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1.  Comparison of select cancer biomarkers in human circulating and bulk tumor cells using magnetic nanoparticles and miniaturized micro-NMR system 
Circulating tumor cells (CTC) harvested from peripheral blood have received significant interest as sources for serial sampling to gauge treatment efficacy. Nanotechnology and microfluidic based approaches are emerging to facilitate such analyses. While of considerable clinical importance, there is little information on how similar or different CTC are from their shedding bulk tumors. In this clinical study, paired tumor fine needle aspirate and peripheral blood samples were obtained from cancer patients during image guided biopsy. Using targeted magnetic nanoparticles and a point-of-care micro-NMR system, we compared selected biomarkers (EpCAM, EGFR, HER-2 and vimentin) in both CTC and fine needle biopsies of solid epithelial cancers. We show a weak correlation between each paired sample, suggesting that use of CTC as ‘liquid biopsies’ and proxies to metastatic solid lesions could be misleading.
PMCID: PMC4330975  PMID: 23570873
Circulating tumor cells; magnetic nanoparticles; micro-NMR (µNMR); point of care diagnostics
2.  Endoscopic fluorescence imaging of cathepsin activity can discriminate, dysplasia from colitis 
Inflammatory bowel diseases  2013;19(7):1339-1345.
Surveillance colonoscopy using random biopsies to detect colitis-associated cancer (CAC) suffers from poor sensitivity. Although chromo-endoscopy improves detection, acceptance in the community has been slow. Here, we examine the usefulness of near infrared fluorescence (NIRF) endoscopy to image molecular probes for cathepsin activity in colitis-induced dysplasia.
In patient samples, cathepsin activity was correlated with colitis and dysplasia. In mice, cathepsin activity was detected as fluorescent hydrolysis product of substrate based probes (SBPs) after injection into IL-10−/− colitic mice. Fluorescence colonoscopy and colonic whole mount imaging were performed prior to complete sectioning and pathology review of resected colons.
Cathepsin activity was 5 and 8 – fold higher in dysplasia and CAC respectively, compared to areas of mild colitis in patient tissue sections. The signal to noise ratios for dysplastic lesions seen by endoscopy in IL-10−/− mice were 5.2 ±1.3, (P=0.0001). Lesions with increased NIRF emissions were classified as raised or flat dysplasia, lymphoid tissue and ulcers. Using images collected by endoscopy, a receiver operating characteristic curve (ROC) for correctly diagnosing dysplasia was calculated. The area under the curve was 0.927. At a cut off of 1000 MFI, the sensitivity and specificity for detecting dysplasia were 100% and 83% respectively. Analysis revealed that enhanced NIRF emissions derived from increased numbers of infiltrating myeloid derived suppressor cells and macrophages with equivalent cathepsin activity.
These studies indicate that cathepsin SBP imaging correctly identifies dysplastic foci within chronically inflamed colons. Cathepsin-based NIRF endoscopy presents unique advantages that may increase sensitivity and specificity of surveillance colonoscopy in patients with CUC.
PMCID: PMC4306589  PMID: 23591598
Dysplasia; colitis; macrophages; cathepsin activity; fluorescence endoscopy
3.  Fluorescent Exendin-4 Derivatives for Pancreatic β-Cell Analysis 
Bioconjugate chemistry  2013;25(1):171-177.
The ability to reliably identify pancreatic β-cells would have far reaching implications for a greater understanding of β-cell biology, measurement of β-cell mass in diabetes, islet transplantation, and drug development. The glucagon-like peptide-1 receptor (GLP1R) is highly expressed on the surface of insulin producing pancreatic β-cells. Using systematic modifications of the GLP1R ligand, exendin-4, we screened over 25 compounds and identified a palette of fluorescent exendin-4 with high GLP1R binding affinity. We show considerable differences in affinity, as well as utility of the top candidates for flow cytometry and microscopy of β-cells. Some of the developed compounds should be particularly useful for basic and translational β-cell research.
