In vivo imaging of nanoparticle-labeled CAR T cells.

Metastatic osteosarcoma has a poor prognosis with a 2-y, event-free survival rate of ∼15 to 20%, highlighting the need for the advancement of efficacious therapeutics. Chimeric antigen receptor (CAR) T-cell therapy is a potent strategy for eliminating tumors by harnessing the immune system. However, clinical trials with CAR T cells in solid tumors have encountered significant challenges and have not yet demonstrated convincing evidence of efficacy for a large number of patients. A major bottleneck for the success of CAR T-cell therapy is our inability to monitor the accumulation of the CAR T cells in the tumor with clinical-imaging techniques. To address this, we developed a clinically translatable approach for labeling CAR T cells with iron oxide nanoparticles, which enabled the noninvasive detection of the iron-labeled T cells with magnetic resonance imaging (MRI), photoacoustic imaging (PAT), and magnetic particle imaging (MPI). Using a custom-made microfluidics device for T-cell labeling by mechanoporation, we achieved significant nanoparticle uptake in the CAR T cells, while preserving T-cell proliferation, viability, and function. Multimodal MRI, PAT, and MPI demonstrated homing of the T cells to osteosarcomas and off-target sites in animals administered with T cells labeled with the iron oxide nanoparticles, while T cells were not visualized in animals infused with unlabeled cells. This study details the successful labeling of CAR T cells with ferumoxytol, thereby paving the way for monitoring CAR T cells in solid tumors.

Restoring Endogenous Repair Mechanisms to Heal Chronic Wounds with a Multifunctional Wound Dressing.

Current treatment of chronic wounds has been critically limited by various factors, including bacterial infection, biofilm formation, impaired angiogenesis, and prolonged inflammation. Addressing these challenges, we developed a multifunctional wound dressing-based three-pronged approach for accelerating wound healing. The multifunctional wound dressing, composed of nanofibers, functional nanoparticles, natural biopolymers, and selected protein and peptide, can target multiple endogenous repair mechanisms and represents a promising alternative to current wound healing products.

Tracking Stem Cell Implants in Cartilage Defects of Minipigs by Using Ferumoxytol-enhanced MRI.

Background Cartilage repair outcomes of matrix-associated stem cell implants (MASIs) in patients have been highly variable. Conventional MRI cannot help distinguish between grafts that will and grafts that will not repair the underlying cartilage defect until many months after the repair. Purpose To determine if ferumoxytol nanoparticle labeling could be used to depict successful or failed MASIs compared with conventional MRI in a large-animal model. Materials and Methods Between January 2016 and December 2017, 10 Göttingen minipigs (n = 5 male; n = 5 female; mean age, 6 months ± 5.1; age range, 4-20 months) received implants of unlabeled (n = 12) or ferumoxytol-labeled (n = 20) viable and apoptotic MASIs in cartilage defects of the distal femur. All MASIs were serially imaged with MRI on a 3.0-T imaging unit at week 1 and weeks 2, 4, 8, 12, and 24, with calculation of T2 relaxation times. Cartilage regeneration outcomes were assessed by using the MR observation of cartilage repair tissue (MOCART) score (scale, 0-100), the Pineda score, and histopathologic quantification of collagen 2 production in the cartilage defect. Findings were compared by using the unpaired Wilcoxon rank sum test, a linear regression model, the Fisher exact test, and Pearson correlation. Results Ferumoxytol-labeled MASIs showed significant T2 shortening (22.2 msec ± 3.2 vs 27.9 msec ± 1.8; P < .001) and no difference in cartilage repair outcomes compared with unlabeled control MASIs (P > .05). At week 2 after implantation, ferumoxytol-labeled apoptotic MASIs showed a loss of iron signal and higher T2 relaxation times compared with ferumoxytol-labeled viable MASIs (26.6 msec ± 4.9 vs 20.8 msec ± 5.3; P = .001). Standard MRI showed incomplete cartilage defect repair of apoptotic MASIs at 24 weeks. Iron signal loss at 2 weeks correlated with incomplete cartilage repair, diagnosed at histopathologic examination at 12-24 weeks. Conclusion Ferumoxytol nanoparticle labeling can accelerate the diagnosis of successful and failed matrix-associated stem cell implants at MRI in a large-animal model. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Sneag and Potter in this issue.

