Dynamic gadobutrol-enhanced MRI predicts early response to antivascular but not to antiproliferation therapy in a mouse xenograft model.

PURPOSE: Dynamic contrast-enhanced magnetic resonance imaging has been described as a method to assess tumor vascularity and, therefore, is discussed as a noninvasive biomarker for drug response prediction in tumor therapies. Because antiangiogenic and antiproliferative drugs are frequently combined for therapy, the aim was to investigate (1) the early response predictability and (2) the extent to which these therapy types influence dynamic contrast-enhanced magnetic resonance imaging with gadobutrol soon after therapy initiation. METHODS: Mice bearing a KPL-4 tumor were treated with either bevacizumab as an antiangiogenic drug or trastuzumab as a cytotoxic anti-tumor drug. The gadobutrol-contrast agent exposure of the tumor was recorded before and at several time points after therapy initiation to examine the response prediction by dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Both therapies resulted in significant tumor growth attenuation over 30 days of therapy, but the individual response to each therapy was different. Specifically, bevacizumab affected the dynamic gadobutrol-enhanced MRI-derived area under the curve at early time points (≤8 days), while trastuzumab did not. CONCLUSION: The area under the curve obtained from dynamic gadobutrol-enhanced MRI predicted early tumor response to the antiangiogenic drug bevacizumab, but not to the anti-tumor cell drug trastuzumab. This indicates that the area under the curve may be useful for assessing early antiangiogenic but not antiproliferative drug effects.

Studying the effect of particle size and coating type on the blood kinetics of superparamagnetic iron oxide nanoparticles.

PURPOSE: Magnetic resonance imaging (MRI), one of the most powerful imaging techniques available, usually requires the use of an on-demand designed contrast agent to fully exploit its potential. The blood kinetics of the contrast agent represent an important factor that needs to be considered depending on the objective of the medical examination. For particulate contrast agents, such as superparamagnetic iron oxide nanoparticles (SPIOs), the key parameters are particle size and characteristics of the coating material. In this study we analyzed the effect of these two properties independently and systematically on the magnetic behavior and blood half-life of SPIOs. METHODS: Eleven different SPIOs were synthesized for this study. In the first set (a), seven carboxydextran (CDX)-coated SPIOs of different sizes (19-86 nm) were obtained by fractionating a broadly size-distributed CDX-SPIO. The second set (b) contained three SPIOs of identical size (50 nm) that were stabilized with different coating materials, polyacrylic acid (PAA), poly-ethylene glycol, and starch. Furthermore, small PAA-SPIOs (20 nm) were synthesized to gain a global insight into the effects of particle size vs coating characteristics. Saturation magnetization and proton relaxivity were determined to represent the magnetic and imaging properties. The blood half-life was analyzed in rats using MRI, time-domain nuclear magnetic resonance, and inductively coupled plasma optical emission spectrometry. RESULTS: By changing the particle size without modifying any other parameters, the relaxivity r(2) increased with increasing mean particle diameter. However, the blood half-life was shorter for larger particles. The effect of the coating material on magnetic properties was less pronounced, but it had a strong influence on blood kinetics depending on the ionic character of the coating material. CONCLUSION: In this report we systematically demonstrated that both particle size and coating material influence blood kinetics and magnetic properties of SPIO independently. These data provide key information for the selection of a contrast agent for a defined application and are additionally valuable for other nano areas, such as hyperthermia, drug delivery, and nanotoxicology.

Current limitations of molecular magnetic resonance imaging for tumors as evaluated with high-relaxivity CD105-specific iron oxide nanoparticles.

