We have developed a multifaceted highly specific reporter for multimodal in vivo imaging and applied it for detection of brain tumors. A metabolically biotinylated, membrane-bound form of Gaussia luciferase was synthesized, termed mbGluc-biotin. We engineered glioma cells to express this reporter and showed that brain tumor formation can be temporally imaged by bioluminescence following systemic administration of coelenterazine. Brain tumors expressing this reporter had high sensitivity for detection by magnetic resonance and fluorescence tomographic imaging upon injection of streptavidin conjugated to magnetic nanoparticles or fluorophore, respectively. Moreover, single photon emission computed tomography showed enhanced imaging of these tumors upon injection with streptavidin complexed to 111In-DTPA-biotin. This work shows for the first time a single small reporter ( 40 kDa) which can be monitored with most available molecular imaging modalities and can be extended for single cell imaging using intravital microscopy, allowing real-time tracking of any cell expressing it in vivo.
The ability to detect rare cells (< 100 cells per ml of whole blood) and obtain quantitative measurements of specific biomarkers on single cells is increasingly important in basic biomedical research. Implementing such methodology for widespread use in the clinic, however, has been hampered by low cell density, small sample sizes, and requisite sample purification. To overcome these challenges, we have developed a microfluidic chip-based micro-Hall detector (μHD), which can directly measure single, immunomagnetically tagged cells in whole blood. The μHD can detect single cells even in the presence of vast numbers of blood cells and unbound reactants, and does not require any washing or purification steps. In addition, the high bandwidth and sensitivity of the semiconductor technology used in the μHD enables high-throughput screening (currently ~107 cells/min). The clinical utility of the μHD chip was demonstrated by detecting circulating tumor cells in whole blood of 20 ovarian cancer patients at higher sensitivity than currently possible with clinical standards. Furthermore, the use of a panel of magnetic nanoparticles, distinguished with unique magnetization properties and bio-orthogonal chemistry, allowed simultaneous detection of the biomarkers EpCAM, HER2/neu, and EGFR on individual cells. This cost-effective, single-cell analytical technique is well-suited to perform molecular and cellular diagnosis of rare cells in the clinic.
Observing drug responses in the tumor microenvironment in vivo can be technically challenging. As a result, cellular responses to molecularly targeted cancer drugs are often studied in cell culture, which does not accurately represent the behavior of cancer cells growing in vivo. Using high resolution microscopy and fluorescently labeled genetic reporters for apoptosis, we developed an approach to visualize drug-induced cell death at single cell resolution in vivo. Stable expression of the mitochondrial intermembrane protein IMS-RP was established in human breast and pancreatic cancer cells. Automated image analysis was then used to quantify release of IMS-RP into the cytoplasm upon apoptosis and irreversible mitochondrial permeabilization. Both breast and pancreatic cancer cells showed higher basal apoptotic rates in vivo than in culture. To study drug-induced apoptosis, we exposed tumor cells to navitoclax (ABT-263), an inhibitor of Bcl-2, Bcl-xL, and Bcl-w, both in vitro and in vivo. Although the tumors responded to Bcl-2 inhibition in vivo, inducing apoptosis in around 20% of cancer cells, the observed response was much higher in cell culture. Together, our findings demonstrate an imaging technique that can be used to directly visualize cell death within the tumor microenvironment in response to drug treatment.
apoptosis; chemotherapy; imaging; breast cancer; pancreatic cancer
Myeloid cell content in atherosclerotic plaques associates with rupture and thrombosis. Thus, imaging of lesional monocyte and macrophages (Mo/Mϕ) could serve as a biomarker of disease progression and therapeutic intervention.
To noninvasively assess plaque inflammation with dextran nanoparticle-facilitated hybrid PET/MR imaging.