The Protein Corona around Nanoparticles Facilitates Stem Cell Labeling for Clinical MR Imaging.

Purpose To evaluate if the formation of a protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for in vivo tracking with magnetic resonance (MR) imaging. Materials and Methods Ferumoxytol was incubated in media containing human serum (group 1), fetal bovine serum (group 2), StemPro medium (group 3), protamine (group 4), and protamine plus heparin (group 5). Formation of a protein corona was characterized by means of dynamic light scattering, ζ potential, and liquid chromatography-mass spectrometry. Iron uptake was evaluated with 3,3'-diaminobenzidine-Prussian blue staining, lysosomal staining, and inductively coupled plasma spectrometry. To evaluate the effect of a protein corona on stem cell labeling, human mesenchymal stem cells (hMSCs) were labeled with the above formulations, implanted into pig knee specimens, and investigated with T2-weighted fast spin-echo and multiecho spin-echo sequences on a 3.0-T MR imaging unit. Data in different groups were compared by using a Kruskal-Wallis test. Results Compared with bare nanoparticles, all experimental groups showed significantly increased negative ζ values (from -37 to less than -10; P = .008). Nanoparticles in groups 1-3 showed an increased size because of the formation of a protein corona. hMSCs labeled with group 1-5 media showed significantly shortened T2 relaxation times compared with unlabeled control cells (P = .0012). hMSCs labeled with group 3 and 5 media had the highest iron uptake after cells labeled with group 1 medium. After implantation into pig knees, hMSCs labeled with group 1 medium showed significantly shorter T2 relaxation times than hMSCs labeled with group 2-5 media (P = .0022). Conclusion The protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for clinical cell tracking with MR imaging. © RSNA, 2017 Online supplemental material is available for this article.

Detection of Stem Cell Transplant Rejection with Ferumoxytol MR Imaging: Correlation of MR Imaging Findings with Those at Intravital Microscopy.

Purpose To determine whether endogenous labeling of macrophages with clinically applicable nanoparticles enables noninvasive detection of innate immune responses to stem cell transplants with magnetic resonance (MR) imaging. Materials and Methods Work with human stem cells was approved by the institutional review board and the stem cell research oversight committee, and animal experiments were approved by the administrative panel on laboratory animal care. Nine immunocompetent Sprague-Dawley rats received intravenous injection of ferumoxytol, and 18 Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice received rhodamine-conjugated ferumoxytol. Then, 48 hours later, immune-matched or mismatched stem cells were implanted into osteochondral defects of the knee joints of experimental rats and calvarial defects of Jax mice. All animals underwent serial MR imaging and intravital microscopy (IVM) up to 4 weeks after surgery. Macrophages of Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice express enhanced green fluorescent protein (GFP), which enables in vivo correlation of ferumoxytol enhancement at MR imaging with macrophage quantities at IVM. All quantitative data were compared between experimental groups by using a mixed linear model and t tests. Results Immune-mismatched stem cell implants demonstrated stronger ferumoxytol enhancement than did matched stem cell implants. At 4 weeks, T2 values of mismatched implants were significantly lower than those of matched implants in osteochondral defects of female rats (mean, 10.72 msec for human stem cells and 11.55 msec for male rat stem cells vs 15.45 msec for sex-matched rat stem cells; P = .02 and P = .04, respectively) and calvarial defects of recipient mice (mean, 21.7 msec vs 27.1 msec, respectively; P = .0444). This corresponded to increased recruitment of enhanced GFP- and rhodamine-ferumoxytol-positive macrophages into stem cell transplants, as visualized with IVM and histopathologic examination. Conclusion Endogenous labeling of macrophages with ferumoxytol enables noninvasive detection of innate immune responses to stem cell transplants with MR imaging. © RSNA, 2017 Online supplemental material is available for this article.

Iron administration before stem cell harvest enables MR imaging tracking after transplantation.

PURPOSE: To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. RESULTS: In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. CONCLUSION: Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.

Intravenous ferumoxytol allows noninvasive MR imaging monitoring of macrophage migration into stem cell transplants.

