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1.
Methods ; 99: 99-111, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26546729

ABSTRACT

Magnetic Resonance Imaging (MRI) is a commonly used, non-invasive imaging technique that provides visualization of soft tissues with high spatial resolution. In both a research and clinical setting, the major challenge has been identifying a non-invasive and safe method for longitudinal tracking of delivered cells in vivo. The labeling and tracking of contrast agent labeled cells using MRI has the potential to fulfill this need. Contrast agents are often used to enhance the image contrast between the tissue of interest and surrounding tissues with MRI. The most commonly used MRI contrast agents contain Gd(III) ions. However, Gd(III) ions are highly toxic in their ionic form, as they tend to accumulate in the liver, spleen, kidney and bones and block calcium channels. Endohedral metallofullerenes such as trimetallic nitride endohedral metallofullerenes (Trimetasphere®) are one unique class of fullerene molecules where a Gd3N cluster is encapsulated inside a C80 carbon cage referred to as Gd3N@C80. These endohedral metallofullerenes have several advantages over small chelated Gd(III) complexes such as increased stability of the Gd(III) ion, minimal toxic effects, high solubility in water and high proton relativity. In this study, we describe the evaluation of gadolinium-based Trimetasphere® positive contrast agent for the ​in vitro labeling and in vivo tracking of human amniotic fluid-derived stem cells within lung tissue. In addition, we conducted a 'proof-of-concept' experiment demonstrating that this methodology can be used to track the homing of stem cells to injured lung tissue and provide longitudinal analysis of cell localization over an extended time course.


Subject(s)
Contrast Media/chemistry , Fullerenes/chemistry , Lung Injury/diagnostic imaging , Lung/diagnostic imaging , Radiation Injuries, Experimental/diagnostic imaging , Stem Cell Transplantation , Animals , Cell Proliferation , Cell Survival/drug effects , Cell Tracking , Cells, Cultured , Contrast Media/toxicity , Fullerenes/toxicity , Humans , Limit of Detection , Lung/pathology , Lung Injury/therapy , Magnetic Resonance Imaging , Metallocenes , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Phantoms, Imaging , Radiation Injuries, Experimental/therapy , Staining and Labeling , Stem Cells/physiology
2.
Tumour Biol ; 36(2): 1155-62, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25722217

ABSTRACT

Transforming growth factor-ß1 (TGF-ß1) plays a dual role in apoptosis and in proapoptotic responses in the support of survival in a variety of cells. The aim of this study was to determine the function of TGF-ß1 in bladder cancer cells and the relationship with POK erythroid myeloid ontogenic factor (Pokemon). TGF-ß1 and its receptors mediate several tumorigenic cascades that regulate cell proliferation, migration, and survival of bladder cancer cells. Bladder cancer cells T24 were treated with different levels of TGF-ß1. Levels of Pokemon, E-cadherin, Snail, MMP2, MMP9, Twist, VEGF, and ß-catenin messenger RNA (mRNA) and protein were examined by real-time quantitative fluorescent PCR and Western blot analysis, respectively. The effects of TGF-ß1 on epithelial-mesenchymal transition of T24 cells were evaluated with wound-healing assay, proliferation of T24 was evaluated with reference to growth curves with MTT assay, and cell invasive ability was investigated by Transwell assay. Data show that Pokemon was inhibited by TGF-ß1 treatment; the gene and protein of E-cadherin and ß-catenin expression level showed decreased markedly after TGF-ß1 treatment (P < 0.05). While the bladder cancer cell after TGF-ß1 treatment showed a significantly reduced wound-closing efficiency at 6, 12, and 24 h, mechanistic analyses demonstrated that different levels of TGF-ß1 promotes tumor cell growth, migration, and invasion in bladder cancer cells (P < 0.01, P < 0.05, respectively). In summary, our findings suggest that TGF-ß1 may inhibit the expression of Pokemon, ß-catenin, and E-cadherin. The high expression of TGF-ß1 leads to an increase in the phenotype and apical-base polarity of epithelial cells. These changes of cells may result in the recurrence and progression of bladder cancer at last. Related mechanism is worthy of further investigation.