Methods and Results
Using clinically approved building blocks, we systematically developed 13nm polymeric nanoparticles consisting of crosslinked short chain dextrans which were modified with desferoxamine for zirconium-89 radiolabeling (89Zr-DNP) and a near infrared fluorochrome (VT680) for microscopic and cellular validation. Flow cytometry of cells isolated from excised aortas showed DNP uptake predominantly in Mo/Mϕ (76.7%) and lower signal originating from other leukocytes such as neutrophils and lymphocytes (11.8% and 0.7%, p<0.05 versus Mo/Mϕ). DNP colocalized with the myeloid cell marker CD11b on immunohistochemistry. PET/MRI revealed high uptake of 89Zr-DNP in the aortic root of ApoE−/− mice (standard uptake value, ApoE−/− mice versus wild type controls, 1.9±0.28 versus 1.3±0.03, p<0.05), corroborated by ex vivo scintillation counting and autoradiography. Therapeutic silencing of the monocyte-recruiting receptor CCR2 with siRNA decreased 89Zr-DNP plaque signal (p<0.05) and inflammatory gene expression (p<0.05).
Hybrid PET/MR imaging with a 13nm DNP enables noninvasive assessment of inflammation in experimental atherosclerotic plaques and reports on therapeutic efficacy of anti-inflammatory therapy.
PET/MRI; inflammation; atherosclerosis; molecular imaging; nanoparticles
Oligonucleotide hybridization was used as a cell-labeling method to significantly amplify the loading of magnetic probes onto target cells. The method utilized short oligonucleotides as the binding agents between antibodies and superparamagnetic iron oxide. This method not only enabled multiplexed analysis, but also allowed detection of multiple markers on a single sample containing only scant cell numbers.
magnetic labeling; oligonucleotide hybridization; magnetic resonance; multiplexed analysis; signal amplification
Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single cell pharmacokinetic imaging of PARP-1 inhibitors (PARPi) and model drug behavior under varying conditions. We visualize intracellular kinetics of PARPi distribution in real time, showing that PARPi reaches its cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modeling. Our theoretical and experimental data indicate that tumor cells are exposed to sufficiently high PARPi concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA repair inhibition. This suggests that single cell pharmacokinetic imaging and derived modeling improves our understanding of drug action at single cell resolution in vivo.
Mutually orthogonal tetrazine–transcyclooctene and azide–cyclooctyne cycloaddition reactions were used simultaneously for the bioorthogonal labeling of two different live cell populations in the same culture. These small-molecule probes show good chemical reactivity and can be readily incorporated into biological systems.
azides; bioorthogonal reactions; cycloaddition; imaging; tetrazines
During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaque in the arterial wall and cause its rupture, thus triggering myocardial infarction and stroke. Survivors of acute coronary syndromes have a high risk of recurrent events for unknown reasons. Here we show that the systemic response to ischemic injury aggravates chronic atherosclerosis. After myocardial infarction or stroke, apoE−/− mice developed larger atherosclerotic lesions with a more advanced morphology. This disease acceleration persisted over many weeks and was associated with markedly increased monocyte recruitment. When seeking the source of surplus monocytes in plaque, we found that myocardial infarction liberated hematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signaling. The progenitors then seeded the spleen yielding a sustained boost in monocyte production. These observations provide new mechanistic insight into atherogenesis and provide a novel therapeutic opportunity to mitigate disease progression.
Atherosclerotic lesions are believed to grow via the recruitment of bone marrow-derived monocytes. Among the known murine monocyte subsets, Ly-6Chigh monocytes are inflammatory, accumulate in lesions preferentially, and differentiate. Here we hypothesized that the bone marrow outsources the production of Ly-6Chigh monocytes during atherosclerosis.
Methods and Results
Using murine models of atherosclerosis and fate-mapping approaches, we show that hematopoietic stem and progenitor cells (HSPC) progressively relocate from the bone marrow to the splenic red pulp where they encounter GM-CSF and IL-3, clonally expand, and differentiate to Ly-6Chigh monocytes. Monocytes born in such extramedullary niches intravasate, circulate, and accumulate abundantly in atheromata. Upon lesional infiltration, Ly-6Chigh monocytes secrete inflammatory cytokines, reactive oxygen species, and proteases. Eventually, they ingest lipids and become foam cells.
Our findings indicate that extramedullary sites supplement the bone marrow’s hematopoietic function by producing circulating inflammatory cells that infiltrate atherosclerotic lesions.