PURPOSE: To develop a clinically applicable imaging technique for monitoring differential migration of macrophages into viable and apoptotic matrix-associated stem cell implants (MASIs) in arthritic knee joints. MATERIALS AND METHODS: With institutional animal care and use committee approval, six athymic rats were injected with intravenous ferumoxytol (0.5 mmol iron per kilogram of body weight) to preload macrophages of the reticuloendothelial system with iron oxide nanoparticles. Forty-eight hours later, all animals received MASIs of viable adipose-derived stem cells (ADSCs) in an osteochondral defect of the right femur and mitomycin-pretreated apoptotic ADSCs in an osteochondral defect of the left femur. One additional control animal each received intravenous ferumoxytol and bilateral scaffold-only implants (without cells) or bilateral MASIs without prior ferumoxytol injection. All knees were imaged with a 7.0-T magnetic resonance (MR) imaging unit with T2-weighted fast spin-echo sequences immediately after, as well as 2 and 4 weeks after, matrix-associated stem cell implantation. Signal-to-noise ratios (SNRs) of viable and apoptotic MASIs were compared by using a linear mixed-effects model. MR imaging data were correlated with histopathologic findings. RESULTS: All ADSC implants showed a slowly decreasing T2 signal over 4 weeks after matrix-associated stem cell implantation. SNRs decreased significantly over time for the apoptotic implants (SNRs on the day of matrix-associated stem cell implantation, 2 weeks after the procedure, and 4 weeks after the procedure were 16.9, 10.9, and 6.7, respectively; P = .0004) but not for the viable implants (SNRs on the day of matrix-associated stem cell implantation, 2 weeks after the procedure, and 4 weeks after the procedure were 17.7, 16.2, and 15.7, respectively; P = .2218). At 4 weeks after matrix-associated stem cell implantation, SNRs of apoptotic ADSCs were significantly lower than those of viable ADSCs (mean, 6.7 vs 15.7; P = .0013). This corresponded to differential migration of iron-loaded macrophages into MASIs. CONCLUSION: Iron oxide loading of macrophages in the reticuloendothelial system by means of intravenous ferumoxytol injection can be utilized to monitor differential migration of bone marrow macrophages into viable and apoptotic MASIs in a rat model.

Mechanoporation enables rapid and efficient radiolabeling of stem cells for PET imaging.

Regenerative medicine uses the patient own stem cells to regenerate damaged tissues. Molecular imaging techniques are commonly used to image the transplanted cells, either right after surgery or at a later time. However, few techniques are fast or straightforward enough to label cells intraoperatively. Adipose tissue-derived stem cells (ADSCs) were harvested from knee joints of minipigs. The cells were labeled with PET contrast agent by flowing mechanoporation using a microfluidic device. While flowing through a series of microchannels, cells are compressed repeatedly by micro-ridges, which open transient pores in their membranes and induce convective transport, intended to facilitate the transport of 68Ga-labeled and lipid-coated mesoporous nanoparticles (MSNs) into the cells. This process enables cells to be labeled in a matter of seconds. Cells labeled with this approach were then implanted into cartilage defects, and the implant was imaged using positron emission tomography (PET) post-surgery. The microfluidic device can efficiently label millions of cells with 68Ga-labeled MSNs in as little as 15 min. The method achieved labeling efficiency greater than 5 Bq/cell on average, comparable to 30 min-long passive co-incubation with 68Ga-MSNs, but with improved biocompatibility due to the reduced exposure to ionizing radiation. Labeling time could also be accelerated by increasing throughput through more parallel channels. Finally, as a proof of concept, ADSCs were labeled with 68Ga-MSNs and quantitatively assessed using clinical PET/MR in a mock transplant operation in pig knee joints. MSN-assisted mechanoporation is a rapid, effective and straightforward approach to label cells with 68Ga. Given its high efficiency, this labeling method can be used to track small cells populations without significant effects on viability. The system is applicable to a variety of cell tracking studies for cancer therapy, regenerative therapy, and immunotherapy.

Ascorbic Acid and Iron Supplement Treatment Improves Stem Cell-Mediated Cartilage Regeneration in a Minipig Model.

