AC133+ progenitor cells as gene delivery vehicle and cellular probe in subcutaneous tumor models: a preliminary study

<p>Abstract</p> <p>Background</p> <p>Despite enormous progress in gene therapy for breast cancer, an optimal systemic vehicle for delivering gene products to the target tissue is still lacking. The purpose of this study was to determine whether AC133+ progenitor cells (APC) can be used as both gene delivery vehicles and cellular probes for magnetic resonance imaging (MRI). In this study, we used superparamagentic iron oxide (SPIO)-labeled APCs to carry the human sodium iodide symporter (hNIS) gene to the sites of implanted breast cancer in mouse model. In vivo real time tracking of these cells was performed by MRI and expression of hNIS was determined by Tc-99m pertechnetate (Tc-99m) scan.</p> <p>Results</p> <p>Three million human breast cancer (MDA-MB-231) cells were subcutaneously implanted in the right flank of nude mice. APCs, isolated from fresh human cord blood, were genetically transformed to carry the hNIS gene using adenoviral vectors and magnetically labeled with ferumoxides-protamine sulfate (FePro) complexes. Magnetically labeled genetically transformed cells were administered intravenously in tumor bearing mice when tumors reached 0.5 cm in the largest dimension. MRI and single photon emission computed tomography (SPECT) images were acquired 3 and 7 days after cell injection, with a 7 Tesla animal MRI system and a custom built micro-SPECT using Tc-99m, respectively. Expression of hNIS in accumulated cells was determined by staining with anti-hNIS antibody. APCs were efficiently labeled with ferumoxide-protamine sulfate (FePro) complexes and transduced with hNIS gene. Our study showed not only the accumulation of intravenously administered genetically transformed, magnetically labeled APCs in the implanted breast cancer, but also the expression of hNIS gene at the tumor site. Tc-99m activity ratio (tumor/non-tumor) was significantly different between animals that received non-transduced and transduced cells (P < 0.001).</p> <p>Conclusion</p> <p>This study indicates that genetically transformed, magnetically labeled APCs can be used both as delivery vehicles and cellular probes for detecting <it>in vivo </it>migration and homing of cells. Furthermore, they can potentially be used as a gene carrier system for the treatment of tumor or other diseases.</p

Optimization and Validation of FePro Cell Labeling Method

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12–16 hours) incubation time and uses relatively high dose of Pro (5–6 µg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 µg/ml and Pro 0.75 to 3 µg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached ∼30–35 pg-iron/cell at 24 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved ∼10 pg-iron/cell at 48 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 µg/ml of Fe and 3 µg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 µg/ml of Fe and 3 µg/ml of Pro is effective in labeling cells for cellular MRI

MRI to assess chemoprevention in transgenic adenocarcinoma of mouse prostate (TRAMP)

<p>Abstract</p> <p>Background</p> <p>The current method to determine the efficacy of chemoprevention in TRAMP mouse model of carcinoma of prostate (CaP) is by extracting and weighing the prostate at different time points or by immunohistochemistry analysis. Non-invasive determination of volumes of prostate glands and seminal vesicles before, during and after treatment would be valuable in investigating the efficacy of newer chemopreventive agents in CaP. The purpose of this study was to determine whether <it>in vivo </it>magnetic resonance imaging (MRI) using a 3 tesla clinical MRI system can be used to follow the effect of chemoprevention in TRAMP model of mouse CaP.</p> <p>Methods</p> <p>Mice were randomized into control and treated groups. The animals in treated group received 10 µmol/kg of CDDO, 5 days a week for 20 weeks. Animals underwent <it>in vivo </it>MRI of prostate gland and seminal vesicles by a clinical 3 Tesla MRI system just before (at 5 weeks), during and at the end of treatment, at 25 weeks. T1-weighted and fat saturation (FATSAT) multiecho fast spin echo T2- weighted images (T2WI) were acquired. Volume of the prostate glands and seminal vesicles was determined from MR images. T2 signal intensity changes in the seminal vesicles were determined by subtracting higher echo time (TE) from lower TE T2WI. Following treatments all animals were sacrificed, prostate and seminal vesicles collected, and the tissues prepared for histological staining. All data were expressed as mean ± 1 standard deviation. Two-way or multivariate analysis of variance followed by post-hoc test was applied to determine the significant differences. A p-value of <0.05 was considered significant.</p> <p>Results</p> <p>Histological analysis indicated tumor in 100% of control mice, whereas 10% of the treated mice showed tumor in prostate gland. Both MRI and measured prostate weights showed higher volume/weight in control mouse group. MRI showed significantly higher volume of seminal vesicles in control animals and T2 signal intensity changes in seminal vesicles of control mice indicating higher number of tumor foci, which was also proven by histology.</p> <p>Conclusions</p> <p><it>In vivo </it>MRI is helpful in determining the efficacy of chemoprevention of prostate cancer in TRAMP mice.</p

