Magnetic Silica-Coated Fluorescent Microspheres (MagSiGlow) for Simultaneous Detection of Tumor-Associated Proteins.

Multiplexed bead assays for solution-phase biosensing often encounter cross-over reactions during signal amplification steps, leading to unwanted false positive and high background signals. Current solutions involve complex custom-designed and costly equipment, limiting their application in simple laboratory setup. In this study, we introduce a straightforward protocol to adapt a multiplexed single-bead assay to standard fluorescence imaging plates, enabling the simultaneous analysis of thousands of reactions per plate. This approach focuses on the design and synthesis of bright fluorescent and magnetic microspheres (MagSiGlow) with multiple fluorescent wavelengths serving as unique detection markers. The imaging-based, single-bead assay, combined with a scripted algorithm, allows the detection, segmentation, and co-localization on average of 7500 microspheres per field of view across five imaging channels in less than one second. We demonstrate the effectiveness of this method with remarkable sensitivity at low protein detection limits (100 pg/mL). This technique showed over 85 % reduction in signal cross-over to the solution-based method after the concurrent detection of tumor-associated protein biomarkers. This approach holds the promise of substantially enhancing high throughput biosensing for multiple targets, seamlessly integrating with rapid image analysis algorithms.

Pharmacological Polarization of Tumor-Associated Macrophages Toward a CXCL9 Antitumor Phenotype.

Tumor-associated macrophages (TAM) are a diverse population of myeloid cells that are often abundant and immunosuppressive in human cancers. CXCL9Hi TAM has recently been described to have an antitumor phenotype and is linked to immune checkpoint response. Despite the emerging understanding of the unique antitumor TAM phenotype, there is a lack of TAM-specific therapeutics to exploit this new biological understanding. Here, the discovery and characterization of multiple small-molecule enhancers of chemokine ligand 9 (CXCL9) and their targeted delivery in a TAM-avid systemic nanoformulation is reported. With this strategy, it is efficient encapsulation and release of multiple drug loads that can efficiently induce CXCL9 expression in macrophages, both in vitro and in vivo in a mouse tumor model. These observations provide a window into the molecular features that define TAM-specific states, an insight a novel therapeutic anticancer approach is used to discover.

Perfusion Window Chambers Enable Interventional Analyses of Tumor Microenvironments.

Intravital microscopy (IVM) allows spatial and temporal imaging of different cell types in intact live tissue microenvironments. IVM has played a critical role in understanding cancer biology, invasion, metastases, and drug development. One considerable impediment to the field is the inability to interrogate the tumor microenvironment and its communication cascades during disease progression and therapeutic interventions. Here, a new implantable perfusion window chamber (PWC) is described that allows high-fidelity in vivo microscopy, local administration of stains and drugs, and longitudinal sampling of tumor interstitial fluid. This study shows that the new PWC design allows cyclic multiplexed imaging in vivo, imaging of drug action, and sampling of tumor-shed materials. The PWC will be broadly useful as a novel perturbable in vivo system for deciphering biology in complex microenvironments.

Hybrid LNP Prime Dendritic Cells for Nucleotide Delivery.

The efficient activation of professional antigen-presenting cells-such as dendritic cells (DC)-in tumors and lymph nodes is critical for the design of next-generation cancer vaccines and may be able to provide anti-tumor effects by itself through immune stimulation. The challenge is to stimulate these cells without causing excessive toxicity. It is hypothesized that a multi-pronged combinatorial approach to DC stimulation would allow dose reductions of innate immune receptor-stimulating TLR3 agonists while enhancing drug efficacy. Here, a hybrid lipid nanoparticle (LNP) platform is developed and tested for double-stranded RNA (polyinosinic:polycytidylic acid for TLR3 agonism) and immune modulator (L-CANDI) delivery. This study shows that the ≈120 nm hybrid nanoparticles-in-nanoparticles effectively eradicate tumors by themselves and generate long-lasting, durable anti-tumor immunity in mouse models.

Highly Active Myeloid Therapy for Cancer.

Tumor-associated macrophages (TAM) interact with cancer and stromal cells and are integral to sustaining many cancer-promoting features. Therapeutic manipulation of TAM could therefore improve clinical outcomes and synergize with immunotherapy and other cancer therapies. While different nanocarriers have been used to target TAM, a knowledge gap exists on which TAM pathways to target and what payloads to deliver for optimal antitumor effects. We hypothesized that a multipart combination involving the Janus tyrosine kinase (JAK), noncanonical nuclear factor kappa light chain enhancer of activated B cells (NF-κB), and toll-like receptor (TLR) pathways could lead to a highly active myeloid therapy (HAMT). Thus, we devised a screen to determine drug combinations that yield maximum IL-12 production from myeloid cells to treat the otherwise highly immunosuppressive myeloid environments in tumors. Here we show the extraordinary efficacy of a triple small-molecule combination in a TAM-targeted nanoparticle for eradicating murine tumors, jumpstarting a highly efficient antitumor response by adopting a distinctive antitumor TAM phenotype and synergizing with other immunotherapies. The HAMT therapy represents a recently developed approach in immunotherapy and leads to durable responses in murine cancer models.

Light-Deactivated Fluorescent Probes (FLASH-Off) for Multiplexed Imaging.

Highly multiplexed, cyclic fluorescence imaging has advanced our understanding of the biology, evolution, and complexity of human diseases. Currently available cyclic methods still have considerable limitations including the need for long quenching times and extensive wash steps. Here, we report a new series of fluorochromes that can be efficiently inactivated by a single light pulse (∼405 nm) by means of a photo-immolating triazene linker. Upon UV-light irradiation, the rhodamines are cleaved off from the antibody conjugates and undergo a fast intramolecular spirocyclization that inherently switches off their fluorescence emission without the need to wash or add exogenous chemicals. We show that these switch-off probes are fast, highly controllable, biocompatible, and allow spatiotemporal quenching control of live and fixed samples.