BACKGROUND: The transplantation of mesenchymal stem cells (MSCs) into cartilage defects has led to variable cartilage repair outcomes. Previous in vitro studies have shown that ascorbic acid and reduced iron independently can improve the chondrogenic differentiation of MSCs. However, the combined effect of ascorbic acid and iron supplementation on MSC differentiation has not been investigated. PURPOSE: To investigate the combined in vivo effects of ascorbic acid and a US Food and Drug Administration (FDA)-approved iron supplement on MSC-mediated cartilage repair in mature Göttingen minipigs. STUDY DESIGN: Controlled laboratory study. METHODS: We pretreated bone marrow-derived MSCs with ascorbic acid and the FDA-approved iron supplement ferumoxytol and then transplanted the MSCs into full-thickness cartilage defects in the distal femurs of Göttingen minipigs. Untreated cartilage defects served as negative controls. We evaluated the cartilage repair site with magnetic resonance imaging at 4 and 12 weeks after MSC implantation, followed by histological examination and immunofluorescence staining at 12 weeks. RESULTS: Ascorbic acid plus iron-pretreated MSCs demonstrated a significantly better MOCART (magnetic resonance observation of cartilage repair tissue) score (73.8 ± 15.5), better macroscopic cartilage regeneration score according to the International Cartilage Repair Society (8.6 ± 2.0), better Pineda score (2.9 ± 0.8), and larger amount of collagen type II (28,469 ± 21,313) compared with untreated controls (41.3 ± 2.5, 1.8 ± 2.9, 12.8 ± 1.9, and 905 ± 1326, respectively). The obtained scores were also better than scores previously reported in the same animal model for MSC implants without ascorbic acid. CONCLUSION: Pretreatment of MSCs with ascorbic acid and an FDA-approved iron supplement improved the chondrogenesis of MSCs and led to hyaline-like cartilage regeneration in the knee joints of minipigs. CLINICAL RELEVANCE: Ascorbic acid and iron supplements are immediately clinically applicable. Thus, these results, in principle, could be translated into clinical applications.

Can the biomolecular corona induce an allergic reaction?-A proof-of-concept study.

Ferumoxytol nanoparticles are being used clinically for the treatment of anemia and molecular imaging in patients. It is well documented that while most patients tolerate ferumoxytol well, a small percentage of patients (i.e., 0.01%) develop severe allergic reactions. The purpose of our proof-of-concept study was to determine whether patients with or without hypersensitivity reactions have specific protein corona profiles around ferumoxytol nanoparticles. In a retrospective, institutional review board approved pilot study, we enrolled 13 pediatric patients (5 girls, 8 boys, mean age 16.9 ± 8.2 years) who received a ferumoxytol-enhanced magnetic resonance imaging and who did (group 1, n = 5) or did not (group 2, n = 8) develop an allergic reaction. Blood samples of these patients were incubated with ferumoxytol, and the formation of a hard protein corona around ferumoxytol nanoparticles was measured by dynamic light scattering, zeta potential, and liquid chromatography-mass spectrometry. We also performed in vitro immune response analyses to randomly selected coronas from each group. Our results provide preliminary evidence that ex vivo analysis of the biomolecular corona may provide useful and predictive information on the possibility of severe allergic reactions to ferumoxytol nanoparticles. In the future, patients with predisposition of an allergic reaction to ferumoxytol may be diagnosed based on the proteomic patterns of the corona around ferumoxytol in their blood sample.

Vitamin D status of 6- to 7-year-old children living in Isfahan, Iran.

INTRODUCTION: Vitamin D is essential for the maintenance of good health, and vitamin D deficiency has been reported from many countries, including those with a lot of sunshine. This study was conducted to evaluate the vitamin D status in healthy 6- to 7-year-old children in Isfahan, Iran. MATERIAL AND METHODS: Five hundred and thirteen healthy children were enrolled. Serum PTH and 25-hydroxyvitamin D (25-OHD) were measured. Dietary vitamin D intake, duration of daily sunlight exposure, and percentage of exposed body surface area were determined. 25-OHD levels < 20 ng/mL and < 10 ng/mL were defined as mild and severe vitamin D deficiency, respectively. The ROC curve was utilized to obtain a local cut-off point of vitamin D deficiency. RESULTS: 25-OHD was < 20 ng/mL in 3% and < 33 ng/mL (local cut-off point of vitamin D deficiency) in 26% of subjects. Duration of sunlight exposure and daily intake of vitamin D had significant effects on serum level of vitamin D. CONCLUSIONS: A high prevalence of vitamin D deficiency in Isfahan children was observed in this study. Improvements in duration of sunlight exposure and daily intake of vitamin D can prevent vitamin D deficiency in these children.