Human Cord Blood-Derived AC133+ Progenitor Cells Preserve Endothelial Progenitor Characteristics after Long Term In Vitro Expansion

Stem cells/progenitors are central to the development of cell therapy approaches for vascular ischemic diseases. The crucial step in rescuing tissues from ischemia is improvement of vascularization that can be achieved by promoting neovascularization. Endothelial progenitor cells (EPCs) are the best candidates for developing such an approach due to their ability to self-renew, circulate and differentiate into mature endothelial cells (ECs). Studies showed that intravenously administered progenitors isolated from bone marrow, peripheral or cord blood home to ischemic sites. However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients. Hence, the ability to amplify the numbers of autologous EPCs by long term in vitro expansion while preserving their angiogenic potential is critically important for developing EPC based therapies. Therefore, the objective of this study was to evaluate the capacity of cord blood (CB)-derived AC133+ cells to differentiate, in vitro, towards functional, mature endothelial cells (ECs) after long term in vitro expansion.We systematically characterized the properties of CB AC133+ cells over the 30 days of in vitro expansion. During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers. Flow cytometry and immunocytochemistry demonstrated that CB AC133+ cells' expression of endothelial progenitor markers was not affected by long term in vitro culturing. After culturing under EC differentiation conditions, cells exhibited high expression of mature ECs markers, such as CD31, VEGFR-2 and von Willebrand factor, as well as the morphological changes indicative of differentiation towards mature ECs. In addition, throughout the 30 day culture cells preserved their functional capacity that was demonstrated by high uptake of DiI fluorescently conjugated-acetylated-low density lipoprotein (DiI-Ac-LDL), in vitro and in vivo migration towards chemotactic stimuli and in vitro tube formation.These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage

Glucocorticoid-Induced Leucine Zipper Promotes Neutrophil and T-Cell Polarization with Protective Effects in Acute Kidney Injury

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Endothelial Progenitor Cells (EPCs) as Gene Carrier System for Rat Model of Human Glioma

Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1) intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS) to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2) whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities.Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m) in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS). Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors.EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for glioma therapy as well as imaging probes

Inhibitor of DNA binding 1 as a secreted angiogenic transcription factor in rheumatoid arthritis

Abstract Introduction Rheumatoid arthritis (RA) is characterized by enhanced blood vessel development in joint synovium. This involves the recruitment of endothelial progenitor cells (EPCs), allowing for de novo vessel formation and pro-inflammatory cell infiltration. Inhibitor of DNA Binding 1 (Id1) is a transcription factor characteristic of EPCs that influences cell maturation. Method Enzyme-linked immunosorbant assay (ELISA) and polymerase chain reaction (PCR) were used to examine Id1 levels in synovial fluid (SF) and endothelial cells (ECs), respectively. Immunohistology was used to determine the expression of Id1 in synovial tissue (ST). Human dermal microvascular EC (HMVEC) migration and tube forming assays were used to determine if recombinant human Id1 (rhuId1) and/or RA SF immunodepleted Id1 showed angiogenic activity. We also utilized the RA ST severe combined immunodeficient (SCID) mouse chimera to examine if Id1 recruits EPCs to RA synovium. Results ST samples immunostained for Id1 showed heightened expression in RA compared to osteoarthritis (OA) and normal (NL) ST. By immunofluorescence staining, we found significantly more Id1 in RA compared to OA and NL vasculature, showing that Id1 expressing cells, and therefore EPCs, are most active in vascular remodeling in the RA synovium. We also detected significantly more Id1 in RA compared to OA and other arthritis SFs by ELISA, which correlates highly with Chemokine (C-X-C motif) ligand 16 (CXCL16) levels. In vitro chemotaxis assays showed that Id1 is highly chemotactic for HMVECs and can be attenuated by inhibition of Nuclear Factor κB and phosphoinositide 3-kinase. Using in vitro Matrigel assays, we found that HMVECs form tubes in response to rhuId1 and that Id1 immunodepleted from RA SF profoundly decreases tube formation in Matrigel in vitro. PCR showed that Id1 mRNA could be up-regulated in EPCs compared to HMVECs in response to CXCL16. Finally, using the K/BxN serum induced arthritis model, we found that EC CXCR6 correlated with Id1 expression by immunohistochemistry. Conclusions We conclude that Id1 correlates highly with CXCL16 expression, EPC recruitment, and blood vessel formation in the RA joint, and that Id1 is potently angiogenic and can be up-regulated in EPCs by CXCL16.http://deepblue.lib.umich.edu/bitstream/2027.42/110805/1/13075_2013_Article_4258.pd

