Moritz F. Kircher

Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease

Monocytes participate importantly in the pathogenesis of atherosclerosis, but their spatial and temporal recruitment from circulation remains uncertain. This study tests the hypothesis that monocyte accumulation in atheroma correlates with the extent of disease by using a sensitive and simple quantitative assay that allows tracking of highly enriched populations of blood monocytes. A two-step isolation method yielded viable and functionally intact highly enriched peripheral blood monocyte populations (>90%). Recipient mice received syngeneic monocytes labeled in two ways: by transgenically expressing EGFP or with a radioactive tracer [111In]oxine. After 5 days, more labeled cells accumulated in the aorta, principally the aortic root and ascending aorta, of 10-wk-old ApoE−/− compared with 10-wk-old C57BL/6 mice (223 ± 3 vs. 87 ± 22 cells per aorta). Considerably more monocytes accumulated in 20-wk-old ApoE−/− mice on either chow (314 ± 41 cells) or high-cholesterol diet (395 ± 53 cells). Fifty-week-old ApoE−/− mice accumulated even more monocytes in the aortic root, ascending aorta, and thoracic aorta after both chow (503 ± 67 cells) or high-cholesterol diet (648 ± 81 cells). Labeled monocyte content in the aorta consistently correlated with lesion surface area. These data indicate that monocytes accumulate continuously during atheroma formation, accumulation increases in proportion to lesion size, and recruitment is augmented with hypercholesterolemia. These results provide insights into mechanisms of atherogenesis and have implications for the duration of therapies directed at leukocyte recruitment.

Noninvasive cell-tracking methods

Cell-based therapies, such as adoptive immunotherapy and stem-cell therapy, have received considerable attention as novel therapeutics in oncological research and clinical practice. The development of effective therapeutic strategies using tumor-targeted cells requires the ability to determine in vivo the location, distribution, and long-term viability of the therapeutic cell populations as well as their biological fate with respect to cell activation and differentiation. In conjunction with various noninvasive imaging modalities, cell-labeling methods, such as exogenous labeling or transfection with a reporter gene, allow visualization of labeled cells in vivo in real time, as well as monitoring and quantifying cell accumulation and function. Such cell-tracking methods also have an important role in basic cancer research, where they serve to elucidate novel biological mechanisms. In this Review, we describe the basic principles of cell-tracking methods, explain various approaches to cell tracking, and highlight recent examples for the application of such methods in animals and humans.

Molecular imaging for personalized cancer care.

Molecular imaging is rapidly gaining recognition as a tool with the capacity to improve every facet of cancer care. Molecular imaging in oncology can be defined as in vivo characterization and measurement of the key biomolecules and molecularly based events that are fundamental to the malignant state. This article outlines the basic principles of molecular imaging as applied in oncology with both established and emerging techniques. It provides examples of the advantages that current molecular imaging techniques offer for improving clinical cancer care as well as drug development. It also discusses the importance of molecular imaging for the emerging field of theranostics and offers a vision of how molecular imaging may one day be integrated with other diagnostic techniques to dramatically increase the efficiency and effectiveness of cancer care.

Surface-Enhanced Resonance Raman Scattering Nanostars for High Precision Cancer Imaging

Surface-enhanced resonance Raman scattering gold nanostars allow detection of macro- and microscopic foci of premalignant and cancerous lesions in vivo. Seeing Nanostars Microscopic tumors may be difficult for the naked eye to see, but they are no match for nanosized imaging agents, which home in on cancerous tissues to signal the presence of disease. Harmsen and colleagues created a new generation of cancer imaging agents, called “surface-enhanced resonance Raman scattering (SERRS) nanostars” −75-nm star-shaped gold cores wrapped in Raman reporter molecule-containing silica. When hit by a near-infrared laser, these nanostars emit a unique photonic signature (Raman “fingerprint”). The authors used a new silica encapsulation method and a reporter molecule that was “in resonance” with the laser, which meant that they shone nearly 400 times brighter than their “nonresonant” counterparts during Raman imaging. The SERRS nanostars were used to image macro- and microscopic malignant lesions in animal models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma with high precision. As endoscopic and handheld Raman imaging devices are further developed for the clinic, the SERRS nanostars are sure to find a place in human tumor detection. The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread, and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the true extent of tumors is desired clinically and surgically. We show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal locoregional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed SERRS nanostars. The SERRS nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, and in one human sarcoma xenograft model, SERRS nanostars enabled accurate detection of macroscopic malignant lesions, as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 fM limit of detection) of SERRS nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS nanostars a promising imaging agent for more precise cancer imaging and resection.