PMCID: PMC4016126  PMID: 24328216
pancreatic beta cells; GLP1R; fluorochromes; flow cytometry
4.  Miniaturized Nuclear Magnetic Resonance Platform for Detection and Profiling of Circulating Tumor Cells 
Lab on a chip  2013;14(1):10.1039/c3lc50621e.
Accurate detection and profiling of circulating tumor cells (CTCs) is a highly sought after technology to improve cancer management. Such “liquid biopsies” could offer a non-invasive, repeatable window into each patient’s tumor, facilitating early cancer diagnosis and treatment monitoring. The rarity of CTCs, approximated at 1 CTC for every billion peripheral blood cells, however, poses significant challenges to sensitive and reliable detection. We have recently developed a new biosensor platform, namely a micro-nuclear magnetic resonance (µNMR). Through the synergistic integration of microfabrication, nanosensors, and novel chemistries, the µNMR platform offers high detection sensitivity and point-of-care operation, overcoming technical barriers in CTC research. We herein review the µNMR technology with emphasis on its application on CTC detection. Recent advances in the sensing technology will be summarized, followed by the description on the dynamic interplay between preclinical and clinical CTC studies.
PMCID: PMC3844052  PMID: 23835814
5.  Chronic variable stress activates hematopoietic stem cells 
Nature medicine  2014;20(7):754-758.
Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis1,2. While incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known crosstalk between the brain and immune system includes the hypothalamic–pituitary–adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic–adrenal–medullary axis, which controls stress–induced catecholamine release in support of the fight–or–flight reflex3,4. It remains unknown however if chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive progenitors, giving rise to higher levels of disease–promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Sympathetic nerve fibers release surplus noradrenaline, which uses the β3 adrenergic receptor to signal bone marrow niche cells to decrease CXCL12 levels. Consequently, elevated hematopoietic stem cell proliferation increases output of neutrophils and inflammatory monocytes. When atherosclerosis–prone ApoE−/− mice encounter chronic stress, accelerated hematopoiesis promotes plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.
PMCID: PMC4087061  PMID: 24952646
6.  Magnetic Nanosensor for Detection and Profiling of Erythrocyte-Derived Microvesicles 
ACS nano  2013;7(12):11227-11233.
During the course of their lifespan, erythrocytes actively shed phospholipid-bound, microvesicles (MVs). In stored blood, the number of these erythrocyte-derived MVs have been observed to increase over time, suggesting their potential value as a quality metric for blood products. The lack of sensitive, standardized MV assays, however, poses a significant barrier to implementing MV analyses into clinical settings. Here, we report on a new nanotechnology platform capable of rapid and sensitive MV detection in packed red blood cell (pRBC) units. A filter-assisted microfluidic device was designed to enrich MVs directly from pRBC units, and label them with target-specific magnetic nanoparticles. Subsequent detection using a miniaturized nuclear magnetic resonance system enabled accurate MV quantification as well as the detection of key molecular markers (CD44, CD47, CD55). By applying the developed platform, MVs in stored blood units could also be monitored longitudinally. Our results showed that MV counts increase over time, and thus could serve as an effective metric of blood aging. Furthermore, our studies found that MVs have the capacity to generate oxidative stress and consume nitric oxide. By advancing our understanding of MV biology, we expect that the developed platform will lead to improved blood product quality and transfusion safety.
PMCID: PMC3898036  PMID: 24295203
nanotechnology; biosensor; microvesicles; microfluidics; blood; transfusion
7.  In Vivo Imaging of Multidrug Resistance Using a Third Generation MDR1 Inhibitor 
Bioconjugate chemistry  2014;25(6):1137-1142.
Cellular up-regulation of multidrug resistance protein 1 (MDR1) is a common cause for resistance to chemotherapy; development of third generation MDR1 inhibitors—several of which contain a common 6,7-dimethoxy-2-phenethyl-1,2,3,4- tetrahydroisoquinoline substructure—are underway. Efficacy of these agents has been difficult to ascertain, partly due to a lack of pharmacokinetic reporters for quantifying inhibitor localization and transport dynamics. Some of the recent third generation inhibitors have pendant heterocycles, for example, a chromone moiety, which we hypothesized could be converted to a fluorophore. Following synthesis and teasing of a small set of analogs, we identified one lead compound that can be used as a cellular imaging agent that exhibits structural similarity and behavior akin to the latest generation of MDR1 inhibitors.