Atherosclerosis; Imaging; Immune System; Immunology; Macrophage
Tissue macrophages play a critical role both in normal physiology as well in disease states. However, due to a lack of specific imaging agents, we continue to have a poor understanding of their absolute numbers, flux rates and functional states in different tissues. Here, we describe a new macrophage specific PET imaging agent, labeled with zirconium-89 (89Zr), that was based on a crosslinked, short-chain dextran nanoparticle (13 nm). Following systemic administration, the particle demonstrated a vascular half-life of 3.9 hours, and was found to locate primarily to tissue resident macrophages rather than to other white blood cells. Subsequent imaging of the probe using a xenograft mouse model of cancer allowed for quantitation of tumor associated macrophage (TAM) numbers, which are of major interest in emerging molecular targeting strategies. It is likely that the material described, which enables the visualization of macrophage biology in vivo, will likewise be useful for a multitude of human applications.
Nanoparticles; Macrophage; 89Zr; PET imaging
The development of faster and more sensitive detection methods capable of identifying specific bacterial types and strains has remained a longstanding clinical challenge. Thus to date, the diagnosis of bacterial infections continues to rely on the performance of time-consuming cultures. Here, we demonstrate the use of bioorthogonal chemistry for magnetically labeling specific pathogens to enable their subsequent detection by nuclear magnetic resonance. Antibodies against a bacterial target of interest were first modified with trans-cyclooctene and then coupled to tetrazine-modified magnetic nanoprobes, directly on the bacteria. This labeling method was verified using surface plasmon resonance as well as by using a miniaturized diagnostic magnetic resonance device capable of highly specific detection of Staphylococcus aureus. Compared to other copper-free bioorthogonal chemistries, the cycloaddition reaction described displayed faster kinetics and yielded higher labeling efficiency. Considering the short assay times and the portability of the necessary instrumentation, it is feasible that this approach could be adapted for clinical use in resource-limited settings.
Glioblastomas shed large quantities of small, membrane-bound microvesicles (MVs) into the circulation. While these hold promise as potential biomarkers of therapeutic response, their identification and quantitation remain challenging. Here, we describe a highly sensitive and rapid analytical technique for profiling circulating MVs directly from blood samples of glioblastoma patients. MVs, introduced onto a dedicated microfluidic chip, are labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system. Compared with current methods, this integrated system has a much higher detection sensitivity, and can differentiate glioblastoma multiforme (GBM) MVs from non-tumor host cell-derived MVs. We also show that circulating GBM MVs can serve as a surrogate for primary tumor mutations and a predictive metric of treatment-induced changes. This platform could provide both an earlier indicator of drug efficacy and a potential molecular stratifier for human clinical trials.
Imaging has become an indispensable tool in the study of cancer biology and in clinical prognosis and treatment. The rapid advances in high resolution fluorescent imaging at single cell level and MR/PET/CT image registration, combined with new molecular probes of cell types and metabolic states, will allow the physical scales imaged by each to be bridged. This holds the promise of translation of basic science insights at the single cell level to clinical application. In this article, we describe the recent advances in imaging at the macro- and micro-scale and how these advances are synergistic with new imaging agents, reporters, and labeling schemes. Examples of new insights derived from the different scales of imaging and relevant probes are discussed in the context of cancer progression and metastasis.
Multi-photon microscopy and other techniques can be used to monitor the behavior and fate of tumor cells in vivo. This can be correlated with gene expression profiles to define “invasion signatures” and new prognostic markers.
Many cancers shed malignant cells into the circulation. Albeit their rare frequency, these cancer cells serve both diagnostic and therapeutic purposes. However, their purification, quantification and characterization remain challenging. Here, we present a low-cost, rapid microfluidic cell sorter (μFCS) device for the detection and molecular analysis of circulating tumor cells (CTCs). The μFCS employs a weir-shaped microfluidic structure to separate and capture CTCs from unprocessed whole blood cells based on their size difference. The system further allows on-chip culture and molecular profiling of captured cancer cells, and provides easy cell retrieval for subsequent proteomic and genetic analyses. Using a mouse model of cancer metastasis, we show that the μFCS can enrich CTCs from whole blood to unmask their cancer genetic signature. With its rapid processing speed and versatility for downstream analyses, this platform could have a wide range of potential applications in clinical cancer diagnosis.