Brain iron deposition after Ferumoxytol-enhanced MRI: A study of Porcine Brains.

Recent evidence of gadolinium deposition in the brain has raised safety concerns. Iron oxide nanoparticles are re-emerging as promising alternative MR contrast agents, because the iron core can be metabolized. However, long-term follow up studies of the brain after intravenous iron oxide administration have not been reported thus far. In this study, we investigated, if intravenously administered ferumoxytol nanoparticles are deposited in porcine brains. Methods: In an animal care and use committee-approved prospective case-control study, ten Göttingen minipigs received either intravenous ferumoxytol injections at a dose of 5 mg Fe/kg (n=4) or remained untreated (n=6). Nine to twelve months later, pigs were sacrificed and the brains of all pigs underwent ex vivo MRI at 7T with T2 and T2*-weighted sequences. MRI scans were evaluated by measuring R2* values (R2*=1000/T2*) of the bilateral caudate nucleus, lentiform nucleus, thalamus, dentate nucleus, and choroid plexus. Pig brains were sectioned and stained with Prussian blue and evaluated for iron deposition using a semiquantitative scoring system. Data of ferumoxytol exposed and unexposed groups were compared with an unpaired t-test and a Mann-Whitney U test. Results: T2 and T2* signal of the different brain regions was not visually different between ferumoxytol exposed and unexposed controls. There were no significant differences in R2* values of the different brain regions in the ferumoxytol exposed group compared to controls (p>0.05). Prussian blue stains of the same brain regions, scored according to a semiquantitative score, were not significantly different either between the ferumoxytol exposed group and unexposed controls (p>0.05). Conclusions: Our study shows that intravenous ferumoxytol doses of 5-10 mg Fe/kg do not lead to iron deposition in the brain of pigs. We suggest iron oxide nanoparticles as a promising alternative for gadolinium-enhanced MRI.

Instant labeling of therapeutic cells for multimodality imaging.

Autologous therapeutic cells are typically harvested and transplanted in one single surgery. This makes it impossible to label them with imaging biomarkers through classical transfection techniques in a laboratory. To solve this problem, we developed a novel microfluidic device, which provides highly efficient labeling of therapeutic cells with imaging biomarkers through mechanoporation. Methods: Studies were performed with a new, custom-designed microfluidic device, which contains ridges, which compress adipose tissue-derived stem cells (ADSCs) during their device passage. Cell relaxation after compression leads to cell volume exchange for convective transfer of nanoparticles and nanoparticle uptake into the cell. ADSCs were passed through the microfluidic device doped with iron oxide nanoparticles and 18F-fluorodeoxyglucose (FDG). The cellular nanoparticle and radiotracer uptake was evaluated with DAB-Prussian blue, fluorescent microscopy, and inductively coupled plasma spectrometry (ICP). Labeled and unlabeled ADSCs were imaged in vitro as well as ex vivo in pig knee specimen with magnetic resonance imaging (MRI) and positron emission tomography (PET). T2 relaxation times and radiotracer signal were compared between labeled and unlabeled cell transplants using Student T-test with p<0.05. Results: We report significant labeling of ADSCs with iron oxide nanoparticles and 18F-FDG within 12+/-3 minutes. Mechanoporation of ADSCs with our microfluidic device led to significant nanoparticle (> 1 pg iron per cell) and 18F-FDG uptake (61 mBq/cell), with a labeling efficiency of 95%. The labeled ADSCs could be detected with MRI and PET imaging technologies: Nanoparticle labeled ADSC demonstrated significantly shorter T2 relaxation times (24.2±2.1 ms) compared to unlabeled cells (79.6±0.8 ms) on MRI (p<0.05) and 18F-FDG labeled ADSC showed significantly higher radiotracer uptake (614.3 ± 9.5 Bq / 1×104 cells) compared to controls (0.0 ± 0.0 Bq/ 1×104 cells) on gamma counting (p<0.05). After implantation of dual-labeled ADSCs into pig knee specimen, the labeled ADSCs revealed significantly shorter T2 relaxation times (41±0.6 ms) compared to unlabeled controls (90±1.8 ms) (p<0.05). Conclusion: The labeling of therapeutic cells with our new microfluidic device does not require any chemical intervention, therefore it is broadly and immediately clinically applicable. Cellular labeling using mechanoporation can improve our understanding of in vivo biodistributions of therapeutic cells and ultimately improve long-term outcomes of therapeutic cell transplants.