Measurement of rat brain tumor kinetics using an intravascular MR contrast agent and DCE‐MRI nested model selection

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109323/1/jmri24469.pd

Differentiation of Glioma and Radiation Injury in Rats Using In Vitro Produce Magnetically Labeled Cytotoxic T-Cells and MRI

A limitation with current imaging strategies of recurrent glioma undergoing radiotherapy is that tumor and radiation injury cannot be differentiated with post contrast CT or MRI, or with PET or other more complex parametric analyses of MRI data. We propose to address the imaging limitation building on emerging evidence indicating that effective therapy for recurrent glioma can be attained by sensitized T-cells following vaccination of primed dendritic cells (DCs). The purpose of this study was to determine whether cord blood T-cells can be sensitized against glioma cells (U-251) and if these sensitized cytotoxic T-cells (CTLs) can be used as cellular magnetic resonance imaging probes to identify and differentiate glioma from radiation necrosis in rodent models.Cord blood T and CD14+ cells were collected. Isolated CD14+ cells were then converted to dendritic cells (DCs), primed with glioma cell lysate and used to sensitize T-cells. Phenotypical expression of the generated DCs were analyzed to determine the expression level of CD14, CD86, CD83 and HLA-DR. Cells positive for CD25, CD4, CD8 were determined in generated CTLs. Specificity of cytotoxicity of the generated CTLs was also determined by lactate dehydrogenase (LDH) release assay. Secondary proliferation capacity of magnetically labeled and unlabeled CTLs was also determined. Generated CTLs were magnetically labeled and intravenously injected into glioma bearing animals that underwent MRI on days 3 and 7 post- injection. CTLs were also administered to animals with focal radiation injury to determine whether these CTLs accumulated non-specifically to the injury sites. Multi-echo T2- and T2*-weighted images were acquired and R2 and R2* maps created. Our method produced functional, sensitized CTLs that specifically induced U251 cell death in vitro. Both labeled and unlabeled CTLs proliferated equally after the secondary stimulation. There were significantly higher CD25 positive cells (p = <0.006) in CTLs. In addition, T2- and T2*-weighted MR images showed increased low signal intensity areas in animals that received labeled CTLs as compared to the images from animals that received control cells. Histological analysis confirmed the presence of iron positive cells in sites corresponding to MRI low signal intensity regions. Significant differences (p = <0.001) in tumor R2 and R2* values were observed among the groups of animals. Animals with radiation injury exhibited neither MRI hypointense areas nor presence of iron positive cells.Our results indicate that T-cells can be effectively sensitized by in vitro methods and used as cellular probes to identify and differentiate glioma from radiation necrosis

Effects of Ferumoxides – Protamine Sulfate Labeling on Immunomodulatory Characteristics of Macrophage-like THP-1 Cells

Superparamagnetic Iron Oxide (SPIO) complexed with cationic transfection agent is used to label various mammalian cells. Labeled cells can then be utilized as an in vivo magnetic resonance imaging (MRI) probes. However, certain number of in vivo administered labeled cells may be cleared from tissues by the host's macrophages. For successful translation to routine clinical application of SPIO labeling method it is important that this mode of in vivo clearance of iron does not elicit any diverse immunological effects. The purpose of this study was to demonstrate that SPIO agent ferumoxides-protamine sulfate (FePro) incorporation into macrophages does not alter immunological properties of these cells with regard to differentiation, chemotaxis, and ability to respond to the activation stimuli and to modulate T cell response. We used THP-1 cell line as a model for studying macrophage cell type. THP-1 cells were magnetically labeled with FePro, differentiated with 100 nM of phorbol ester, 12-Myristate-13-acetate (TPA) and stimulated with 100 ng/ml of LPS. The results showed 1) FePro labeling had no effect on the changes in morphology and expression of cell surface proteins associated with TPA induced differentiation; 2) FePro labeled cells responded to LPS with slightly higher levels of NFκB pathway activation, as shown by immunobloting; TNF-α secretion and cell surface expression levels of CD54 and CD83 activation markers, under these conditions, were still comparable to the levels observed in non-labeled cells; 3) FePro labeling exhibited differential, chemokine dependent, effect on THP-1 chemotaxis with a decrease in cell directional migration to MCP-1; 4) FePro labeling did not affect the ability of THP-1 cells to down-regulate T cell expression of CD4 and CD8 and to induce T cell proliferation. Our study demonstrated that intracellular incorporation of FePro complexes does not alter overall immunological properties of THP-1 cells. The described experiments provide the model for studying the effects of in vivo clearance of iron particles via incorporation into the host's macrophages that may follow after in vivo application of any type of magnetically labeled mammalian cells. To better mimic the complex in vivo scenario, this model may be further exploited by introducing additional cellular and biological, immunologically relevant, components