biotechnology; cellular profiling; circulating tumor cells; genetic analysis; microfluidics
1,2,4,5-Tetrazines have been established as effective dienes for inverse electron demand [4 + 2] Diels-Alder cycloaddition reactions with strained alkenes for over fifty years. Recently, this reaction pair combination has been applied to bioorthogonal labeling and cell detection applications; however, to date there has been no detailed examination and optimization of tetrazines for use in biological experiments. Here we report the synthesis and characterization of twelve conjugatable tetrazines. The tetrazines were all synthesized in a similar fashion and were screened in parallel to identify candidates most ideally suited for biological studies. In depth follow up studies revealed compounds with varying degrees of stability and reactivity that could each be useful in different bioorthogonal applications. One promising, highly stable and water soluble derivative was used in pre-targeted cancer cell labeling studies, confirming its utility as a bioorthogonal moiety.
Pedal to the metal: Using inverse Diels-Alder catalyst free TCO/Tz cycloadditions, we were able to quickly and selectively generate an 18F-labeled AZD2281-derivative from multiple different scaffolds (A–E). Excess cold material was removed within minutes using a TCO scavenger resin. This protocol allows the parallel synthesis of a library of potential PET imaging agents in a short time, increasing the efficiency of lead compound detection. The novel PET probe was successfully tested in biological assays and its potency and targeted accumulation was confirmed in vivo.
PARP1; cancer; bioorthogonal conjugation; 18F PET; in vivo imaging; scavenger resin
Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible KrasG12D-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on KrasG12D expression. Transcriptome and metabolomic analyses indicate that KrasG12D serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that KrasG12D drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.
All juvenile NOD mice exhibit insulitis, but there is substantial variation in their progression to diabetes. We demonstrate that a patient-validated magnetic-resonance-imaging (MRI) strategy to non-invasively visualize local effects of pancreatic-islet inflammation can predict diabetes onset in NOD mice. MRI signals acquired during a narrow early time-window allowed pre-sorting into disease-progressors and -nonprogressors and an estimate of time-to-diabetes. We exploited this capability to identify novel elements correlated with disease protection, including CRIg (complement receptor of the immunoglobulin superfamily), which marked a subset of macrophages associated with diabetes resistance. Administration of CRIg-Fc depressed MRI signals and diabetes incidence. In addition to identifying regulators of disease progression, this study shows that diabetes is set at an early age in NOD mice.
A nuclear protein target, polo-like kinase 1 (PLK1) was imaged using a biocompatible bioorthogonal ligation between a specific drug and a fluorescent dye in live cells. Colocalization of the dye and the protein target was confirmed by antibody staining and by expressing a GFP construct of PLK1. The two-step PLK1 imaging procedure was used to quantify PLK1 expression levels in cancer cell lines of various tissue origins.
cancer; cell cycle; cycloaddition; live cell imaging; polo-like kinase
Histone deacetylases (HDACs) are a group of enzymes that modulate gene expression and cell state by deacetylation of both histone and non-histone proteins. A variety of HDAC inhibitors (HDACi) have already undergone clinical testing in cancer. Real-time in vivo imaging of HDACs and their inhibition would be invaluable; however, the development of appropriate imaging agents has remained a major challenge. Here, we describe the development and evaluation of 18F-suberoylanilide hydroxamic acid (18F-SAHA 1a), a close analog of the most clinically relevant HDACi, suberoylanilide hydroxamic acid (SAHA). We demonstrate that 1a has near identical biochemical activity profiles to SAHA, and report findings from pharmacokinetic studies. Using a murine ovarian cancer model, we likewise show that HDACi target binding efficacy can be quantitated within 24 hours of administration. 1a thus represents the first 18F-positron emission tomography (PET) HDAC imaging agent, which also exhibits low nanomolar potency and is pharmacologically analogous to a clinically relevant HDACi.
