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DiR-labeled Embryonic Stem Cells for Targeted Imaging of in vivo Gastric Cancer Cells.

ABSTRACT: Embryonic stem (ES) cells have great potential in applications such as disease modeling, pharmacological screening and stem cell therapies. Up to date, there is no related report on the use of ES cells as tracking and contrast reagents of cancer cells in vivo. Herein we report that DiR-labeled murine ES cells can recognize and target gastric cancer cells in vivo. DiR-labeled murine ES (mES) cells (5×10(6)) were intravenously injected into gastric tumor-bearing mice. The biodistribution of DiR-labeled mES cells was monitored by IVIS imaging within 24 h. Major organs were harvested and analyzed by immunofluorescence staining and Western blotting. Chemotaxis assay was employed to investigate the chemotaxis of ES cells tracking cancer cells. Fluorescent imaging results showed that DiR-labeled mES cells targeted gastric cancer tissue in vivo as early as 10 min post-injection, reaching a peak at 2h post-injection. Immunofluorescence staining and Western blotting results showed gastric cancer tissues specifically expressed SSEA-1. In vitro migration tests confirmed that mES cells actively moved to test sites with different concentration of CXCL12 in a dose-dependent manner. In conclusion, DiR-labeled mES cells may be used for gastric cancer targeted imaging in vivo, and have great potential in applications such as identifying and imaging of early gastric cancer in near future.

SUBMITTER: Ruan J

PROVIDER: S-EPMC3388594 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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DiR-labeled Embryonic Stem Cells for Targeted Imaging of in vivo Gastric Cancer Cells.

Ruan Jing J Song Hua H Li Chao C Bao Chenchen C Fu Hualin H Wang Kan K Ni Jian J Cui Daxiang D

Theranostics 20120615 6

Embryonic stem (ES) cells have great potential in applications such as disease modeling, pharmacological screening and stem cell therapies. Up to date, there is no related report on the use of ES cells as tracking and contrast reagents of cancer cells in vivo. Herein we report that DiR-labeled murine ES cells can recognize and target gastric cancer cells in vivo. DiR-labeled murine ES (mES) cells (5×10(6)) were intravenously injected into gastric tumor-bearing mice. The biodistribution of DiR-la ...[more]

PMID: 22768029

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Project description:OBJECTIVES:The aim of the current study is to test the ability to label and detect murine embryonic stem cell-derived cardiovascular progenitor cells (ES-CPC) with cardiac magnetic resonance (CMR) using the novel contrast agent Gadofluorine M-Cy3 (GdFM-Cy3). BACKGROUND:Cell therapy shows great promise for the treatment of cardiovascular disease. An important limitation to previous clinical studies is the inability to accurately identify transplanted cells. GdFM-Cy3 is a lipophilic paramagnetic contrast agent that contains a perfluorinated side chain and an amphiphilic character that allows for micelle formation in an aqueous solution. Previous studies reported that it is easily taken up and stored within the cytosol of mesenchymal stem cells, thereby allowing for paramagnetic cell labeling. Investigators in our laboratory have recently developed techniques for the robust generation of ES-CPC. We reasoned that GdFM-Cy3 would be a promising agent for the in vivo detection of these cells after cardiac cell transplantation. METHODS:ES-CPC were labeled with GdFM-Cy3 by incubation. In vitro studies were performed to assess the impact of GdFM-Cy3 on cell function and survival. A total of 500,000 GdFM-Cy3-labeled ES-CPC or control ES-CPC were injected into the myocardium of mice with and without myocardial infarction. Mice were imaged (9.4-T) before and over a 2-week time interval after stem cell transplantation. Mice were then euthanized, and their hearts were sectioned for fluorescence microscopy. RESULTS:In vitro studies demonstrated that GdFM-Cy3 was easily transfectable, nontoxic, stayed within cells after labeling, and could be visualized using CMR and fluorescence microscopy. In vivo studies confirmed the efficacy of the agent for the detection of cells transplanted into the hearts of mice after myocardial infarction. A correspondence between CMR and histology was observed. CONCLUSIONS:The results of the current study suggest that it is possible to identify and potentially track GdFM-Cy3-labeled ES-CPC in murine infarct models via CMR.

Project description:Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 ?g/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI.The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts, highlighting its utility in future cardiac stem cell therapy trials.

Project description:BackgroundThere is increasing evidence that solid cancers contain cancer-initiating cells (CICs) that are capable of regenerating a tumor that has been surgically removed and/or treated with chemotherapy and/or radiation therapy. Currently, cell surface markers, like CD133 or CD44, are used to identify CICs in vitro; however, these markers cannot be used to identify and track CICs in vivo. The 26S proteasome is the main regulator of many processes within a proliferating cell, and its activity may be altered depending on the phenotype of a cell.MethodsHuman glioma and breast cancer cells were engineered to stably express ZsGreen fused to the carboxyl-terminal degron of ornithine decarboxylase, resulting in a fluorescent fusion protein that accumulates in cells in the absence of 26S proteasome activity; activities of individual proteases were monitored in a plate reader by detecting the cleavage of fluorogenic peptide substrates. Proteasome subunit expression in cells expressing the fusion protein was assessed by quantitative reverse transcription-polymerase chain reaction, and the stem cell phenotype of CICs was assessed by a sphere formation assay, by immunohistochemical staining for known stem cell markers in vitro, and by analyzing their tumorigenicity in vivo. CICs were tracked by in vivo fluorescence imaging after radiation treatment of tumor-bearing mice and targeted specifically via a thymidine kinase-degron fusion construct. All P values were derived from two-sided tests.ResultsCancer cells grown as sphere cultures in conditions, which enrich for cancer stem cells (CSCs), had decreased proteasome activity relative to the respective monolayers (percent decrease in chymotryptic-like activity of sphere cultures relative to monolayers--U87MG: 26.64%, 95% confidence interval [CI] = 10.19 to 43.10, GL261, 52.91%, 95% CI = 28.38 to 77.43). The cancer cells with low proteasome activity can thus be monitored in vitro and in vivo by the accumulation of a fluorescent protein (ZsGreen) fused to a degron that targets it for 26S proteasome degradation. In vitro, ZsGreen-positive cells had increased sphere-forming capacity, expressed CSC markers, and lacked differentiation markers compared with ZsGreen-negative cells. In vivo, ZsGreen-positive cells were approximately 100-fold more tumorigenic than ZsGreen-negative cells when injected into nude mice (ZsGreen positive, 30 mice per group; ZsGreen negative, 31 mice per group), and the number of CICs in tumors increased after 72 hours post radiation treatment. CICs were selectively targeted via a proteasome-dependent suicide gene, and their elimination in vivo led to tumor regression.ConclusionOur results demonstrate that reduced 26S proteasome activity is a general feature of CICs that can easily be exploited to identify, track, and target them in vitro and in vivo.