PMCID: PMC4098115  PMID: 24806886
Tariquidar; MDR1; imaging; multidrug resistance
8.  Photoactivatable Drug-Caged Fluorophore Conjugate Allows Direct Quantification of Intracellular Drug Transport 
We report here a method that utilizes photoactivatable drug-caged fluorophore conjugate to quantify intracellular drug trafficking processes at single cell resolution. Photoactivation is performed in labeled cellular compartments to visualize intracellular drug exchange at physiologic conditions, without the need for washing, facilitating its translation to in vivo cancer models.
PMCID: PMC3856557  PMID: 24135896
9.  Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis 
The Journal of Experimental Medicine  2014;211(6):1243-1256.
In response to lung infection, pleural innate response activator B cells produce GM-CSF–dependent IgM and ensure a frontline defense against bacterial invasion.
Pneumonia is a major cause of mortality worldwide and a serious problem in critical care medicine, but the immunophysiological processes that confer either protection or morbidity are not completely understood. We show that in response to lung infection, B1a B cells migrate from the pleural space to the lung parenchyma to secrete polyreactive emergency immunoglobulin M (IgM). The process requires innate response activator (IRA) B cells, a transitional B1a-derived inflammatory subset which controls IgM production via autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. The strategic location of these cells, coupled with the capacity to produce GM-CSF–dependent IgM, ensures effective early frontline defense against bacteria invading the lungs. The study describes a previously unrecognized GM-CSF-IgM axis and positions IRA B cells as orchestrators of protective IgM immunity.
PMCID: PMC4042649  PMID: 24821911
10.  Epigenetic modulation of type-1 diabetes via a dual effect on pancreatic macrophages and β cells 
eLife  null;3:e04631.
Epigenetic modifiers are an emerging class of anti-tumor drugs, potent in multiple cancer contexts. Their effect on spontaneously developing autoimmune diseases has been little explored. We report that a short treatment with I-BET151, a small-molecule inhibitor of a family of bromodomain-containing transcriptional regulators, irreversibly suppressed development of type-1 diabetes in NOD mice. The inhibitor could prevent or clear insulitis, but had minimal influence on the transcriptomes of infiltrating and circulating T cells. Rather, it induced pancreatic macrophages to adopt an anti-inflammatory phenotype, impacting the NF-κB pathway in particular. I-BET151 also elicited regeneration of islet β-cells, inducing proliferation and expression of genes encoding transcription factors key to β-cell differentiation/function. The effect on β cells did not require T cell infiltration of the islets. Thus, treatment with I-BET151 achieves a ‘combination therapy’ currently advocated by many diabetes investigators, operating by a novel mechanism that coincidentally dampens islet inflammation and enhances β-cell regeneration.
eLife digest
The DNA inside a cell is often tightly wrapped around proteins to form a compact structure called chromatin. Chemical groups added to the chromatin can encourage nearby genes to either be switched on or off; and several enzymes and other proteins help to read, add, or remove these marks from the chromatin. If these chromatin modifications (or the related enzymes and proteins) are disturbed it can lead to diseases like cancer. It has also been suggested that similar changes may influence autoimmune diseases, in which the immune system attacks the body's own tissues.
Drugs that target the proteins that read, add, or remove these chromatin modifications are currently being developed to treat cancer. For example, drugs that inhibit one family of these proteins called BET have helped to treat tumors in mice that have cancers of the blood or lymph nodes. However, because these drugs target pathways involved in the immune system they may also be useful for treating autoimmune diseases.
Now Fu et al. have tested whether a BET inhibitor might be a useful treatment for type-1 diabetes. In patients with type-1 diabetes, the cells in the pancreas that produce the insulin hormone are killed off by the immune system. Without adequate levels of insulin, individuals with type-1 diabetes may experience dangerous highs and lows in their blood sugar levels and must take insulin and sometimes other medications.