Dual Immunostimulatory Pathway Agonism through a Synthetic Nanocarrier Triggers Robust Anti-Tumor Immunity in Murine Glioblastoma.

Myeloid cells are abundant, create a highly immunosuppressive environment in glioblastoma (GBM), and thus contribute to poor immunotherapy responses. Based on the hypothesis that small molecules can be used to stimulate myeloid cells to elicit anti-tumor effector functions, a synthetic nanoparticle approach is developed to deliver dual NF-kB pathway-inducing agents into these cells via systemic administration. Synthetic, cyclodextrin-adjuvant nanoconstructs (CANDI) with high affinity for tumor-associated myeloid cells are dually loaded with a TLR7 and 8 (Toll-like receptor, 7 and 8) agonist (R848) and a cIAP (cellular inhibitor of apoptosis protein) inhibitor (LCL-161) to dually activate these myeloid cells. Here CANDI is shown to: i) readily enter the GBM tumor microenvironment (TME) and accumulate at high concentrations, ii) is taken up by tumor-associated myeloid cells, iii) potently synergize payloads compared to monotherapy, iv) activate myeloid cells, v) fosters a "hot" TME with high levels of T effector cells, and vi) controls the growth of murine GBM as mono- and combination therapies with anti-PD1. Multi-pathway targeted myeloid stimulation via the CANDI platform can efficiently drive anti-tumor immunity in GBM.

In Vivo Click Chemistry Enables Multiplexed Intravital Microscopy.

The ability to observe cells in live organisms is essential for understanding their function in complex in vivo milieus. A major challenge today has been the limited ability to perform higher multiplexing beyond four to six colors to define cell subtypes in vivo. Here, a click chemistry-based strategy is presented for higher multiplexed in vivo imaging in mouse models. The method uses a scission-accelerated fluorophore exchange (SAFE), which exploits a highly efficient bioorthogonal mechanism to completely remove fluorescent signal from antibody-labeled cells in vivo. It is shown that the SAFE-intravital microscopy imaging method allows 1) in vivo staining of specific cell types in dorsal and cranial window chambers of mice, 2) complete un-staining in minutes, 3) in vivo click chemistries at lower (µm) and thus non-toxic concentrations, and 4) the ability to perform in vivo cyclic imaging. The potential utility of the method is demonstrated by 12 color imaging of immune cells in live mice.

Dual-Activatable Cell Tracker for Controlled and Prolonged Single-Cell Labeling.

Cell trackers are fluorescent chemical tools that facilitate imaging and tracking cells within live organisms. Despite their versatility, these dyes lack specificity, tend to leak outside of the cell, and stain neighboring cells. Here, we report a dual-activatable cell tracker for increased spatial and temporal staining control, especially for single-cell tracking. This probe overcomes the typical problems of current cell trackers: off-target staining, high background signal, and leakage from the intracellular medium. Staining with this dye is not cytotoxic, and it can be used in sensitive primary cells. Moreover, this dye is resistant to harsh fixation and permeabilization conditions and allows for multiwavelength studies with confocal microscopy and fluorescence-activated cell sorting. Using this cell tracker, we performed in vivo homing experiments in mice with primary splenocytes and tracked a single cell in a heterogeneous, multicellular culture environment for over 20 h. These experiments, in addition to comparative proliferation studies with other cell trackers, demonstrated that the signal from this dye is retained in cells for over 72 h after photoactivation. We envision that this type of probes will facilitate the analysis of single-cell behavior and migration in cell culture and in vivo experiments.

Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching.

Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

A Photoactivatable Probe for Super-Resolution Imaging of Enzymatic Activity in Live Cells.

A dual-activatable, fluorogenic probe was developed to sense esterase activity with single-molecule resolution. Without enzymatic pre-activation, the diazoindanone-based probe has an electron-poor core and, upon irradiation, undergoes Wolff rearrangement to give a ring-expanded xanthene core that is nonemissive. If the probe is pre-activated by carboxylesterases, the tricyclic core becomes electron-rich, and the photoinduced Wolff rearrangement produces a highly emissive rhodol dye. Live-cell and solution studies confirmed the selectivity of the probe and revealed that the photoactivated dye does not diffuse away from the original location of activation because the intermediate ketene forms a covalent bond with surrounding macromolecules. Single-molecule localization microscopy was used to reconstruct a super-resolved image of esterase activity. These single-molecule images of enzymatic activity changed significantly upon treatment of the cells with inhibitors of human carboxylesterase I and II, both in terms of total number of signals and intracellular distribution. This proof-of-principle study introduces a sensing mechanism for single-molecule detection of enzymatic activity that could be applied to many other biologically relevant targets.

Identification and characterisation of the phenolics of Ilex glabra L. Gray (Aquifoliaceae) leaves by liquid chromatography tandem mass spectrometry.

The phenolics of the leaves of Ilex glabra L. Gray (Aquifoliaceae) were investigated qualitatively by LC-MS(n). Thirty-two phenolics were detected and characterised on the basis of their unique fragmentation pattern in the negative ion mode tandem MS spectra. All of them were extracted for the first time from this source and fifteen of them were not reported previously in nature. For the positive identification of phenolic glucosides by LC-MS(n) a series of authentic standards and experiments were carried out. This is the first report of a full characterisation of 3,4-dihydroxybenzoyl glucosides, 3,4-dihydroxybenzyl glucosides, 4-hydroxybenzoyl glucosides, chlorogenic acid glucosides and vanillic acid glucosides by LC-MS(2-4).