Project description:BackgroundGastric cancer is 2th most common cancer in China, and is still the second most common cause of cancer-related death in the world. How to recognize early gastric cancer cells is still a great challenge for early diagnosis and therapy of patients with gastric cancer. This study is aimed to develop one kind of multifunctional nanoprobes for in vivo targeted magnetofluorescent imaging of gastric cancer.MethodsBRCAA1 monoclonal antibody was prepared, was used as first antibody to stain 50 pairs of specimens of gastric cancer and control normal gastric mucous tissues, and conjugated with fluorescent magnetic nanoparticles with 50 nm in diameter, the resultant BRCAA1-conjugated fluorescent magnetic nanoprobes were characterized by transmission electron microscopy and photoluminescence spectrometry, as-prepared nanoprobes were incubated with gastric cancer MGC803 cells, and were injected into mice model loaded with gastric cancer of 5 mm in diameter via tail vein, and then were imaged by fluorescence optical imaging and magnetic resonance imaging, their biodistribution was investigated. The tissue slices were observed by fluorescent microscopy, and the important organs such as heart, lung, kidney, brain and liver were analyzed by hematoxylin and eosin (HE) stain method.ResultsBRCAA1 monoclonal antibody was successfully prepared, BRCAA1 protein exhibited over-expression in 64% gastric cancer tissues, no expression in control normal gastric mucous tissues, there exists statistical difference between two groups (P < 0.01). The BRCAA1-conjugated fluorescent magnetic nanoprobes exhibit very low-toxicity, lower magnetic intensity and lower fluorescent intensity with peak-blue-shift than pure FMNPs, could be endocytosed by gastric cancer MGC803 cells, could target in vivo gastric cancer tissues loaded by mice, and could be used to image gastric cancer tissues by fluorescent imaging and magnetic resonance imaging, and mainly distributed in local gastric cancer tissues within 12 h post-injection. HE stain analysis showed that no obvious damages were observed in important organs.ConclusionsThe high-performance BRCAA1 monoclonal antibody-conjugated fluorescent magnetic nanoparticles can target in vivo gastric cancer cells, can be used for simultaneous magnetofluorescent imaging, and may have great potential in applications such as dual-model imaging and local thermal therapy of early gastric cancer in near future.

Project description:UnlabelledOur understanding of estrogen (17β-estradiol, E2) receptor biology has evolved in recent years with the discovery and characterization of a 7-transmembrane-spanning G protein-coupled estrogen receptor (GPER/GPR30) and the development of GPER-selective functional chemical probes. GPER is highly expressed in certain breast, endometrial, and ovarian cancers, establishing the importance of noninvasive methods to evaluate GPER expression in vivo. Here, we developed (99m)Tc-labeled GPER ligands to demonstrate the in vivo status of GPER as an estrogen receptor (ER) and for GPER visualization in whole animals. A series of (99m)Tc(I)-labeled nonsteroidal tetrahydro-3H-cyclopenta[c]quinolone derivatives was synthesized utilizing pyridin-2-yl hydrazine and picolylamine chelates. Radioligand receptor binding studies revealed binding affinities in the 10 to 30 nmol/L range. Cell signaling assays previously demonstrated that derivatives retaining a ketone functionality displayed agonist properties, whereas those lacking such a hydrogen bond acceptor were antagonists. In vivo biodistribution and imaging studies performed on mice bearing human endometrial and breast cancer cell xenografts yielded significant tumor uptake (0.4-1.1%ID/g). Blocking studies revealed specific uptake in multiple organs (adrenals, uterus, and mammary tissue), as well as tumor uptake with similar levels of competition by E2 and G-1, a GPER-selective agonist. In conclusion, we synthesized and evaluated a series of first-generation (99m)Tc-labeled GPER-specific radioligands, demonstrating GPER as an estrogen-binding receptor for the first time in vivo using competitive binding principles, and establishing the utility of such ligands as tumor imaging agents. These results warrant further investigation into the role of GPER in estrogen-mediated carcinogenesis and as a target for diagnostic/therapeutic/image-guided drug delivery.ImplicationsThese studies provide a molecular basis to evaluate GPER expression and function as an ER through in vivo imaging.

Dataset Information

DiR-labeled Embryonic Stem Cells for Targeted Imaging of in vivo Gastric Cancer Cells.

Publications

DiR-labeled Embryonic Stem Cells for Targeted Imaging of in vivo Gastric Cancer Cells.

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