Histone Deacetylase; Epigenetics; PET; imaging; in vivo; inhibitor
Current thrombolytic therapies rely upon exogenous plasminogen activators (PA) to effectively lyse clots, thereby restoring blood flow and preventing tissue and organ death. Yet, these PAs may also impair normal hemostasis which may lead to life-threatening bleeding, including intracerebral hemorrhage. Thus, the aim of this current study is to develop new thrombus-targeted fibrinolytic agents that harness the multifunctional theranostic capabilities of nanomaterials, potentially allowing for the generation of efficacious thrombolytics while minimizing deleterious side effects.
Materials and Methods
A thrombus-targeted nano-fibrinolytic agent (CLIO-FXIII-PEG-tPA) was synthesized using a magnetofluorescent crosslinked dextran-coated iron oxide (CLIO) nanoparticle platform that was conjugated to recombinant tissue plasminogen activator (tPA). Thrombus-targeting was achieved by derivatizing the nanoparticle with an activated factor XIII (FXIIIa)-sensitive peptide based on the amino terminus of α2-antiplasmin. Human plasma clot binding ability of the targeted and control agents was assessed by fluorescence reflectance imaging. Next, the in vitro enzymatic activity of the agents was assessed by S2288-based amidolytic activity, and an ELISA D-dimer assay for fibrinolysis. In vivo targeting of the nanoagent was next examined by intravital fluorescence microscopy of murine arterial and venous thrombosis. The fibrinolytic activity of the targeted nanoagent compared to free tPA was then evaluated in vivo in murine pulmonary embolism.
In vitro, the targeted thrombolytic nanoagent demonstrated binding to fresh frozen plasma (FFP) clots superior to control nanoagents (ANOVA p < 0.05). On a weight (mg) basis, the S2288 amidolytic efficiency of the targeted nanoagent was approximately 15% reduced compared to free tPA. When normalized by S2288-based activity, targeted, control, and free tPA samples demonstrated equivalent in vitro fibrinolytic activity against human plasma clots, as determined by ELISA D-dimer assays. The FXIIIa targeted fibrinolytic nanoagent efficiently bound the margin of intravascular thrombi as detected by IVFM. In in vivo fibrinolysis studies normalized for activity, the FXIIIa-targeted agent lysed pulmonary emboli with similar efficacy as free tPA (p>0.05).
The applicability of a FXIIIa-targeted thrombolytic nanoagent in the treatment of thromboembolism was demonstrated in vitro and in vivo. Future studies are planned to investigate the safety profile and overall efficacy of this class of nanoagents, and to further optimize their thrombus-targeting profile and lytic action.
Fibrinolytic; iron oxide; therapy; thrombosis; multimodal; theranostic; imaging
IL-1b signaling augments continued splenic monocyte supply during acute inflammation.
Monocytes (Mo) and macrophages (MΦ) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MΦ and their effector functions without compromising innate immunity’s critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MΦ in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (20 h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by IL-1β; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MΦ exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke. These findings expand our knowledge of Mo/MΦ flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure.
Novel therapeutic agents combined with innovative modes of delivery and non-invasive imaging of drug delivery, pharmacokinetics and efficacy are crucial in developing effective clinical anti-cancer therapies. In this study, we have created and characterized multiple novel variants of anti-angiogenic protein thrombospondin (aaTSP-1) that were comprised of unique regions of 3 type-I-repeats of TSP-1 and employed engineered human neural stem cells (hNSC) to provide sustained on-site delivery of secretable aaTSP-1 to tumor-vasculature. We show that hNSC-aaTSP-1 has anti-angiogenic effect on human brain and dermal microvascular endothelial cells co-cultured with established glioma cells and CD133+ glioma-initiating-cells. Using human glioma cells and hNSC engineered with different combinations of fluorescent and bioluminescent marker proteins and employing bioluminescence imaging and intravital-scanning microscopy, we show that aaTSP-1 targets the vascular-component of gliomas and a single administration of hNSC-aaTSP-1 markedly reduces tumor vessel-density that results in inhibition of tumor-progression and increased survival in mice bearing highly malignant human gliomas. We also show that therapeutic hNSC do not proliferate and remain in an un-differentiated state in the brains of glioma bearing mice. This study provides a platform for accelerated development of future cell based therapies for cancer.
‘in vivo imaging’; ‘glioma’; ‘human neural stem cells’; TSP-1’; ‘endothelial cells’; ‘angiogenesis’