Using mice that spontaneously develop type-1 diabetes when still relatively young, Fu et al. tested what would happen if the mice received a BET inhibitor for just 2 weeks early on in life. Treated mice were protected from developing type-1 diabetes for the rest of their lives. Specifically, the treatment protected the insulin-producing cells and allowed them to continue producing insulin. The drug reduced inflammation in the pancreas and increased the expression of genes that promote the regeneration of insulin-producing cells.
Diabetes researchers have been searching for drug combinations that protect the insulin-producing cells and boost their regeneration. As such, Fu et al. suggest that these findings justify further studies to see if BET inhibitors may help to treat or prevent type-1 diabetes in humans.
PMCID: PMC4270084  PMID: 25407682
autoimmune diabetes; bromodomain inhibitor; NF-KB; mouse
11.  Effect of Small Molecule Modification on Single Cell Pharmacokinetics of PARP Inhibitors 
Molecular cancer therapeutics  2014;13(4):986-995.
The heterogeneous delivery of drugs in tumors is an established process contributing to variability in treatment outcome. Despite the general acceptance of variable delivery, the study of the underlying causes is challenging given the complex tumor microenvironment including intra- and inter-tumor heterogeneity. The difficulty in studying this distribution is even more significant for small molecule drugs where radiolabeled compounds or mass spectrometry detection lack the spatial and temporal resolution required to quantify the kinetics of drug distribution in vivo. In this work, we take advantage of the synthesis of fluorescent drug conjugates that retain their target binding but are designed with different physiochemical and thus pharmacokinetic properties. Using these probes, we followed the drug distribution in cell culture and tumor xenografts with temporal resolution of seconds and subcellular spatial resolution. These measurements, including in vivo permeability of small molecule drugs, can be used directly in predictive pharmacokinetic models for the design of therapeutics and companion imaging agents as demonstrated by a finite element model.
PMCID: PMC3981972  PMID: 24552776
Drug distribution; tumor heterogeneity; finite element modeling; drug permeability; intracellular imaging agent
12.  Bioorthogonal approach to identify unsuspected drug targets in live cells 
Angewandte Chemie (International ed. in English)  2013;52(40):10.1002/anie.201304096.
PMCID: PMC3856564  PMID: 23960025
bioorthogonal; proteomics; PARP1; cleavable linkers; secondary drug targets
13.  A Pretargeted PET Imaging Strategy Based on Bioorthogonal Diels–Alder Click Chemistry 
The specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacologic half-lives necessitate the use of radioisotopes with long physical halsives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodology for pretargeted PET imaging based on the bioorthogonal Diels–Alder click reaction between tetrazine and transcyclooctene.
A proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and 64Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with 64Cu. Pretargeted in vivo biodistribution and PET imaging experiments were performed with athymic nude mice bearing A33 antigen–expressing, SW1222 colorectal cancer xenografts.
The huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the 64Cu-NOTA–labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9–10 MBq/μg. For in vivo experiments, mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with 64Cu-NOTA–labeled tetrazine for PET imaging and biodistribution experiments. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per gram), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution experiments performed using A33 directly labeled with either 64Cu or 89Zr revealed that although absolute tumor uptake was higher with the directly radiolabeled antibodies, the pre-targeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calculations revealed that the 64Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with 89Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, respectively).
The high quality of the images produced by this pretargeting approach, combined with the ability of the methodology to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clinical translation.
PMCID: PMC4151562  PMID: 23708196
positron emission tomography (PET); pretargeting; click chemistry; antibody; colorectal cancer
14.  Cancer cell profiling by barcoding allows multiplexed protein analysis in fine needle aspirates 
Science translational medicine  2014;6(219):219ra9.
Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. Here, we introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine needle aspirates (FNA), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing where barcodes can be photo-cleaved and digitally detected without any amplification steps. Following extensive benchmarking in cell lines, this method showed high reproducibility and achieved single cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.