OBJECTIVE: Tumor imaging via molecular magnetic resonance imaging (MRI) that uses specific superparamagnetic iron oxide particles (SPIOs) has been addressed in the literature several times in the last 20 years. To our knowledge, none of the reported approaches is currently used for routine clinical diagnostic evaluation, nor are any in clinical development. This raises questions as to whether SPIO-enhanced molecular MRI is sensitive and specific enough for use in clinical practice. The aim of our preclinical study was to investigate the minimum requirements for obtaining sensitive molecular MRI for use in tumor evaluations under optimal conditions. The well-vascularized F9 teratocarcinoma tumor model, which exhibits high levels of the highly accessible target CD105 (endoglin), was used to compare the accumulation and visualization of target-specific SPIOs by MRI. MATERIAL AND METHODS: Superparamagnetic iron oxide particles were optimized in the following ways: (a) proton relaxivity was increased for higher imaging sensitivity, (b) a coating material was used for optimal loading density of the αCD105 antibody, and (c) binding activity to the target CD105 was increased. Binding activity and specificity were confirmed in vitro using enzyme-linked immunosorbent assay and in vivo using pharmacokinetic and biodistribution studies of 11 F9 teratoma-bearing mice together with micro-autoradiography. CD105 target expression was determined using immunohistochemistry and quantitative enzyme-linked immunosorbent assay. The transverse relaxation rate R2* was quantified by 3.0-T MRI in the tumors, kidneys, and muscles before and up to 60 minutes after injection in 11 mice. The use of [Fe]-labeled SPIOs for all in vivo experiments allowed for the direct correlation of the imaging results with SPIO accumulation. RESULTS: High-relaxivity αCD105-polyacrylic acid-SPIOs (r2 up to 440 L mmol Fe s) with strong binding activity accumulated specifically in tumors (1.4% injected dose/g) and kidneys (4.1% injected dose/g) in a manner dependent on the target concentration. The accumulation occurred within the first 3 minutes after injection. Visualization of specific SPIOs was accomplished with MRI. In contrast to the successful use of MRI in all examined kidneys (mean ± SEM ΔR2*, 61 ± 11 s), only 6 of 11 tumors (mean ± SEM ΔR2*, 15 ± 7 s) showed a clear signal when compared with the control even though optimal conditions were used. CONCLUSION: The accumulation of CD105-specific SPIOs in F9 mouse teratomas was robust. However, visualization of the specifically accumulated SPIOs by MRI was not reliable because of its limited signal detection sensitivity. We postulate that it will be challenging to improve the imaging properties of targeted SPIOs further. Therefore, molecular MRI by targeted SPIOs is currently not suitable for clinical tumor imaging using routinely applicable sequences and field strength.

Substantial changes of cellular iron homeostasis during megakaryocytic differentiation of K562 cells.

We investigated the remodeling of iron metabolism during megakaryocytic development of K562 cells. Differentiation was successfully verified by increase of the megakaryocytic marker CD61 and concomitant decrease of the erythroid marker γ-globin. The reduction of erythroid properties was accompanied by changes in the cellular iron content and in the expression of proteins regulating cellular iron homeostasis. Independent of available inorganic or transferrin-bound extracellular iron, total intracellular iron increases while the iron-to-protein ratio decreases. The iron exporter ferroportin is downregulated within 1-6 h, followed by downregulation of transferrin receptor-1 (TfR1) and ferritin heavy chain (H-ferritin) mainly after 24-48 h. The hemochromatosis protein-1, a ligand of TfR1, peaked after 24 h. All effects were independent of iron supply with the exception of H-ferritin, which was restored by excess iron. While alterations of CD61, TfR1 and ferritin expression were revoked by a protein kinase C inhibitor, downregulation of ferroportin remained unaffected.

Chimeric toxins inhibit growth of primary oral squamous cell carcinoma cells.