Cavernous angioma: a clinical study of 35 cases with review of the literature.

BACKGROUND: Cavernous angioma is a vascular malformation which can be found in any region within the central nervous system. OBJECTIVES: There are few clinical and demographic cavernous angioma studies with large sample sizes. Therefore, the present study was designed to provide further information on the clinical and demographic characteristics of cavernous angioma using a relatively large sample of Persian patients. METHODS: Patients with cavernous angioma were recruited from the outpatient neurology clinics in Isfahan, Iran, from October 2003 to October 2006. RESULTS: In all cases, the diagnosis of cavernous angioma was based on brain magnetic resonance imaging. There were 35 patients (female/male: 17 : 18) identified with cavernous angioma. The mean age at presentation was 28.8 years. Initial manifestations included seizures in 16, headache in 11 and intracranial hemorrhage in eight patients. During follow-up, all patients experienced seizures and 19 developed headaches. Depression, vertigo, nausea, vomiting, disequilibrium, loss of consciousness and sensorimotor symptoms were also observed. CONCLUSION: Some of the findings of the present study were in accordance with previous studies. However, more of our patients with positive family history had solitary rather than multiple lesions, and more of our patients had generalized tonic-clonic seizures rather than partial seizures. Moreover, our data demonstrated that if there is a history of cavernous angioma with intracranial hemorrhage in family members, the presenting cavernous angioma patient is more prone to intracranial hemorrhage.

Magnetic resonance imaging of stem cell-macrophage interactions with ferumoxytol and ferumoxytol-derived nanoparticles.

"Off the shelf" allogeneic stem cell transplants and stem cell nano-composites are being used for the treatment of degenerative bone diseases. However, major and minor histocompatibility antigens of therapeutic cell transplants can be recognized as foreign and lead to their rejection by the host immune system. If a host immune response is identified within the first week post-transplant, immune modulating therapies could be applied to prevent graft failure and support engraftment. Ferumoxytol (Feraheme™) is an FDA approved iron oxide nanoparticle preparation for the treatment of anemia in patients. Ferumoxytol can be used "off label" as an magnetic resonance (MR) contrast agent, as these nanoparticles provide measurable signal changes on magnetic resonance imaging (MRI). In this focused review article, we will discuss three methods to localize and identify innate immune responses to stem cell transplants using ferumoxytol-enhanced MRI, which are based on tracking stem cells, tracking macrophages or detecting mediators of cell death: (a) monitor MRI signal changes of ferumoxytol-labeled stem cells in the presence or absence of innate immune responses, (b) monitor influx of ferumoxytol-labeled macrophages into stem cell implants, and (c) monitor apoptosis of stem cell implants with caspase-3 activatable nanoparticles. These techniques can detect transplant failure at an early stage, when immune-modulating interventions can potentially preserve the viability of the cell transplants and thereby improve bone and cartilage repair outcomes. Approaches 1 and 2 are immediately translatable to clinical practice. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Cells at the Nanoscale Diagnostic Tools > Biosensing.

Association of Tumor [18F]FDG Activity and Diffusion Restriction with Clinical Outcomes of Rhabdomyosarcomas.

PURPOSE: To evaluate whether the extent of restricted diffusion and 2-deoxy-2-[18F] fluoro-D-glucose ([18F]FDG) uptake of pediatric rhabdomyosarcomas (RMS) on positron emission tomography (PET)/magnetic resonance (MR) images provides prognostic information. PROCEDURE: In a retrospective, IRB-approved study, we evaluated [18F]FDG PET/CT and diffusion-weighted (DW) MR imaging studies of 21 children and adolescents (age 1-20 years) with RMS of the head and neck. [18F]FDG PET and DW MR scans at the time of the initial tumor diagnosis were fused using MIM software. Quantitative measures of the tumor mass with restricted diffusion, [18F]FDG hypermetabolism, or both were dichotomized at the median and tested for significance using Gray's test. Data were analyzed using a survival analysis and competing risk model with death as the competing risk. RESULTS: [18F]FDG PET/MR images demonstrated a mismatch between tumor areas with increased [18F]FDG uptake and restricted diffusion. The DWI, PET, and DWI + PET tumor volumes were dichotomized at their median values, 23.7, 16.4, and 9.5 cm3, respectively, and were used to estimate survival. DWI, PET, and DWI + PET overlap tumor volumes above the cutoff values were associated with tumor recurrence, regardless of post therapy COG stage (p = 0.007, p = 0.04, and p = 0.07, respectively). CONCLUSION: The extent of restricted diffusion within RMS and overlap of hypermetabolism plus restricted diffusion predict unfavorable clinical outcomes.

