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Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p = 0.013) and inversely correlated with patient disease-free survival rates (p = 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p

Targeted therapy for breast cancer stem cell (BCSC): A novel liposomal system (APTEDB -LS-siRNA(EDB) ) that enables simultaneous targeting and knockdown of extra domain B of fibronectin (EDB-FN) shows potent therapeutic efficacy in the BCSC-derived tumors in vivo.

Molecular imaging can be a breakthrough tool for the investigation of the behavior and ultimate feasibility of transplanted human mesenchymal stem cells (hMSCs) inside the body, and for the development of guidelines and recommendations based on the treatment and evaluation of stem cell therapy for patients. The goals of this study were to evaluate the multilineage differentiation ability of hMSCs expressing an MRI reporter, human ferritin heavy chain (FTH) and to investigate the feasibility of using FTH-based MRI to provide noninvasive imaging of transplanted hMSCs. The transduction of FTH and green fluorescence protein (GFP) did not influence the expression of the mesenchymal stem cell surface markers (CD29+/CD105+/CD34–/CD45–) or the self-renewal marker genes [octamer-binding transcription factor 4 (OCT-4) and SRY (sex determining region Y)-box 2 (Sox-2)], cell viability, migration ability and the release of cytokines [interleukin-5 (IL-5), IL-10, IL-12p70, tumor necrosis factor-α (TNF-α)]. FTH-hMSCs retained the capacity to differentiate into adipogenic, chondrogenic, osteogenic and neurogenic lineages. The transduction of FTH led to a significant enhancement in cellular iron storage capacity and caused hypointensity and a significant increase in R2* values of FTH-hMSC-collected phantoms and FTH-hMSC-transplanted sites of the brain, as shown by in vitro and in vivo MRI performed at 9.4 T, compared with control hMSCs. This study revealed no differences in biological characteristics between hMSCs and FTH-hMSCs and, therefore, these cells could be used for noninvasive monitoring with MRI during stem cell therapy for brain injury. Our study suggests the use of FTH for in vivo long-term tracking and ultimate fate of hMSCs without alteration of their characteristics and multidifferentiation potential. Copyright © 2014 John Wiley & Sons, Ltd.

This study was undertaken to investigate the effect of a magnetic resonance imaging (MRI) contrast agent, superparamagnetic iron oxide nanoparticle (SPIO), on signal transduction by glucose and insulin in pancreatic β-cells. INS-1 cells were labeled in culture medium containing clinically approved SPIO for 24 h. Labeled and unlabeled cells were stimulated with glucose (25 mM) or insulin (0.1-1 µM) for 12 h. The phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase B (AKT) and intracellular insulin protein levels were assessed by Western blotting. After labeling with increasing amounts of SPIO, cytotoxicity was not observed, yet the intracellular iron concentration increased in a dose-dependent manner. SPIO labeling (200 µg Fe ml(-1)) induced a significant increase in ERK1/2 and AKT phosphorylation (labeled vs unlabeled, p < 0.05), but significantly reduced the glucose-stimulated phosphorylation of ERK1/2 and AKT and insulin-stimulated phosphorylation of AKT (labeled vs unlabeled, p < 0.05). The level of intracellular insulin protein was found to be lower in labeled cells than unlabeled cells (labeled vs unlabeled, p < 0.05). This study demonstrates that SPIO labeling alters some fundamental functional variables, at least in INS-1 cells, through modulation of the glucose- or insulin-induced activation of ERK1/2 and AKT, which leads to insulin biosynthesis.

Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest. This study demonstrates the feasibility of using the MR reporter ferritin for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice. A B16F10 murine melanoma cell line expressing human ferritin heavy chain (hFTH) and green fluorescent protein (GFP) was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4-T MRI in vitro and in vivo, yielding significant changes in T2* relative to control cells. In BALB/c nude mice, the presence of hFTH- and GFP-expressing metastatic melanoma cells in deep-seated axillary LNs was demonstrated as areas of low T2* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T2* MRI data, correlated well with tumor burden, which was determined by histology (r = −0.8773, p = 0.0001). This study is the first to use MRI and an MR reporter gene for both the visualization and quantification of metastatic cancer cells in LNs. Copyright © 2011 John Wiley & Sons, Ltd.

The control of dendritic cell (DC) migration into lymph nodes (LNs) is important for the development of more effective DC-based immunotherapies. This study was undertaken to evaluate, dynamically and noninvasively, prostaglandin E2 (PGE2)-enhanced migration of DCs using a 1.5 T clinical MR scanner. DC2.4 cells were labeled with superparamagnetic iron oxide (SPIO), a clinically approved MRI contrast agent. DCs were stimulated with tumor necrosis factor-α and interferon-γ in the presence or absence of PGE2. Before and after subcutaneous injection of labeled DCs into the hind leg footpads of mice, MRI detailing the extent of DC migration into popliteal LNs was performed using a 1.5 T clinical MR scanner. SPIO labeling did not influence the viability, endocytic activity, migratory ability and/or co-stimulatory molecule expression of DCs. PGE2 enhanced significantly chemokine receptor-7 expression and the migration of DCs (p < 0.05). After subcutaneous injection of DCs, there were decreases in MR signal intensity in popliteal LNs at 24 h post-injection; in PGE2-treated cells, the MR signal intensity was significantly lower (decrease of 86.6 ± 2.5%) than in PGE2-untreated cells (decrease of 70.0 ± 4.2%) (p < 0.05). Histological analyses with the conventionally used Prussian blue stain demonstrated that the PGE2-treated DCs migrated more deeply into the center of LNs. PGE2-enhanced migration of SPIO-labeled DCs into LNs can be detected using a 1.5 T clinical MR scanner. Our results suggest that in vivo MRI of DC migration is a useful imaging method to predict DC therapy with a high rate of efficacy and to improve DC-based immunotherapy, thereby reducing costs compared with current treatments in clinical trials.