Treatment of oral squamous cell carcinoma (OSCC) is currently based on surgery and radiotherapy. Prolongation of the survival time of patients with progressing tumors is infrequently achieved. To improve the therapeutic options, targeted therapies are a favorable alternative. Therefore, we analyzed the effect of a chimeric toxin (CT) named SE consisting of the epidermal growth factor and the plant protein toxin saporin from Saponaria officinalis. A second construct (SA2E) additionally contains a peptidic adapter designed to enhance efficacy of the CT in vivo and to reduce side effects. The IC(50) values for an OSCC cell line (BHY) were 0.27 nM and 0.73 nM for SE and SA2E, respectively, while fibroblasts remained unaffected. To investigate primary tumor cells, we developed a technique to analyze freshly prepared OSCC cells of 28 patients in a stem cell assay directly after surgery. Cells were treated for 1 h with the CTs, subsequently seeded into soft agar and colony growth determined after 1-2 weeks In spite of the short time of CT incubation, the amount of colonies was reduced to about 78% by 10 nM and to 69% by 100 nM of either toxin. A combined application of 10 nM SA2E with a saponin from Gypsophila paniculata reduced the amount of surviving cells to 68%. The results demonstrate the impact of the CTs on OSCC cells and depict that the stem cell assay is suitable to determine the potential of anti-tumor drugs before studies in vivo will be initiated.

Release of the soluble transferrin receptor is directly regulated by binding of its ligand ferritransferrin.

The human transferrin receptor (TfR) is shed by an integral metalloprotease releasing a soluble form (sTfR) into serum. The sTfR reflects the iron demand of the body and is postulated as a regulator of iron homeostasis via binding to the hereditary hemochromatosis protein HFE. To study the role of transferrin in this process, we investigated TfR shedding in HL60 cells and TfR-deficient Chinese hamster ovary cells transfected with human TfR. Independent of TfR expression, sTfR release decreases with increasing ferritransferrin concentrations, whereas apo-transferrin exhibits no inhibitory effect. To investigate the underlying mechanism, we generated several TfR mutants with different binding affinities for transferrin. Shedding of TfR mutants in transfected cells correlates exactly with their binding affinity, implying that the effect of ferritransferrin on TfR shedding is mediated by a direct molecular interaction. Analysis of sTfR release from purified microsomal membranes revealed that the regulation is independent from intracellular trafficking or cellular signaling events. Our results clearly demonstrated that sTfR does not only reflect the iron demand of the cells but also the iron availability in the bloodstream, mirrored by iron saturation of transferrin, corroborating the important potential function of sTfR as a regulator of iron homeostasis.

Mutational suppression of transferrin receptor shedding can be compensated by distinct metalloproteases acting on alternative sites.

The human transferrin receptor (TfR) is proteolytically cleaved at R100 within the juxtamembrane stalk and to a lesser extent at an alternative site. We examined the effect of stalk mutations on human TfR shedding in transfected CHO cells. Point mutations at R100 led to an increase in alternative shedding while the R100 cleavage product was undetectable. Replacing the TfR-stalk by the corresponding sequences from tumor necrosis factor-alpha or interleukin-6 receptor also led to TfR ectodomain shedding. These results show that cleavage at alternative sites can compensate for suppressed cleavage at the major site and inhibitor studies reveal that at least three metalloproteases are involved in the shedding process.

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G.

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.

Shedding of the transferrin receptor is mediated constitutively by an integral membrane metalloprotease sensitive to tumor necrosis factor alpha protease inhibitor-2.

The transferrin receptor (TfR) is a transmembrane protein that mediates cellular uptake of iron. Although the serum concentration of the soluble TfR (sTfR) is altered in several diseases and used for diagnostic purposes, the identity and regulation of the shedding protease is unknown. In this study we quantified sTfR release from microsomal membranes and leukocytic cell lines in the presence of numerous protease inhibitors and cell activating compounds. We show that sTfR release is mediated by an integral membrane metalloprotease and can be inhibited by matrix metalloproteinase inhibitor 2 and tumor necrosis factor alpha protease inhibitor-2 (TAPI-2). Cleavage is also inhibited by a specific furin inhibitor, indicating that the protease is activated by a furin-like proprotein convertase. Whereas stimulation of the cells by the ectodomain shedding activator phorbol 12-N-myristate 13-acetate did not alter sTfR release significantly, the phosphatase inhibitor pervanadate led to an increase of TfR shedding in several leukocytic cell lines. Our results suggest that TfR shedding is constitutively mediated by a member of the metalloprotease family known as ADAM (for a disintegrin and metalloprotease).