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Find video protocols related to scientific articles indexed in Pubmed.
Selective methioninase-induced trap of cancer cells in S/G2 phase visualized by FUCCI imaging confers chemosensitivity.
Oncotarget
PUBLISHED: 09-20-2014
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A major impediment to the response of tumors to chemotherapy is that the large majority of cancer cells within a tumor are quiescent in G0/G1, where cancer cells are resistant to chemotherapy. To attempt to solve this problem of quiescent cells in a tumor, cancer cells were treated with recombinant methioninase (rMETase), which selectively traps cancer cells in S/G2. The cell cycle phase of the cancer cells was visualized with the fluorescence ubiquitination-based cell cycle indicator cell cycle indicator (FUCCI). At the time of rMETase-induced S/G2-phase blockage, identified by the cancer cells' green fluorescence by FUCCI imaging, the cancer cells were administered S/G2-dependent chemotherapy drugs, which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil. Treatment of cancer cells with drugs only, without rMETase-induced S/G2 phase blockage, led to the majority of the cancer-cell population being blocked in G0/G1 phase, identified by the cancer cells becoming red fluorescent in the FUCCI system. The G0/G1 blocked cells were resistant to the chemotherapy. In contrast, trapping of cancer cells in S/G2 phase by rMETase treatment followed by FUCCI-imaging-guided chemotherapy was highly effective in killing the cancer cells.
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Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX) and cell line mouse models.
Oncotarget
PUBLISHED: 09-17-2014
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The aim of the present study was to examine the efficacy of tumor-targeting Salmonella typhimurium A1-R treatment following anti-vascular endothelial growth factor (VEGF) therapy on VEGF-positive human pancreatic cancer. A pancreatic cancer patient-derived orthotopic xenograft (PDOX) that was VEGF-positive and an orthotopic VEGF-positive human pancreatic cancer cell line (MiaPaCa-2-GFP) as well as a VEGF-negative cell line (Panc-1) were tested. Nude mice with these tumors were treated with gemcitabine (GEM), bevacizumab (BEV), and S. typhimurium A1-R. BEV/GEM followed by S. typhimurium A1-R significantly reduced tumor weight compared to BEV/GEM treatment alone in the PDOX and MiaPaCa-2 models. Neither treatment was as effective in the VEGF-negative model as in the VEGF-positive models. These results demonstrate that S. typhimurium A1-R following anti-angiogenic therapy is effective on pancreatic cancer including the PDOX model, suggesting its clinical potential.
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Tumor-targeting Salmonella typhimurium A1-R prevents experimental human breast cancer bone metastasis in nude mice.
Oncotarget
PUBLISHED: 09-13-2014
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Bone metastasis is a lethal and morbid late stage of breast cancer that is currently treatment resistant. More effective mouse models and treatment are necessary. High bone-metastatic variants of human breast cancer cells were selected in nude mice by cardiac injection. After cardiac injection of a high bone-metastatic variant of breast cancer, all untreated mice had bone metastases compared to only 20% with parental cells. Treatment with tumor-targeting Salmonella typhimurium A1-R completely prevented the appearance of bone metastasis of the high metastatic variant in nude mice (P < 0.001). After injection of the highly bone-metastatic breast cancer variant to the tibia of nude mice, S. typhimurium A1-R treatment significantly reduced tumor growth in the bone (P < 0.001). These data indicated that S. typhimurium A1-R is useful to prevent and inhibit breast cancer bone metastasis and should be of future clinical use for breast cancer in the adjuvant setting.
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In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.
J Fluoresc
PUBLISHED: 08-22-2014
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In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.
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Fluorescence-guided surgery improves outcome in an orthotopic osteosarcoma nude-mouse model.
J. Orthop. Res.
PUBLISHED: 08-19-2014
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In order to develop a model for fluorescence-guided surgery (FGS), 143B human osteosarcoma cells expressing red fluorescent protein (RFP) were injected into the intramedullary cavity of the tibia in nude mice. The fluorescent areas of residual tumors after bright-light surgery (BLS) and FGS were 10.2?±?2.4?mm(2) and 0.1?±?0.1?mm(2) , respectively (p?
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The tumor-educated-macrophage increase of malignancy of human pancreatic cancer is prevented by zoledronic acid.