Noninvasive In Vivo Imaging of Monocyte Trafficking to Atherosclerotic Lesions

Background— Monocytes play a key role in atherogenesis, but their participation has been discerned largely via ex vivo analyses of atherosclerotic lesions. We sought to establish a noninvasive technique to determine monocyte trafficking to atherosclerotic lesions in live animals. Methods and Results— Using a micro–single-photon emission computed tomography small-animal imaging system and a Food and Drug Administration–approved radiotracer ([indium 111] oxyquinoline, 111In-oxine), we demonstrate here that monocyte recruitment to atherosclerotic lesions can be visualized in a noninvasive, dynamic, and 3-dimensional fashion in live animals. We show in vivo that monocytes are recruited avidly to plaques within days of adoptive transfer. Using micro–single-photon emission computed tomography imaging as a screening tool, we were able to investigate modulatory effects on monocyte recruitment in live animals. We found that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors rapidly and substantially reduce monocyte recruitment to existing atherosclerotic lesions, as imaged here in vivo. Conclusions— This novel approach to track monocytes to atherosclerotic plaques in vivo should have broad applications and create new insights into the pathogenesis of atherosclerosis and other inflammatory diseases.

Murine B16 melanomas expressing high levels of the chemokine stromal-derived factor-1/CXCL12 induce tumor-specific T cell chemorepulsion and escape from immune control.

The chemokine, stromal-derived factor-1/CXCL12, is expressed by normal and neoplastic tissues and is involved in tumor growth, metastasis, and modulation of tumor immunity. T cell-mediated tumor immunity depends on the migration and colocalization of CTL with tumor cells, a process regulated by chemokines and adhesion molecules. It has been demonstrated that T cells are repelled by high concentrations of the chemokine CXCL12 via a concentration-dependent and CXCR4 receptor-mediated mechanism, termed chemorepulsion or fugetaxis. We proposed that repulsion of tumor Ag-specific T cells from a tumor expressing high levels of CXCL12 allows the tumor to evade immune control. Murine B16/OVA melanoma cells (H2b) were engineered to constitutively express CXCL12. Immunization of C57BL/6 mice with B16/OVA cells lead to destruction of B16/OVA tumors expressing no or low levels of CXCL12 but not tumors expressing high levels of the chemokine. Early recruitment of adoptively transferred OVA-specific CTL into B16/OVA tumors expressing high levels of CXCL12 was significantly reduced in comparison to B16/OVA tumors, and this reduction was reversed when tumor-specific CTLs were pretreated with the specific CXCR4 antagonist, AMD3100. Memory OVA-specific CD8+ T cells demonstrated antitumor activity against B16/OVA tumors but not B16/OVA.CXCL12-high tumors. Expression of high levels of CXCL12 by B16/OVA cells significantly reduced CTL colocalization with and killing of target cells in vitro in a CXCR4-dependent manner. The repulsion of tumor Ag-specific T cells away from melanomas expressing CXCL12 confirms the chemorepellent activity of high concentrations of CXCL12 and may represent a novel mechanism by which certain tumors evade the immune system.

Dynamic Magnetic Fields Remote Control Apoptosis Via Nanoparticle Rotation

The ability to control the movement of nanoparticles remotely and with high precision would have far-reaching implications in many areas of nanotechnology. We have designed a unique dynamic magnetic field (DMF) generator that can induce rotational movements of superparamagnetic iron oxide nanoparticles (SPIONs). We examined whether the rotational nanoparticle movement could be used for remote induction of cell death by injuring lysosomal membrane structures. We further hypothesized that the shear forces created by the generation of oscillatory torques (incomplete rotation) of SPIONs bound to lysosomal membranes would cause membrane permeabilization, lead to extravasation of lysosomal contents into the cytoplasm, and induce apoptosis. To this end, we covalently conjugated SPIONs with antibodies targeting the lysosomal protein marker LAMP1 (LAMP1-SPION). Remote activation of slow rotation of LAMP1-SPIONs significantly improved the efficacy of cellular internalization of the nanoparticles. LAMP1-SPIONs then preferentially accumulated along the membrane in lysosomes in both rat insulinoma tumor cells and human pancreatic beta cells due to binding of LAMP1-SPIONs to endogenous LAMP1. Further activation of torques by the LAMP1-SPIONs bound to lysosomes resulted in rapid decrease in size and number of lysosomes, attributable to tearing of the lysosomal membrane by the shear force of the rotationally activated LAMP1-SPIONs. This remote activation resulted in an increased expression of early and late apoptotic markers and impaired cell growth. Our findings suggest that DMF treatment of lysosome-targeted nanoparticles offers a noninvasive tool to induce apoptosis remotely and could serve as an important platform technology for a wide range of biomedical applications.