This study was undertaken to compare the labeling efficiencies of three iron-oxide based MRI contrast agents [Feridex, Resovist and monocrystalline iron oxide (MION)] and to evaluate their effects on the biological properties of human mesenchymal stem cells (hMSCs). The hMSCs were cultivated for 1 and 7 days after 24-h labeling with iron oxide nanoparticles (12.5 microg Fe/mL) in the presence of poly-L-lysine (0.75 microg/mL). The hMSCs were labeled more efficiently with use of Feridex, Resovist as compared to MION. No significant differences were observed in terms of viability and proliferation of labeled hMSCs. The level of Oct-4 mRNA increased in labeled hMSCs at day 1 and the cellular phenotype changed from CD45-/CD44+/CD29+ to CD45low/CD44+/CD29+ at day 7, which closely resembles the phenotype of fresh bone marrow-derived hMSCs. Our study has demonstrated that the Feridex or Resovist is the preferred labeling agent for hMSCs. There was a change in Oct-4 and CD45 expression after labeling.

Increased modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) has been implicated in the development of diabetic cardiomyopathy. We used the well-characterized ES cells (Nkx2.5GFP knock-in ES cells), to investigate the role of O-GlcNAcylation in cardiomyocyte development. O-GlcNAcylation decreased in differentiating ES cells, as did the expression of O-GlcNAc transferase. Increasing O-GlcNAcylation with glucosamine or by inhibiting N-acetylglucosaminidase (streptozotocin or PUGNAc) decreased the number of cardiomyocyte precursors and cardiac-specific gene expression. On the other hand, decreasing O-GlcNAcylation with an inhibitor of glutamine fructose-6-phosphate amidotransferase (6-diazo-5-oxo-norleucine) increased cardiomyocyte precursors. These results suggest that excessive O-GlcNAcylation impairs cardiac cell differentiation in ES cells.

This study was undertaken to investigate the in vitro effect of islet labeling with iron oxide nanoparticles for MRI on islet viability, insulin secretion, and gene expression. Isolated rat islets were labeled with Resovist (25–200 µg Fe/mL, a clinically approved MRI contrast agent) in the presence or absence of poly-l-Lysine (PLL, 1.5 µg/mL) for 48 h. The iron content of labeled islets was found to increase in a dose-dependent manner. More than 90% of the islets were labeled with 100 µg Fe/mL. We confirmed the localizations of iron oxide nanoparticles within islet β-cells by insulin immunostaining. As the concentration of Resovist increased, T2 values as determined by T2-weighted MRI on a 1.5 Tesla MR scanner decreased. Labeling of 100 islets in a medium containing 100 µg Fe/mL of Resovist in the absence of PLL provided sufficient contrast for islet visualization on T2-weighted MRI. MTT assays showed that the viability of labeled islets was not different from that of unlabeled islets. No statistical difference was observed between labeled (2.91 ± 0.36) and unlabeled islets (2.83 ± 0.61) in terms of the ability to secrete insulin, as determined by the glucose stimulation index. We also evaluated the effect of iron oxide incorporation on the gene expressions in islet cells using RT-PCR (reverse transcriptase PCR). Insulin expression in labeled islets was significantly elevated (1.83 ± 0.25 fold vs. unlabeled; p = 0.005), but not the expression of somatostatin (1.39 ± 0.18 fold vs. unlabeled; p = 0.085) or glucagons (1.28 ± 0.13 fold vs. unlabeled; p = 0.09). Expression of an important transcription factor for insulin gene transcription, BETA2 (beta-cell E-box trans-activator), was increased in labeled islets (1.67 ± 0.15 fold vs. unlabeled; p = 0.029). The findings of this study indicate that Resovist provides a satisfactory means to image islets and has no deleterious effect on islet function or gene expression. Copyright © 2009 John Wiley & Sons, Ltd.

Embryonic stem (ES) cells are a useful system to study cardiac differentiation in vitro. It has been difficult, however, to track the fates of chamber-specific cardiac lineages, since differentiation is induced within the embryoid body. We have established an in vitro culture system to track Nkx2.5(+) cell lineages during mouse ES cell differentiation by using green fluorescent protein (GFP) as a reporter. Nkx2.5/GFP(+) cardiomyocytes purified from embryoid bodies express sarcomeric tropomyosin and myosin heavy chain and heterogeneously express cardiac troponin I (cTnI), myosin light chain 2v (MLC2v) and atrial natriuretic peptide (ANP). After 4-week culture, GFP(+) cells exhibited electrophysiological characteristics specific to sinoatrial (SA) node, atrial, or ventricular type. Furthermore, we found that administration of 10(-7) M retinoic acid (RA) to embryoid bodies increased the percentage of MLC2v(-)ANP(+) cells; this also increased the expression of atrial-specific genes in the Nkx2.5/GFP(+) fraction, in a time- and dose-dependent fashion. These results suggest that Nkx2.5(+) lineage cells possess the potential to differentiate into various cardiomyocyte cell types and that RA can modify the differentiation potential of Nkx2.5(+) cardiomyocytes at an early stage.