Subject(s)
DNA-Binding Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Transcription Factors/biosynthesis , Transforming Growth Factor beta1/genetics , Urinary Bladder Neoplasms/genetics , Apoptosis/genetics , Cadherins/biosynthesis , Cell Line, Tumor , Cell Proliferation/genetics , DNA-Binding Proteins/metabolism , Epithelial-Mesenchymal Transition/genetics , Humans , RNA, Messenger/biosynthesis , Transcription Factors/metabolism , Transforming Growth Factor beta1/administration & dosage , Transforming Growth Factor beta1/metabolism , Urinary Bladder Neoplasms/pathology , Wound Healing , beta Catenin/biosynthesis
3.
Front Immunol ; 5: 67, 2014.
Article in English | MEDLINE | ID: mdl-24605110

ABSTRACT

Mucin 1 (MUC1) is a transmembrane mucin glycoprotein that is over-expressed and aberrantly glycosylated in >80% of human pancreatic ductal adenocarcinoma (PDA) and is associated with poor prognosis. To understand the role of MUC1 in PDA, we have recently developed two mouse models of spontaneous PDA, one that expresses full-length human MUC1 transgene (KCM mice) and one that is null for MUC1 (KCKO mice). We have previously reported that KCM mice express high levels of myeloid derived suppressor cells (MDSCs) in their tumors and develop highly aggressive PDA. To further understand the underlying mechanism for high MDSC levels in KCM-tumors, we generated primary cell lines from KCM and KCKO-tumors. In this study, we report that MDSCs derived using KCM cells express significantly higher levels of arginase 1 and inducible nitric oxide synthase (markers associated with immune suppression) and lower levels of CD115 (a marker associated with maturation of myeloid cells) as compared to KCKO-derived MDSCs. Functionally, KCM-derived MDSCs secrete significantly higher levels of urea and nitric oxide (NO) when co-cultured with normal splenic cells as compared to KCKO-derived MDSCs. Data indicates that KCM-derived MDSCs remain immature and are more suppressive as compared to KCKO-derived MDSCs. This was further corroborated in vivo where MDSCs isolated from KCM-tumor-bearing mice retained their immature state and were highly suppressive as compared to MDSCs derived from KCKO-tumor-bearing mice. Finally, we show that KCM cells secrete significantly higher levels of prostaglandin E2 (PGE2), a COX-2 metabolite and a known driver of suppressive MDSCs as compared to KCKO cells. Thus, inhibiting PGE2 with a specific COX-2 inhibitor reverses the immunosuppressive and immature phenotype of KCM-derived MDSCs. This is the first report that clearly suggests a functional role of pancreatic tumor-associated MUC1 in the development of functional MDSCs.

4.
J Interferon Cytokine Res ; 33(11): 672-81, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23777205

ABSTRACT

Breast tumor cells alter their microenvironment in part through the expression of protumor molecules that influence macrophages during tumor progression and metastasis. Macrophage recruitment is stimulated by chemotactic factors, including tumor necrosis factor alpha (TNF-α), which also stimulates the cytotoxic/tumor cell killing macrophage phenotype. Through TNF-α converting enzyme (TACE/ADAM17) activities, breast tumor cells shed membrane-bound proteins, including their TNF receptors (sTNFR1/2), which serve as decoys sequestering TNF-α and preventing TNF-α-driven apoptosis of tumor cells, thereby decreasing TNF-α bioavailability. Here we investigated the levels of sTNFRs shed by breast tumor cells and determined the effects of shed sTNFRs on macrophage migration toward TNF-α. TNF-α and sTNFRs concentrations were measured in murine normal epithelial, stromal, and mammary tumor cells. The migration of murine macrophages towards TNF-α in the presence of tumor derived soluble factors (TDSFs) shed by TACE was determined. TNF-α concentrations secreted by tumor and normal epithelial cells were below the detection limit contrasting with stromal cells, especially macrophages, which expressed higher levels of TNF-α (P<0.001). Regardless of the cell tested, treatment with the TACE inhibitor TAPI-0 led to a significant decrease in sTNFR2 shed (P<0.05). The dose-dependent macrophage migration toward TNF-α prevented by incubation with TDSFs was not observed with TDSFs collected following TAPI-0 treatment (P<0.05). Furthermore, the TNF-α-driven increased pAkt expression in macrophage was inhibited by TACE shed TDSFs (P<0.05). These results highlight the role of tumor-shed sTNFRs in TNF-α -driven macrophage chemotaxis.