PLoS ONE
PUBLISHED: 08-12-2014
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We previously defined macrophages harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as "tumor-educated-macrophages" (Edu) and macrophages harvested from mice without tumors as "naïve-macrophages" (Naïve), and demonstrated that Edu-macrophages promoted tumor growth and metastasis. In this study, Edu- and Naïve-macrophages were compared for their ability to enhance pancreatic cancer malignancy at the cellular level in vitro and in vivo. The inhibitory efficacy of Zoledronic acid (ZA) on Edu-macrophage-enhanced metastasis was also determined. XPA1 human pancreatic cancer cells in Gelfoam co-cultured with Edu-macrophages proliferated to a greater extent compared to XPA1 cells cultured with Naïve-macrophages (P = 0.014). XPA1 cells exposed to conditioned medium harvested from Edu culture significantly increased proliferation (P = 0.016) and had more migration stimulation capability (P<0.001) compared to cultured cancer cells treated with the conditioned medium from Naïve. The mitotic index of the XPA1 cells, expressing GFP in the nucleus and RFP in the cytoplasm, significantly increased in vivo in the presence of Edu- compared to Naïve-macrophages (P = 0.001). Zoledronic acid (ZA) killed both Edu and Naïve in vitro. Edu promoted tumor growth and metastasis in an orthotopic mouse model of the XPA1 human pancreatic cancer cell line. ZA reduced primary tumor growth (P = 0.006) and prevented metastasis (P = 0.025) promoted by Edu-macrophages. These results indicate that ZA inhibits enhanced primary tumor growth and metastasis of human pancreatic cancer induced by Edu-macrophages.
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Ratiometric Activatable Cell-Penetrating Peptides Label Pancreatic Cancer, Enabling Fluorescence-Guided Surgery, Which Reduces Metastases and Recurrence in Orthotopic Mouse Models.
Ann. Surg. Oncol.
PUBLISHED: 08-11-2014
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The aim of this study was to evaluate the efficacy of using matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9)-cleavable ratiometric activatable cell-penetrating peptides (RACPPs) conjugated to Cy5 and Cy7 fluorophores to accurately label pancreatic cancer for fluorescence-guided surgery (FGS) in an orthotopic mouse model.
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Transgenic nude mice ubiquitously expressing fluorescent proteins for color-coded imaging of the tumor microenvironment.
Methods Mol. Biol.
PUBLISHED: 07-28-2014
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We have developed a transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the ?-actin promoter drives GFP expression in essentially all tissues. In the adult mice, many organs brightly expressed GFP, including the spleen, heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum as well as the circulatory system. The liver expressed GFP at a lesser level. The red fluorescent protein (RFP) transgenic nude mouse was obtained by crossing non-transgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives RFP (DsRed2) expression in essentially all tissues. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, liver, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. The cyan fluorescent protein (CFP) nude mouse was developed by crossing nontransgenic nude mice with the transgenic CK/ECFP mouse in which the ?-actin promoter drives expression of CFP in almost all tissues. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescence signals of all internal organs, which vary in intensity. The GFP, RFP, and CFP nude mice when transplanted with cancer cells of another color are powerful models for color-coded imaging of the tumor microenvironment (TME) at the cellular level.
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Selective efficacy of zoledronic acid on metastasis in a patient-derived orthotopic xenograph (PDOX) nude-mouse model of human pancreatic cancer.
J Surg Oncol
PUBLISHED: 07-13-2014
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Patient-derived orthotopic xenograft (PDOX) nude-mouse models replicate the behavior of clinical cancer, including metastasis. The objective of the study was to determine the efficacy of zoledronic acid (ZA) on metastasis of a patient-derived orthotopic xenograft (PDOX) nude-mouse model of pancreatic cancer.
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Protein-tyrosine pseudokinase 7 (PTK7) directs cancer cell motility and metastasis.
J. Biol. Chem.
PUBLISHED: 07-08-2014
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It is well established that widely expressed PTK7 is essential for vertebrate tissue morphogenesis. In cancer, the functionality of PTK7 is selectively regulated by membrane type-1 matrix metalloproteinase (MT1-MMP), ADAMs (a disintegrin domain and metalloproteinases), and ?-secretase proteolysis. Here, we established that the full-length membrane PTK7, its Chuzhoi mutant with the two functional MT1-MMP cleavage sites, and its L622D mutant with the single inactivated MT1-MMP cleavage site differentially regulate cell motility in a two-dimensional versus three-dimensional environment. We also demonstrated that in polarized cancer cells, the levels of PTK7 expression and proteolysis were directly linked to the structure and kinetics of cell protrusions, including lamellipodia and invadopodia. In the functionally relevant and widely accepted animal models of metastasis, mouse and chick embryo models, both the overexpression and knock-out of PTK7 in HT1080 cells abrogated metastatic dissemination. Our analysis of human tissue specimens confirmed intensive proteolysis of PTK7 in colorectal cancer tumors, but not in matching normal tissue. Our results provide convincing evidence that both PTK7 expression and proteolysis, rather than the level of the cellular full-length PTK7 alone, contribute to efficient directional cell motility and metastasis in cancer.