PMCID: PMC4063286  PMID: 24431113
15.  Superbright Bioorthogonal Turn-on Probes** 
Angewandte Chemie (International ed. in English)  2013;52(27):10.1002/anie.201301100.
The fastest and the brightest: A new design that intimately connects tetrazine to a BODIPY fluorophore enables exceptionally efficient energy transfer and quenching. Upon reaction of the tetrazine, the brightness of the fluorophore increases more than a thousand-fold, a fluorogenic activation up to two orders of magnitude greater than previously described.
PMCID: PMC3875324  PMID: 23712730
click chemistry; fluorescent probes; fluorogenic; tetrazine; through-bond energy transfer
16.  Factor XIII Deficiency Causes Cardiac Rupture, Impairs Wound Healing, and Aggravates Cardiac Remodeling in Mice With Myocardial Infarction 
Circulation  2006;113(9):1196-1202.
Identification of key molecular players in myocardial healing could lead to improved therapies, reduction of scar formation, and heart failure after myocardial infarction (MI). We hypothesized that clotting factor XIII (FXIII), a transglutaminase involved in wound healing, may play an important role in MI given prior clinical and mouse model data.
Methods and Results
To determine whether a truly causative relationship existed between FXIII activity and myocardial healing, we prospectively studied myocardial repair in FXIII-deficient mice. All FXIII−/− and FXIII−/+ (FXIII activity <5% and 70%) mice died within 5 days after MI from left ventricular rupture. In contradistinction, FXIII−/− mice that received 5 days of intravenous FXIII replacement therapy had normal survival rates; however, cardiac MRI demonstrated worse left ventricular remodeling in these reconstituted FXIII−/− mice. Using a FXIII-sensitive molecular imaging agent, we found significantly greater FXIII activity in wild-type mice and FXIII−/− mice receiving supplemental FXIII than in FXIII−/− mice (P<0.05). In FXIII−/− but not in reconstituted FXIII−/− mice, histology revealed diminished neutrophil migration into the MI. Reverse transcriptase–polymerase chain reaction studies suggested that the impaired inflammatory response in FXIII−/− mice was independent of intercellular adhesion molecule and lipopolysaccharide-induced CXC chemokine, both important for cell migration. After MI, expression of matrix metalloproteinase-9 was 650% higher and collagen-1 was 53% lower in FXIII−/− mice, establishing an imbalance in extracellular matrix turnover and providing a possible mechanism for the observed cardiac rupture in the FXIII−/− mice.
These data suggest that FXIII has an important role in murine myocardial healing after infarction.
PMCID: PMC4066325  PMID: 16505171
factor XIII; healing; heart failure; myocardial infarction; remodeling
17.  Myeloperoxidase-targeted imaging of active inflammatory lesions in murine experimental autoimmune encephalomyelitis 
Brain : a journal of neurology  2008;131(0 4):1123-1133.
Inflammatory demyelinating plaques are the pathologic hallmark of active multiple sclerosis and often precede clinical manifestations. Noninvasive early detection of active plaques would thus be crucial in establishing presymptomatic diagnosis and could lead to early preventive treatment strategies. Using murine experimental autoimmune encephalomyelitis as a model of multiple sclerosis, we demonstrate that a prototype paramagnetic myeloperoxidase (MPO) sensor can detect and confirm more, smaller, and earlier active inflammatory lesions in living mice by in vivo magnetic resonance imaging (MRI). We show that MPO expression corresponded with areas of inflammatory cell infiltration and demyelination, and higher MPO activity as detected by MPO imaging, biochemical assays, and histopathological analyses correlated with increased clinical disease severity. Our findings present a potential new translational approach for specific noninvasive inflammatory plaque imaging. This approach could be used in longitudinal studies to identify active demyelinating plaques as well as to more accurately track disease course following treatment in clinical trials.
PMCID: PMC4044727  PMID: 18234693
myeloperoxidase; neuroinflammation; demyelination; targeted imaging; MRI
18.  Single cell imaging of Bruton's Tyrosine Kinase using an irreversible inhibitor 
Scientific Reports  2014;4:4782.
