Daniel Golovko

MRI of Tumor-Associated Macrophages with Clinically Applicable Iron Oxide Nanoparticles

Purpose: The presence of tumor-associated macrophages (TAM) in breast cancer correlates strongly with poor outcome. The purpose of this study was to develop a clinically applicable, noninvasive diagnostic assay for selective targeting and visualization of TAMs in breast cancer, based on magnetic resonanceI and clinically applicable iron oxide nanoparticles. Experimental Design: F4/80-negative mammary carcinoma cells and F4/80-positive TAMs were incubated with iron oxide nanoparticles and were compared with respect to magnetic resonance signal changes and iron uptake. MMTV-PyMT transgenic mice harboring mammary carcinomas underwent nanoparticle-enhanced magnetic resonance imaging (MRI) up to 1 hour and 24 hours after injection. The tumor enhancement on MRIs was correlated with the presence and location of TAMs and nanoparticles by confocal microscopy. Results:In vitro studies revealed that iron oxide nanoparticles are preferentially phagocytosed by TAMs but not by malignant tumor cells. In vivo, all tumors showed an initial contrast agent perfusion on immediate postcontrast MRIs with gradual transendothelial leakage into the tumor interstitium. Twenty-four hours after injection, all tumors showed a persistent signal decline on MRIs. TAM depletion via αCSF1 monoclonal antibodies led to significant inhibition of tumor nanoparticle enhancement. Detection of iron using 3,3′-diaminobenzidine-enhanced Prussian Blue staining, combined with immunodetection of CD68, localized iron oxide nanoparticles to TAMs, showing that the signal effects on delayed MRIs were largely due to TAM-mediated uptake of contrast agent. Conclusion: These data indicate that tumor enhancement with clinically applicable iron oxide nanoparticles may serve as a new biomarker for long-term prognosis, related treatment decisions, and the evaluation of new immune-targeted therapies. Clin Cancer Res; 17(17); 5695–704. ©2011 AACR.

The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells

For in vivo applications of magnetically labeled stem cells, biological effects of the labeling procedure have to be precluded. This study evaluates the effect of different ferucarbotran cell labeling protocols on chondrogenic differentiation of human mesenchymal stem cells (hMSC) as well as their implications for MR imaging. hMSC were labeled with ferucarbotran using various protocols: cells were labeled with 100 microg Fe/ml for 4 and 18 h and additional samples were cultured for 6 or 12 days after the 18 h labeling. Supplementary samples were labeled by transfection with protamine sulfate. Iron uptake was quantified by ICP-spectrometry and labeled cells were investigated by transmission electron microscopy and by immunostaining for ferucarbotran. The differentiation potential of labeled cells was compared with unlabeled controls by staining with Alcian blue and Hematoxylin and Eosin, then quantified by measurements of glucosaminoglycans (GAG). Contrast agent effect at 3 T was investigated on days 1 and 14 of chondrogenic differentiation by measuring signal-to-noise ratios on T(2)-SE and T(2)*-GE sequences. Iron uptake was significant for all labeling protocols (p < 0.05). The uptake was highest after transfection with protamine sulfate (25.65 +/- 3.96 pg/cell) and lowest at an incubation time of 4 h without transfection (3.21 +/- 0.21 pg/cell). While chondrogenic differentiation was decreased using all labeling protocols, the decrease in GAG synthesis was not significant after labeling for 4 h without transfection. After labeling by simple incubation, chondrogenesis was found to be dose-dependent. MR imaging showed markedly lower SNR values of all labeled cells compared with the unlabeled controls. This contrast agent effect persisted for 14 days and the duration of differentiation. Magnetic labeling of hMSC with ferucarbotran inhibits chondrogenesis in a dose-dependent manner when using simple incubation techniques. When decreasing the incubation time to 4 h, inhibition of chondrogenesis was not significant.