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Methods for the development of tumor-targeting bacteria.
Expert Opin Drug Discov
PUBLISHED: 06-21-2014
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For at least two centuries, there have been reports that cancer patients infected with various bacteria had what appeared to be spontaneous remission. In the late nineteenth and early twentieth centuries, W.B. Coley, of what is now the Memorial Sloan-Kettering Cancer Center, pioneered bacterial therapy of cancer in the clinic with considerable success. After Coley died in 1936, bacterial therapy of cancer started to go out of favor. In the current twenty-first century, there is great resurgent interest in developing bacterial therapy for treating cancer using either obligate or facultative anaerobic bacteria. There is also controversy about which bacteria are optimum for cancer treatment and whether bacteria should be used as tumor-targeting vectors, immune stimulators, or for direct tumor killing.
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Overactivated neddylation pathway as a therapeutic target in lung cancer.
J. Natl. Cancer Inst.
PUBLISHED: 06-01-2014
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A number of oncoproteins and tumor suppressors are known to be neddylated, but whether the neddylation pathway is entirely activated in human cancer remains unexplored.
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Efficacy of tumor-targeting Salmonella typhimurium A1-R on nude mouse models of metastatic and disseminated human ovarian cancer.
J. Cell. Biochem.
PUBLISHED: 05-09-2014
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We report here the efficacy of tumor-targeting Salmonella typhimurium A1-R (A1-R) on mouse models of disseminated and metastatic ovarian cancer. The proliferation-inhibitory efficacy of A1-R on human ovarian cancer cell lines (SKOV-3-GFP, OVCAR-3-RFP) was initially demonstrated in vitro. Orthotopic and dissemination mouse models of ovarian cancer were made with the human ovarian cancer cell line SKOV-3-GFP. After tumor implantation, the mice were treated with A1-R (5?×?10(7) ?colony-forming units [CFU], i.v.), and there were no severe adverse events observed. In the orthotopic model, tumor volume after treatment was 276?±?60.8?mm(3), compared to 930?±?342?mm(3) in the untreated control group (P?=?0.022). There was also a significant difference in survival between treated mice and untreated mice in a peritoneal dissemination model (P?=?0.005). The results of this report demonstrate that A1-R is effective for highly aggressive human ovarian cancer in metastatic and dissemination mouse models and suggest its clinical potential for this highly treatment-resistant disease.
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Spatial-temporal FUCCI imaging of each cell in a tumor demonstrates locational dependence of cell cycle dynamics and chemoresponsiveness.
Cell Cycle
PUBLISHED: 05-08-2014
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The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We report here on the results of monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI) before, during, and after chemotherapy. In nascent tumors in nude mice, approximately 30% of the cells in the center of the tumor are in G?/G? and 70% in S/G?/M. In contrast, approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G?/G? phase. Similarly, approximately 75% of cancer cells far from (> 100 µm) tumor blood vessels of an established tumor are in G?/G?. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Our results suggest why most drugs currently in clinical use, which target cancer cells in S/G?/M, are mostly ineffective on solid tumors. The results also suggest that drugs that target quiescent cancer cells are urgently needed.
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Imaging metastatic cell trafficking at the cellular level in vivo with fluorescent proteins.
Methods Mol. Biol.
