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Cancer-associated fibroblasts (CAFs) expressing fibroblast activation protein alpha (FAP) are abundant in tumor microenvironments and represent an emerging target for PET cancer imaging. While different quinolone-based small molecule agents have been developed for whole-body imaging, there is a scarcity of well-validated fluorescent small molecule imaging agents to better study these cells in vivo. Here, we report the synthesis and characterization of a series of fluorescent FAP imaging agents based on the common quinolone azide inhibitor. Our data show excellent performance of some synthesized FAP Targeting Fluorescent probes (FTFs) for both topical application and intravenous delivery to label CAF populations in solid tumors. These results suggest that FTF can be used to study CAF biology and therapeutic targeting in vivo.

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.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Ferumoxytol MRI, and microscopy with tumor-associated macrophage (TAM)-targeted nanoparticle Macrin, showed serine/threonine-protein kinase B-Raf inhibition and immune checkpoint blockade–enhanced local TAM accumulation by 46% ± 21 (standard deviation), which correlated with 43% ± 20 greater uptake of a model drug delivery nanoparticle in a mouse model of anaplastic thyroid cancer.

Nanoparticulate albumin bound paclitaxel (nab-paclitaxel, nab-PTX) is among the most widely prescribed nanomedicines in clinical use, yet it remains unclear how nanoformulation affects nab-PTX behaviour in the tumour microenvironment. Here, we quantified the biodistribution of the albumin carrier and its chemotherapeutic payload in optically cleared tumours of genetically engineered mouse models, and compared the behaviour of nab-PTX with other clinically relevant nanoparticles. We found that nab-PTX uptake is profoundly and distinctly affected by cancer-cell autonomous RAS signalling, and RAS/RAF/MEK/ERK inhibition blocked its selective delivery and efficacy. In contrast, a targeted screen revealed that IGF1R kinase inhibitors enhance uptake and efficacy of nab-PTX by mimicking glucose deprivation and promoting macropinocytosis via AMPK, a nutrient sensor in cells. This study thus shows how nanoparticulate albumin bound drug efficacy can be therapeutically improved by reprogramming nutrient signalling and enhancing macropinocytosis in cancer cells.

The receptor tyrosine kinase inhibitor, Tie2, has significant roles in endothelial signaling and angiogenesis and is relevant in the pathophysiology of several diseases. However, there are relatively few small molecule probes available to study Tie2, making the evaluation of its activity in vivodifficult. Recently, it was discovered that the small molecule rebastinib (DCC-2036) is a potent Tie2 inhibitor. We hypothesized that fluorescent derivatives of rebastinib could be used as imaging agents for Tie2. On the basis of crystallography structures, we synthesized three fluorescent derivatives, which we then evaluated in both in vitroand in vivoassays. We found that the Rebastinib-BODIPY TMR (Reb-TMR) derivative has superior imaging characteristics in vitro, and we successfully labeled endothelial cells in vivo. We propose that this probe could be further used in in vivoapplications for studying the role of Tie2 in disease.

Amiodarone is an effective antiarrhythmic drug used to treat and prevent different types of cardiac arrhythmias. However, amiodarone can have considerable side effects resulting from accumulation in off-target tissues. Cardiac macrophages are highly prevalent tissue-resident immune cells with importance in homeostatic functions, including immune response and modulation of cardiac conduction. We hypothesized that amiodarone could be more efficiently delivered to the heart via cardiac macrophages, an important step toward reducing overall dose and off-target tissue accumulation. Toward this goal, we synthesized a nanoparticle drug carrier composed of l-lysine cross-linked succinyl-β-cyclodextrin that demonstrates amiodarone binding through supramolecular host–guest interaction as well as a high macrophage affinity. Biodistribution analyses at the organ and single-cell level demonstrate accumulation of nanoparticles in the heart resulting from rapid uptake by cardiac macrophages. Nanoparticle assisted delivery of amiodarone resulted in a 250% enhancement in the selective delivery of the drug to cardiac tissue in part due to a concomitant decrease of pulmonary accumulation, the main source of off-target toxicity.