Subject(s)
Breast Neoplasms/metabolism , Chemotaxis , Macrophages/cytology , Receptors, Tumor Necrosis Factor/metabolism , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/metabolism , ADAM17 Protein , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cells, Cultured , Chemotaxis/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/pharmacology , Female , Macrophages/drug effects , Mice , Solubility , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/metabolism
5.
Breast Cancer Res ; 15(2): R32, 2013 Apr 11.
Article in English | MEDLINE | ID: mdl-23577751

ABSTRACT

INTRODUCTION: Breast cancer remains the second leading cause of cancer-related deaths for women in the United States. Metastasis is regulated not only by intrinsic genetic changes in malignant cells, but also by the microenvironment, especially those associated with chronic inflammation. We recently reported that mice with autoimmune arthritis have significantly increased incidence of bone and lung metastasis and decreased survival associated with breast cancer. In this study, we evaluated the mechanism underlying the increased metastasis. METHODS: We used two mouse models; one that develops spontaneous autoimmune arthritis (SKG mice) injected with metastatic breast cancer cells (4T1), and another that develops spontaneous breast cancer (MMTV-PyV MT mice) injected with type II collagen to induce autoimmune arthritis. Mast cell levels and metastasis were monitored. RESULTS: First, we confirmed that breast tumor-bearing arthritic mice have a significantly higher incidence of bone and lung metastasis than do their nonarthritic counterparts. Next, we showed increased recruitment of mast cells within the primary tumor of arthritic mice, which facilitates metastasis. Next, we report that arthritic mice without any tumors have higher numbers of mast cells in the bones and lungs, which may be the underlying cause for the enhanced lung and bone metastases observed in the arthritic mice. Next, we showed that once the tumor cells populate the metastatic niches (bones and lungs), they further increase the mast cell population within the niche and assist in enhancing metastasis. This may primarily be due to the interaction of c-Kit receptor present on mast cells and stem cell factor (SCF, the ligand for ckit) expressed on tumor cells. Finally, we showed that targeting the SCF/cKit interaction with an anti-ckit antibody reduces the differentiation of mast cells and consequently reduces metastasis. CONCLUSION: This is the first report to show that mast cells may play a critical role in remodeling not only the tumor microenvironment but also the metastatic niche to facilitate efficient metastasis through SCF/cKit interaction in breast cancer with arthritis.


Subject(s)
Arthritis, Experimental/physiopathology , Bone Neoplasms/secondary , Breast Neoplasms/pathology , Lung Neoplasms/secondary , Mast Cells/pathology , Proto-Oncogene Proteins c-kit/metabolism , Stem Cell Factor/metabolism , Animals , Apoptosis , Arthritis, Experimental/complications , Blotting, Western , Bone Neoplasms/etiology , Bone Neoplasms/metabolism , Breast Neoplasms/etiology , Breast Neoplasms/metabolism , Cell Movement , Cell Proliferation , Female , Flow Cytometry , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Lung Neoplasms/etiology , Lung Neoplasms/metabolism , Mast Cells/metabolism , Mice , Mice, Inbred BALB C , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tumor Cells, Cultured , Tumor Microenvironment
6.
Cancer Immunol Immunother ; 61(11): 2055-65, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22543528

ABSTRACT

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibody-Dependent Cell Cytotoxicity/immunology , Carcinoma, Pancreatic Ductal/therapy , CpG Islands/immunology , Killer Cells, Natural/immunology , Mucin-1/immunology , Pancreatic Neoplasms/therapy , Adjuvants, Immunologic/therapeutic use , Animals , Carcinoma, Pancreatic Ductal/immunology , Cell Line, Tumor , Female , Humans , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Myeloid Differentiation Factor 88/immunology , Oligodeoxyribonucleotides/immunology , Pancreatic Neoplasms/immunology , Perforin/biosynthesis , Perforin/immunology , Up-Regulation/immunology
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