Relaxation effects of ferucarbotran-labeled mesenchymal stem cells at 1.5T and 3T: Discrimination of viable from lysed cells

Human mesenchymal stem cells (hMSCs) were labeled with Ferucarbotran by simple incubation and cultured for up to 14 d. Iron content was determined by spectrometry and the intracellular localization of the contrast agent uptake was studied by electron and confocal microscopy. At various time points after labeling, ranging from 1 to 14 d, samples with viable or lysed labeled hMSCs, as well as nonlabeled controls, underwent MRI. Spin‐echo (SE) and gradient‐echo (GE) sequences with multiple TRs and TEs were used at 1.5T and 3T on a clinical scanner. Spectrometry showed an initial iron oxide uptake of 7.08 pg per cell. Microscopy studies revealed lysosomal compartmentalization. Contrast agent effects of hMSCs were persistent for up to 14 d after labeling. A marked difference in the T2 effect of compartmentalized iron oxides compared to free iron oxides was found on T2‐weighted sequences, but not on T  *2 ‐weighted sequences. The observed differences may be explained by the loss of compartmentalization of iron oxide particles, the uniformity of distribution, and the subsequent increase in dephasing of protons on SE images. These results show that viable cells with compartmentalized iron oxides may—in principle—be distinguished from lysed cells or released iron oxides. Magn Reson Med, 2009.

Indocyanine green-enhanced imaging of antigen-induced arthritis with an integrated optical imaging/radiography system.

OBJECTIVE To evaluate a combined indocyanine green-enhanced optical imaging/radiography system for the detection of arthritic joints in a rat model of antigen-induced arthritis. METHODS Arthritis of the knee and ankle joints was induced in 6 Harlan rats, using peptidoglycan-polysaccharide polymers. Three rats served as untreated controls. Optical imaging of the knee and ankle joints was done with an integrated optical imaging/radiography system before and up to 24 hours following intravenous injection of 10 mg/kg indocyanine green. The fluorescence signal intensities of arthritic and normal joints were compared for significant differences, using generalized estimating equation models. Specimens of knee and ankle joints were further processed and evaluated by histology. RESULTS Immediately after administration, indocyanine green provided a significant increase in the fluorescence signal of arthritic joints compared with baseline values (P < 0.05). The fluorescence signal of arthritic joints was significantly higher compared with that of nonarthritic control joints at 1-720 minutes after intravenous injection (P < 0.05). Fusion of indocyanine green-enhanced optical imaging and radiography allowed for anatomic coregistration of the inflamed tissue with the associated joint. Hematoxylin and eosin staining confirmed marked synovial inflammation of arthritic joints and the absence of inflammation in control joints. CONCLUSION Indocyanine green-enhanced optical imaging is a clinically applicable tool for detection of arthritic tissue. Using relatively high doses of indocyanine green, long-term enhanced fluorescence of arthritic joints can be achieved. This may facilitate simultaneous evaluations of multiple joints in a clinical setting. Fusion of indocyanine green-enhanced optical imaging scans with radiography increases anatomic resolution.

Detection of synovitis in the hands of patients with rheumatologic disorders: Diagnostic performance of optical imaging in comparison with magnetic resonance imaging

OBJECTIVE To prospectively compare an indocyanine green (ICG)-enhanced optical imaging system with contrast-enhanced magnetic resonance imaging (MRI) for the detection of synovitis in the hands of patients with rheumatologic disorders. METHODS Forty-five patients (30 women [67%], mean ± SD age 52.6 ± 13.4 years) in whom there was a clinical suspicion of an inflammatory arthropathy were examined with a commercially available device for ICG-enhanced optical imaging as well as by contrast-enhanced 3T MRI as the standard of reference. Three independent readers graded the degree of synovitis in the carpal, metacarpophalangeal, proximal interphalangeal, and distal interphalangeal joints of both hands (1,350 joints), using a 4-point ordinate scale (0 = no synovitis, 1 = mild, 2 = moderate, 3 = severe). Statistical analyses were performed using a logistic generalized estimating equation approach. Agreement of optical imaging ratings made by the different readers was estimated with a weighted kappa coefficient. RESULTS When MRI was used as the standard of reference, optical imaging showed a sensitivity of 39.6% (95% confidence interval [95% CI] 31.1-48.7%), a specificity of 85.2% (95% CI 79.5-89.5%), and accuracy of 67.0% (95% CI 61.4-72.1%) for the detection of synovitis in patients with arthritis. Diagnostic accuracy was especially limited in the setting of mild synovitis, while it was substantially better in patients with severely inflamed joints. Moderate interreader and intrareader agreement was observed. CONCLUSION The evaluated ICG-enhanced optical imaging system showed limitations for the detection of inflamed joints of the hand in comparison with MRI.