Tumor-associated macrophages (TAMs) are widely implicated in cancer progression, and TAM levels can influence drug responses, particularly to immunotherapy and nanomedicines. However, it has been difficult to quantify total TAM numbers and their dynamic spatiotemporal distribution in a non-invasive and translationally relevant manner. Here, we address this need by developing a pharmacokinetically optimized, 64Cu-labeled polyglucose nanoparticle (Macrin) for quantitative positron emission tomography (PET) imaging of macrophages in tumors. By combining PET with high-resolution in vivoconfocal microscopy and ex vivoimaging of optically cleared tissue, we found that Macrin was taken up by macrophages with >90% selectivity. Uptake correlated with the content of macrophages in both healthy tissue and tumors (R2> 0.9) and showed striking heterogeneity in the TAM content of an orthotopic and immunocompetent mouse model of lung carcinoma. In a proof-of-principle application, we imaged Macrin to monitor the macrophage response to neo-adjuvant therapy, using a panel of chemotherapeutic and γ-irradiation regimens. Multiple treatments elicited 180–650% increase in TAMs. Imaging identified especially TAM-rich tumors thought to exhibit enhanced permeability and retention of nanotherapeutics. Indeed, these TAM-rich tumors accumulated >700% higher amounts of a model poly(d,l-lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG) therapeutic nanoparticle compared to TAM-deficient tumors, suggesting that imaging may guide patient selection into nanomedicine trials. In an orthotopic breast cancer model, chemoradiation enhanced TAM and Macrin accumulation in tumors, which corresponded to the improved delivery and efficacy of two model nanotherapies, PEGylated liposomal doxorubicin and a TAM-targeted nanoformulation of the toll-like receptor 7/8 agonist resiquimod (R848). Thus, Macrin imaging offers a selective and translational means to quantify TAMs and inform therapeutic decisions.

Prodrug strategies that facilitate localized and controlled activity of small-molecule therapeutics can reduce systemic exposure and improve pharmacokinetics, yet limitations in activation chemistry have made it difficult to assign tunable multifunctionality to prodrugs. Here, we present the design and application of a modular small-molecule caging strategy that couples bioorthogonal cleavage with a self-immolative linker and an aliphatic anchor. This strategy leverages recently discovered in vivocatalysis by a nanoencapsulated palladium compound (Pd-NP), which mediates alloxylcarbamate cleavage and triggers release of the activated drug. The aliphatic anchor enables >90% nanoencapsulation efficiency of the prodrug, while also allowing >104-fold increased cytotoxicity upon prodrug activation. We apply the strategy to a prodrug formulation of monomethyl auristatin E (MMAE), demonstrating its ability to target microtubules and kill cancer cells only after selective activation by Pd-NP. Computational pharmacokinetic modeling provides a mechanistic basis for the observation that the nanotherapeutic prodrug strategy can lead to more selective activation in the tumor, yet in a manner that is more sensitive to variable enhanced permeability and retention (EPR) effects. Combination treatment with the nanoencapsulated MMAE prodrug and Pd-NP safely blocks tumor growth, especially when combined with a local radiation therapy regimen that is known to improve EPR effects, and represents a conceptual step forward in prodrug design.

Interferon regulatory factor 3 (IRF3) and type I interferons (IFNs) protect against infections and cancer, but excessive IRF3 activation and type I IFN production cause autoinflammatory conditions such as Aicardi–Goutières syndrome and STING-associated vasculopathy of infancy (SAVI). Myocardial infarction (MI) elicits inflammation, but the dominant molecular drivers of MI-associated inflammation remain unclear. Here we show that ischemic cell death and uptake of cell debris by macrophages in the heart fuel a fatal response to MI by activating IRF3 and type I IFN production. In mice, single-cell RNA-seq analysis of 4,215 leukocytes isolated from infarcted and non-infarcted hearts showed that MI provokes activation of an IRF3–interferon axis in a distinct population of interferon-inducible cells (IFNICs) that were classified as cardiac macrophages. Mice genetically deficient in cyclic GMP-AMP synthase (cGAS), its adaptor STING, IRF3, or the type I IFN receptor IFNAR exhibited impaired interferon-stimulated gene (ISG) expression and, in the case of mice deficient in IRF3 or IFNAR, improved survival after MI as compared to controls. Interruption of IRF3-dependent signaling resulted in decreased cardiac expression of inflammatory cytokines and chemokines and decreased inflammatory cell infiltration of the heart, as well as in attenuated ventricular dilation and improved cardiac function. Similarly, treatment of mice with an IFNAR-neutralizing antibody after MI ablated the interferon response and improved left ventricular dysfunction and survival. These results identify IRF3 and the type I IFN response as a potential therapeutic target for post-MI cardioprotection.

Here we present the first generation of two-photon beta cell specific in vivoimaging probes based on GLP1R targeting peptides. Among the three compounds of potential interest, we found quite unexpectedly that a squarine-rotaxane conjugate (2PEx-647) had near ideal in vivoimaging characteristics.