Tumor Formation of Adult Stem Cell Transplants in Rodent Arthritic Joints.

PURPOSE: While imaging matrix-associated stem cell transplants aimed for cartilage repair in a rodent arthritis model, we noticed that some transplants formed locally destructive tumors. The purpose of this study was to determine the cause for this tumor formation in order to avoid this complication for future transplants. PROCEDURES: Adipose-derived stem cells (ADSC) isolated from subcutaneous adipose tissue were implanted into 24 osteochondral defects of the distal femur in ten athymic rats and two immunocompetent control rats. All transplants underwent serial magnetic resonance imaging (MRI) up to 6 weeks post-transplantation to monitor joint defect repair. Nine transplants showed an increasing size over time that caused local bone destruction (group 1), while 11 transplants in athymic rats (group 2) and 4 transplants in immunocompetent rats did not. We compared the ADSC implant size and growth rate on MR images, macroscopic features, histopathologic features, surface markers, and karyotypes of these presumed neoplastic transplants with non-neoplastic ADSC transplants. RESULTS: Implants in group 1 showed a significantly increased two-dimensional area at week 2 (p = 0.0092), 4 (p = 0.003), and 6 (p = 0.0205) compared to week 0, as determined by MRI. Histopathological correlations confirmed neoplastic features in group 1 with significantly increased size, cellularity, mitoses, and cytological atypia compared to group 2. Six transplants in group 1 were identified as malignant chondrosarcomas and three transplants as fibromyxoid sarcomas. Transplants in group 2 and immunocompetent controls exhibited normal cartilage features. Both groups showed a normal ADSC phenotype; however, neoplastic ADSC demonstrated a mixed population of diploid and tetraploid cells without genetic imbalance. CONCLUSIONS: ADSC transplants can form tumors in vivo. Preventive actions to avoid in vivo tumor formations may include karyotyping of culture-expanded ADSC before transplantation. In addition, serial imaging of ADSC transplants in vivo may enable early detection of abnormally proliferating cell transplants.

Nanoparticle enhanced MRI can monitor macrophage response to CD47 mAb immunotherapy in osteosarcoma.

CD47 monoclonal antibodies (mAbs) activate tumor-associated macrophages (TAMs) in sarcomas to phagocytose and eliminate cancer cells. Though CD47 mAbs have entered clinical trials, diagnostic tests for monitoring therapy response in vivo are currently lacking. Ferumoxytol is an FDA-approved iron supplement which can be used "off label" as a contrast agent: the nanoparticle-based drug is phagocytosed by TAM and can be detected with magnetic resonance imaging (MRI). We evaluated if ferumoxytol-enhanced MRI can monitor TAM response to CD47 mAb therapy in osteosarcomas. Forty-eight osteosarcoma-bearing mice were treated with CD47 mAb or control IgG and underwent pre- and post-treatment ferumoxytol-MRI scans. Tumor enhancement, quantified as T2 relaxation times, was compared with the quantity of TAMs as determined by immunofluorescence microscopy and flow cytometry. Quantitative data were compared between experimental groups using exact two-sided Wilcoxon rank-sum tests. Compared to IgG-treated controls, CD47 mAb-treated tumors demonstrated significantly shortened T2 relaxation times on ferumoxytol-MRI scans (p < 0.01) and significantly increased F4/80+CD80+ M1 macrophages on histopathology (p < 0.01). CD47 mAb-treated F4/80+ macrophages demonstrated significantly augmented phagocytosis of ferumoxytol nanoparticles (p < 0.01). Thus, we conclude that ferumoxytol-MRI can detect TAM response to CD47 mAb in mouse models of osteosarcoma. The ferumoxytol-MRI imaging test could be immediately applied to monitor CD47 mAb therapies in clinical trials.

Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection.

Purpose: Stem cell transplants are an effective approach to repair large bone defects. However, comprehensive techniques to monitor the fate of transplanted stem cells in vivo are lacking. Such strategies would enable corrective interventions at an early stage and greatly benefit the development of more successful tissue regeneration approaches. In this study, we designed and synthesized a dual-modality imaging probe (Feru-AFC) that can simultaneously localize transplanted stem cells and diagnose immune rejection-induced apoptosis at an early stage in vivo. Methods: We used a customized caspase-3 cleavable peptide-dye conjugate to modify the surface of clinically approved ferumoxytol nanoparticles (NPs) to generate the dual-modality imaging probe with fluorescence "light-up" feature. We labeled both mouse mesenchymal stem cells (mMSCs, matched) and pig mesenchymal stem cells (pMSCs, mismatched) with the probe and transplanted the labeled cells with biocompatible scaffold at the calvarial defects in mice. We then employed intravital microscopy (IVM) and magnetic resonance imaging (MRI) to investigate the localization, engraftment, and viability of matched and mismatched stem cells, followed by histological analyses to evaluate the results obtained from in vivo studies. Results: The Feru-AFC NPs showed good cellular uptake efficiency in the presence of lipofectin without cytotoxicity to mMSCs and pMSCs. The fluorescence of Feru-AFC NPs was turned on inside apoptotic cells due to the cleavage of peptide by activated caspase-3 and subsequent release of fluorescence dye molecules. Upon transplantation at the calvarial defects in mice, the intense fluorescence from the cleaved Feru-AFC NPs in apoptotic pMSCs was observed with a concomitant decrease in the overall cell number from days 1 to 6. In contrast, the Feru-AFC NP-treated mMSCs exhibited minimum fluorescence and the cell number also remained similar. Furthermore, in vivo MRI of the Feru-AFC NP-treated mMSC and pMSCs transplants could clearly indicate the localization of matched and mismatched cells, respectively. Conclusions: We successfully developed a dual-modality imaging probe for evaluation of the localization and viability of transplanted stem cells in mouse calvarial defects. Using ferumoxytol NPs as the platform, our Feru-AFC NPs are superparamagnetic and display a fluorescence "light-up" signature upon exposure to activated caspase-3. The results show that the probe is a promising tool for long-term stem cell tracking through MRI and early diagnosis of immune rejection-induced apoptosis through longitudinal fluorescence imaging.

Tracking Cell Transplants in Femoral Osteonecrosis with Magnetic Resonance Imaging: A Proof-of-Concept Study in Patients.

PURPOSE: Osteonecrosis is a devastating complication of high-dose corticosteroid therapy in patients with cancer. Core decompression for prevention of bone collapse has been recently combined with the delivery of autologous concentrated bone marrow aspirates. The purpose of our study was to develop an imaging test for the detection of transplanted bone marrow cells in osteonecrosis lesions. EXPERIMENTAL DESIGN: In a prospective proof-of-concept clinical trial (NCT02893293), we performed serial MRI studies of nine hip joints of 7 patients with osteonecrosis before and after core decompression. Twenty-four to 48 hours prior to the surgery, we injected ferumoxytol nanoparticles intravenously to label cells in normal bone marrow with iron oxides. During the surgery, iron-labeled bone marrow cells were aspirated from the iliac crest, concentrated, and then injected into the decompression track. Following surgery, patients received follow-up MRI up to 6 months after bone marrow cell transplantation. RESULTS: Iron-labeled cells could be detected in the access canal by a dark (negative) signal on T2-weighted MR images. T2* relaxation times of iron-labeled cell transplants were significantly lower compared with unlabeled cell transplants of control patients who were not injected with ferumoxytol (P = 0.02). Clinical outcomes of patients who received ferumoxytol-labeled or unlabeled cell transplants were not significantly different (P = 1), suggesting that the added ferumoxytol administration did not negatively affect bone repair. CONCLUSIONS: This immediately clinically applicable imaging test could become a powerful new tool to monitor the effect of therapeutic cells on bone repair outcomes after corticosteroid-induced osteonecrosis.