A number of Bruton's tyrosine kinase (BTK) inhibitors are currently in development, yet it has been difficult to visualize BTK expression and pharmacological inhibition in vivo in real time. We synthesized a fluorescent, irreversible BTK binder based on the drug Ibrutinib and characterized its behavior in cells and in vivo. We show a 200 nM affinity of the imaging agent, high selectivity, and irreversible binding to its target following initial washout, resulting in surprisingly high target-to-background ratios. In vivo, the imaging agent rapidly distributed to BTK expressing tumor cells, but also to BTK-positive tumor-associated host cells.
PMCID: PMC3998017  PMID: 24759210
19.  Noise suppressed, multifocus image fusion for enhanced intraoperative navigation 
Journal of biophotonics  2012;6(4):363-370.
Current intraoperative imaging systems are typically not able to provide ‘sharp’ images over entire large areas or entire organs. Distinct structures such as tissue margins or groups of malignant cells are therefore often difficult to detect, especially under low signal-to-noise-ratio conditions. In this report, we introduce a noise suppressed multifocus image fusion algorithm, that provides detailed reconstructions even when images are acquired under sub-optimal conditions, such is the case for real time fluorescence intraoperative surgery. The algorithm makes use of the Anscombe transform combined with a multi-level stationary wavelet transform with individual threshold-based shrinkage. While the imaging system is integrated with a respiratory monitor triggering system, it can be easily adapted to any commercial imaging system. The developed algorithm is made available as a plugin for Osirix.
Intraoperative detection of small malignant fluorescent cells using the proposed noise suppressed multifocus image fusion system. Red/Yellow circles indicate small groups of malignant cells.
PMCID: PMC3779878  PMID: 22887724
20.  Building Blocks for the Construction of Bioorthogonally Reactive Peptides via Solid-Phase Peptide Synthesis 
ChemistryOpen  2014;3(2):48-53.
The need for post-synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents. In this regard, the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far-reaching implications in both the laboratory and the clinic. Herein, we report the synthesis and development of a series of modular and versatile building blocks for inverse electron-demand Diels–Alder copper-free click chemistry: tetrazine-functionalized artificial amino acids. Following the development of a novel peptide coupling protocol for peptide synthesis in the presence of tetrazines, we successfully demonstrated its effectiveness and applicability. This versatile methodology has the potential to have a transformational impact, opening the door for the rapid, facile, and modular synthesis of bioorthogonally reactive peptide probes.
PMCID: PMC4000166  PMID: 24808990
bioorthogonal chemistry; click chemistry; positron emission tomography (PET); solid-phase peptide synthesis; zirconium-89
21.  Automated motion artifact removal for intravital microscopy, without a priori information 
Scientific Reports  2014;4:4507.
Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.
PMCID: PMC3968488  PMID: 24676021
22.  Local proliferation dominates lesional macrophage accumulation in atherosclerosis 
Nature medicine  2013;19(9):1166-1172.
During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall1,2. The observation that circulating monocytes give rise to lesional macrophages3–9 has reinforced the concept that monocyte infiltration dictates macrophage build-up. Recent work indicates, however, that macrophages do not depend on monocytes in some inflammatory contexts10. We therefore revisited the mechanism of macrophage accumulation in atherosclerosis. We show that murine atherosclerotic lesions experience a surprisingly rapid, 4-week, cell turnover. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation via the involvement of scavenger receptor (SR)-A. Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease.
PMCID: PMC3769444  PMID: 23933982
23.  Advanced Motion Compensation Methods for Intravital Optical Microscopy 
Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions.
PMCID: PMC3832946  PMID: 24273405
Intravital microscopy; image stabilization; in vivo imaging; motion artifact and motion compensation
24.  Angiotensin II drives the production of tumor-promoting macrophages 
Immunity  2013;38(2):296-308.
Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that over-production of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P1 signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone’s pathway to stimulate cancer-promoting immunity.
PMCID: PMC3582771  PMID: 23333075
25.  Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells 
PLoS ONE  2014;9(2):e89882.
Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics.
PMCID: PMC3931832  PMID: 24587097

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