Synovitis in Patients with Early Inflammatory Arthritis Monitored with Quantitative Analysis of Dynamic Contrast-enhanced Optical Imaging and MR Imaging

PURPOSE To evaluate quantitative perfusion measurements of dynamic indocyanine green (ICG)-enhanced optical imaging for monitoring synovitis in the hands of patients with inflammatory arthritis compared with dynamic contrast-enhanced (DCE) magnetic resonance (MR) imaging and clinical outcome. MATERIALS AND METHODS This study was approved by the ethics committee at the institution. Individual joints (n = 840) in the hands and wrists of 28 patients (14 women; mean age, 53.3 years) with inflammatory arthritis were examined at three different time points: before start of therapy and 12 and 24 weeks after start of therapy or therapy escalation. Treatment response was assessed by using clinical measures (simple disease activity index [SDAI]), ICG-enhanced optical imaging, and DCE MR imaging. Dynamic images were obtained for optical imaging and DCE MR imaging. The rate of early enhancement (REE) of the perfusion curves of each joint was calculated by using in-house developed software. Correlation coefficients were estimated to evaluate the associations of changes of imaging parameters and SDAI change. RESULTS Quantitative perfusion measurements with optical imaging and MR imaging correctly identified patients who responded (n = 18) and did not respond to therapy (n = 10), as determined by SDAI. The difference of REE after 24 weeks of treatment compared with baseline in responders was significantly reduced in optical imaging and MR imaging (optical imaging: mean, -21.5%; MR imaging: mean, -41.0%; P < .001 for both), while in nonresponders it was increased (optical imaging: mean, 10.8%; P = .075; MR imaging: mean, 8.7%; P = .03). The REE of optical imaging significantly correlated with MR imaging (ρ = 0.80; P < .001) and SDAI (ρ = 0.61; P < .001). CONCLUSION Quantitative analysis of contrast-enhanced optical imaging allows for potential therapeutic monitoring of synovitis in patients with inflammatory arthritis.

MR signal characteristics of viable and apoptotic human mesenchymal stem cells in matrix-associated stem cell implants for treatment of osteoarthritis.

Objective:To compare magnetic resonance (MR) signal characteristics of contrast agent-labeled apoptotic and viable human mesenchymal stem cells (hMSCs) in matrix-associated stem cell implants. Methods:hMSCs were labeled with Food and Drug Administration-approved ferumoxides nanoparticles. One group (A) remained untreated whereas a second group (B) underwent mitomycin C-induced apoptosis induction. Viability of group A and apoptosis of group B was confirmed by caspase-assays and terminal dUTP nick-end labeling (TUNEL) stains. Labeled viable hMSCs, unlabeled viable hMSCs, labeled apoptotic hMSCs, and unlabeled apoptotic hMSCs (n = 7 samples each) in an agarose scaffold were implanted into cartilage defects of porcine patellae specimens and underwent MR imaging at 7 T, using T1-weighted spin-echo sequences, T2-weighted spin-echo sequences, and T2*-weighted gradient-echo sequences. Signal-to-noise ratios (SNR) of the implants were calculated and compared between different experimental groups using linear mixed regression models. Results:Ferumoxides-labeled hMSCs provided a strong negative T2 and T2*-enhancement. Corresponding SNR data of labeled hMSCs were significantly lower compared with unlabeled controls (P < 0.05). Apoptosis induction resulted in a significant signal decline of ferumoxides-labeled hMSC transplants on short echo time T2-weighted spinecho sequences. SNR data of labeled apoptotic hMSCs were significantly lower compared with labeled viable hMSCs (P < 0.05). Conclusion:Apoptosis of transplanted ferumoxides-labeled stem cells in cartilage defects can be visualized noninvasively by a significant signal decline on T2-weighted MR images. The described MR signal characteristics may serve as a noninvasive outcome measure for the assessment of matrix-associated stem cell implants in clinical practice. Additional studies are needed to further enhance the observed differences between viable and apoptotic cells, for example, by further optimizing the applied MR pulse sequence parameters or intracellular contrast agent concentration.

