structure thus maps onto a gradient of photophysical behavior as seen in single-particle imaging, and the temporal evolution of structure maps onto fluctuating emissive behavior, as seen in single-particle spectroscopy. Taken together, this system provides a striking example of how physical structure and electronic properties are intertwined, and a rare opportunity to use one to chart the other.

Single-Molecule Investigation of Initiation Dynamics of an Organometallic Catalyst.

The action of molecular catalysts comprises multiple microscopic kinetic steps whose nature is of central importance in determining catalyst activity and selectivity. Single-molecule microscopy enables the direct examination of these steps, including elucidation of molecule-to-molecule variability. Such molecular diversity is particularly important for the behavior of molecular catalysts supported at surfaces. We present the first combined investigation of the initiation dynamics of an operational palladium cross-coupling catalyst at the bulk and single-molecule levels, including under turnover conditions. Base-initiated kinetics reveal highly heterogeneous behavior indicative of diverse catalyst population. Unexpectedly, this distribution becomes more heterogeneous at increasing base concentration. We model this behavior with a two-step saturation mechanism and identify specific microscopic steps where chemical variability must exist in order to yield observed behavior. Critically, we reveal how structural diversity at a surface translates into heterogeneity in catalyst behavior, while demonstrating how single-molecule experiments can contribute to understanding of molecular catalysts.