Objectives:Creating functional small-diameter tissue-engineered blood vessels has not been successful to date. Moreover, the processes underlying the in vivoremodeling of these grafts and the fate of cells seeded onto scaffolds remain unclear. Here we addressed these unmet scientific needs by using intravital molecular imaging to monitor the development of tissue-engineered vascular grafts (TEVG) implanted in mouse carotid artery.Methods and Results:Green fluorescent protein–labeled human bone marrow–derived mesenchymal stem cells and cord blood–derived endothelial progenitor cells were seeded on polyglycolic acid–poly-L-lactic acid scaffolds to construct small-caliber TEVG that were subsequently implanted in the carotid artery position of nude mice (n= 9). Mice were injected with near-infrared agents and imaged using intravital fluorescence microscope at 0, 7, and 35 days to validate in vivothe TEVG remodeling capability (Prosense680; VisEn, Woburn, MA) and patency (Angiosense750; VisEn). Imaging coregistered strong proteolytic activity and blood flow through anastomoses at both 7 and 35 days postimplantation. In addition, image analyses showed green fluorescent protein signal produced from mesenchymal stem cell up to 35 days postimplantation. Comprehensive correlative histopathological analyses corroborated intravital imaging findings.Conclusions:Multispectral imaging offers simultaneous characterization of in vivoremodeling enzyme activity, functionality, and cell fate of viable small-caliber TEVG.

Cell communication underlies emergent functions in diverse cell types and tissues. Recent evidence suggests that macrophages are organized in communicating networks, but new tools are needed to quantitatively characterize the resulting cellular conversations. Here, we infer cell communication from spatiotemporal correlations of intracellular calcium dynamics that are non-destructively imaged across cell populations expressing genetically encoded calcium indicators. We describe a hematopoietic calcium reporter mouse (Csf1rCreGCaMP5fl) and a computational analysis pipeline for inferring communication between reporter cells based on “excess synchrony.” We observed signals suggestive of cell communication in macrophages treated with immune-stimulatory DNA in vitroand tumor-associated immune cells imaged in a dorsal window chamber model in vivo. Together, the methods described here expand the toolkit for discovery of cell communication events in macrophages and other immune cells.

Targeted inhibition of oncogenic pathways can be highly effective in halting the rapid growth of tumors but often leads to the emergence of slowly dividing persister cells, which constitute a reservoir for the selection of drug-resistant clones. In BRAFV600Emelanomas, RAF and MEK inhibitors efficiently block oncogenic signaling, but persister cells emerge. Here, we show that persister cells escape drug-induced cell-cycle arrest via brief, sporadic ERK pulses generated by transmembrane receptors and growth factors operating in an autocrine/paracrine manner. Quantitative proteomics and computational modeling show that ERK pulsing is enabled by rewiring of mitogen-activated protein kinase (MAPK) signaling: from an oncogenic BRAFV600Emonomer-driven configuration that is drug sensitive to a receptor-driven configuration that involves Ras-GTP and RAF dimers and is highly resistant to RAF and MEK inhibitors. Altogether, this work shows that pulsatile MAPK activation by factors in the microenvironment generates a persistent population of melanoma cells that rewires MAPK signaling to sustain non-genetic drug resistance.

Radiation therapy enhances nanotherapeutic drug delivery in a tumor-associated macrophage–dependent fashion.

Tumor-associated macrophages limit anti–PD-1 effects by removing the antibody from CD8+T cells.

Magnetic nanoparticles predict the efficacy of drug-loaded polymeric nanoparticles in vivo, helping select for tumors more responsive to nanomedicine.

Single-cell pharmacokinetic analysis of a fluorescent eribulin derivative in vivo revealed drug resistance mediated by MDR1-driven efflux, which was overcome by a nanoencapsulated MDR1 inhibitor.

Molecular and cellular mechanisms of atherogenesis and its treatment are largely being unraveled by in vitro techniques. We describe methodology to directly image macrophage cell activity in vivo in a murine model of atherosclerosis using laser scanning fluorescence microscopy (LSFM) and a macrophage-targeted, near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP). Atherosclerotic apolipoprotein E deficient (apoE -/-) mice (n=10) are injected with MFNP or 0.9&percent; saline, and wild-type mice (n=4) are injected with MFNP as additional controls. After 24 h, common carotid arteries are surgically exposed and prepared for LSFM. Multichannel LSFM of MFNP-enhanced carotid atheroma (5×5‐μm in-plane resolution) shows a strong focal NIRF signal, with a plaque target-to-background ratio of 3.9±1.8. Minimal NIRF signal is observed in control mice. Spectrally resolved indocyanine green (ICG) fluorescence angiograms confirm the intravascular location of atheroma. On ex vivo fluorescence reflectance imaging, greater NIRF plaque signal is seen in apoE -/- MFNP mice compared to controls (p<0.01). The NIRF signal correlates well with immunostained macrophages, both by stained surface area (r=0.77) and macrophage number (r=0.86). The validated experimental methodology thus establishes a platform for investigating macrophage activity in atherosclerosis in vivo, and has implications for the detection of clinical vulnerable plaques.