Guiding brain tumor resection using surface-enhanced Raman scattering nanoparticles and a hand-held Raman scanner.

The current difficulty in visualizing the true extent of malignant brain tumors during surgical resection represents one of the major reasons for the poor prognosis of brain tumor patients. Here, we evaluated the ability of a hand-held Raman scanner, guided by surface-enhanced Raman scattering (SERS) nanoparticles, to identify the microscopic tumor extent in a genetically engineered RCAS/tv-a glioblastoma mouse model. In a simulated intraoperative scenario, we tested both a static Raman imaging device and a mobile, hand-held Raman scanner. We show that SERS image-guided resection is more accurate than resection using white light visualization alone. Both methods complemented each other, and correlation with histology showed that SERS nanoparticles accurately outlined the extent of the tumors. Importantly, the hand-held Raman probe not only allowed near real-time scanning, but also detected additional microscopic foci of cancer in the resection bed that were not seen on static SERS images and would otherwise have been missed. This technology has a strong potential for clinical translation because it uses inert gold–silica SERS nanoparticles and a hand-held Raman scanner that can guide brain tumor resection in the operating room.

Raman's “Effect” on Molecular Imaging

Raman spectroscopy is an optical technique that offers unsurpassed sensitivity and multiplexing capabilities to the field of molecular imaging. In the past, Raman spectroscopy had predominantly been used as an analytic tool for routine chemical analysis, but more recently, researchers have been able to harness its unique properties for imaging and spectral analysis of molecular interactions in cell populations and preclinical animal models. Additionally, researchers have already begun to translate this optical technique into a novel clinical diagnostic tool using various endoscopic strategies.

Enhancing Membrane Permeability by Fatty Acylation of Oligoarginine Peptides

The improved bioavailability of drugs in the treatment of diseases produces a prolonged therapeutic effect and, as a consequence, reduced toxicity and cost. Most oligonucleotides, peptides, or proteins are poorly taken up by cells due to their insufficient association with the lipid bilayer of the plasma membrane. In many cases, therapeutic agents need to possess lipophilic properties in order to achieve the desired pharmacokinetic profile. Thus, the lipophilicity of the molecules assists in the penetration of cytoplasmic and intracellular membranes. Traditionally, the incorporation of homologous series of alkyl groups into a drug of interest produced increases in pharmacological effects. More recently, a short peptide (RKKRRQRRR), derived from HIV Tat-protein, as well as other arginine-containing peptides have attracted attention due to their high cellular uptake efficiency. These membrane-penetrating peptides have been applied as delivery vectors for various biological and medical applications. A systematic study of Tat-peptide indicated that the positively charged arginine residues are crucial to its membrane-penetrating ability, a property that has also been mimicked by a hepta-arginine peptide. It has thus been concluded that the cationic guanidine moiety on the arginine side chain provides the exceptional translocation properties that are not observed in peptides containing similar amino acids such as ornithine, lysine, histidine or citrulline. Although this membrane-translocalization phenomenon has been reported for more than a decade, the detailed mechanism still has to be determined. Here we described the systematic exploration of the changes in cell-localizing ability brought about by the modification of oligoarginine peptides with fatty acid analogues. We originally hypothesized that such modifications would lead to enhanced association with lipid membranes and, potentially, improved transmembrane delivery. The 7-mer oligoarginine Tat-peptide mimetic, originally reported by Wender et al. , was selected as the peptide template on which to study the effect of the length of the acyl chain. A series of fatty acid groups, including hexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, and palmitoyl, were attached to the N terminus of the amidated peptide via a b-alanine spacer on solid support by using a modified Schotten±Baumann reaction. Thereafter, the lipopeptides were labeled with fluorescein isothiocyanate