Magnetic Resonance Imaging of Ferumoxide-Labeled Mesenchymal Stem Cells in Cartilage Defects: In Vitro and in Vivo Investigations

The purpose of this study was to (1) compare three different techniques for ferumoxide labeling of mesenchymal stem cells (MSCs), (2) evaluate if ferumoxide labeling allows in vivo tracking of matrix-associated stem cell implants (MASIs) in an animal model, and (3) compare the magnetic resonance imaging (MRI) characteristics of ferumoxide-labeled viable and apoptotic MSCs. MSCs labeled with ferumoxide by simple incubation, protamine transfection, or Lipofectin transfection were evaluated with MRI and histopathology. Ferumoxide-labeled and unlabeled viable and apoptotic MSCs in osteochondral defects of rat knee joints were evaluated over 12 weeks with MRI. Signal to noise ratios (SNRs) of viable and apoptotic labeled MASIs were tested for significant differences using t-tests. A simple incubation labeling protocol demonstrated the best compromise between significant magnetic resonance signal effects and preserved cell viability and potential for immediate clinical translation. Labeled viable and apoptotic MASIs did not show significant differences in SNR. Labeled viable but not apoptotic MSCs demonstrated an increasing area of T2 signal loss over time, which correlated to stem cell proliferation at the transplantation site. Histopathology confirmed successful engraftment of viable MSCs. The engraftment of iron oxide–labeled MASIs by simple incubation can be monitored over several weeks with MRI. Viable and apoptotic MASIs can be distinguished via imaging signs of cell proliferation at the transplantation site.

Labeling human embryonic stem cell-derived cardiomyocytes with indocyanine green for noninvasive tracking with optical imaging: an FDA-compatible alternative to firefly luciferase.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have demonstrated the ability to improve myocardial function following transplantation into an ischemic heart; however, the functional benefits are transient possibly due to poor cell retention. A diagnostic technique that could visualize transplanted hESC-CMs could help to optimize stem cell delivery techniques. Thus, the purpose of this study was to develop a labeling technique for hESCs and hESC-CMs with the FDA-approved contrast agent indocyanine green (ICG) for optical imaging (OI). hESCs were labeled with 0.5, 1.0, 2.0, and 2.5 mg/ml of ICG for 30, 45, and 60 min at 37 degrees C. Longitudinal OI studies were performed with both hESCs and hESC-CMs. The expression of surface proteins was assessed with immunofluorescent staining. hESCs labeled with 2 mg ICG/ml for 60 min achieved maximum fluorescence. Longitudinal studies revealed that the fluorescent signal was equivalent to controls at 120 h postlabeling. The fluorescence signal of hESCs and hESC-CMs at 1, 24, and 48 h was significantly higher compared to precontrast data (p < 0.05). Immunocytochemistry revealed retention of cell-specific surface and nuclear markers postlabeling. These data demonstrate that hESCs and hESC-CMs labeled with ICG show a significant fluorescence up to 48 h and can be visualized with OI. The labeling procedure does not impair the viability or functional integrity of the cells. The technique may be useful for assessing different delivery routes in order to improve the engraftment of transplanted hESC-CMs or other stem cell progenitors.

Monitoring of natural killer cell immunotherapy using noninvasive imaging modalities.

Cancer immunotherapies can be guided by cellular imaging techniques, which can identify the presence or absence of immune cell accumulation in the tumor tissue in vivo and in real time. This review summarizes various new and evolving imaging techniques employed for tracking and monitoring of adoptive natural killer cell immunotherapies.