PUBLISHED: 04-24-2014
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Fluorescent proteins have revolutionized biology, allowing what was formerly invisible to be clearly seen. The Nobel Prize in Chemistry was awarded in 2008 for the discovery and early use of green fluorescent protein (GFP) as a genetic reporter. Our laboratory pioneered the use of GFP for in vivo imaging. In this chapter we review the developments within our research on subcellular imaging of metastatic trafficking of cancer cells carried out in real time in mice. Dual-color fluorescent cells, with one color fluorescent protein in the nucleus and another color fluorescent protein in the cytoplasm, enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. In the dual-color cells, red fluorescent protein (RFP) is expressed in the cytoplasm of cancer cells, and GFP is linked to histone H2B and is expressed in the nucleus. Nuclear GFP expression enables visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression allows visualization of nuclear cytoplasmic ratios in addition to simultaneous cell and nuclear shape changes. With the use of dual-color fluorescent cells, it is possible to achieve subcellular real-time imaging of cancer cell trafficking in live mice. Extravasation can also be imaged in real time. Dual-color imaging has shown that cytoplasmic processes of cancer cells exit the vessels first, with nuclei following along the cytoplasmic projections [Yamauchi et al., Cancer Res 66:4208-4214, 2006]. Dual-color in vivo cellular imaging was used to visualize cancer cell trafficking blood vessels, as well as in the lymphatic systems of the mice. The real-time imaging of cancer cell seeding on the lung has now been achieved with dual-color cells. Subcellular in vivo imaging confers great promise for understanding metastasis at the cellular level in vivo.
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Imageable clinically relevant mouse models of metastasis.
Methods Mol. Biol.
PUBLISHED: 04-24-2014
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In the past 10 years, we have developed a new approach to the development of a clinically accurate rodent model for human cancer based on our invention of surgical orthotopic implantation (SOI). The SOI models have been described in approx. 70 publications and in 4 patents.*SOI allows human tumors of all the major types of human cancer to reproduce clinical like tumor growth and metastasis in the transplanted rodents. The major features of the SOI models are reviewed here and also compared to transgenic mouse models of cancer.
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Comparison of UVB and UVC effects on the DNA damage-response protein 53BP1 in human pancreatic cancer.
J. Cell. Biochem.
PUBLISHED: 04-23-2014
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We have previously demonstrated that ultraviolet (UV) light is effective against a variety of cancer cells expressing fluorescent proteins in vivo as well as in vitro. In the present report, we compared the DNA damage repair (DDR) response of pancreatic cancer cells after UVB or UVC irradiation. The UV-induced DNA damage repair was imaged with green fluorescent protein (GFP) fused to the DDR-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells growing in 3D Gelfoam® histoculture and in superficial tumors grown in nude mice. 53BP1-GFP forms foci during DNA damage repair. A clonogenic assay in 2D monolayer culture initially showed that UVC and UVB inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, with UVC having more efficacy. Three-dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP foci to be observed within 1?h after UV irradiation, indicating the onset of DDR response. UVB-induced 53BP1-GFP focus formation was observed up to a depth of 120?µm in MiaPaCa-2 cells on Gelfoam® compared to 80?µm for UVC. UVB-induced 53BP1-GFP focus formation was observed up to a depth of 80?µm in MiaPaCa-2 cells, implanted within skin flaps in mice, at a significantly greater extent than UVC. MiaPaCa-2 cells irradiated by UVB or UVC in the skin-flap mouse model had a significant decrease in tumor growth compared to untreated controls with UVB having more efficacy than UVC. Our results demonstrate that UVB has greater tissue penetration than UVC because of its longer wavelength and has clinical potential for eradicating superficial cancer.
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Specificity of lectin-immobilized fluorescent nanospheres for colorectal tumors in a mouse model which better resembles the clinical disease.
Contrast Media Mol Imaging
PUBLISHED: 03-17-2014
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We have been investigating an imaging agent that enables real-time and accurate diagnosis of early colorectal cancer at the intestinal mucosa by colonoscopy. The imaging agent is peanut agglutinin-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) chains encapsulating coumarin 6. Intracolonically-administered lectin-immobilized fluorescent nanospheres detect tumor-derived changes through molecular recognition of lectin for the terminal sugar of cancer-specific antigens on the mucosal surface. The focus of the present study was to evaluate imaging abilities of the nanospheres in animal models that reflect clinical environments. We previously developed an orthotopic mouse model with human colorectal tumors growing on the mucosa of the descending colon to better resemble the clinical disease. The entire colon of the mice in the exposed abdomen was monitored in real time with an in vivo imaging apparatus. Fluorescence from the nanospheres was observed along the entire descending colon after intracolonical administration from the anus. When the luminal side of the colon was washed with phosphate-buffered saline, most of the nanospheres were flushed. However, fluorescence persisted in areas where cancer cells were implanted. Histological evaluation demonstrated that tumors were present in the mucosal epithelia where the nanospheres fluoresced. In contrast, no fluorescence was observed when control mice, without tumors were tested. The lectin-immobilized fluorescent nanospheres were tumor-specific and remained bound to tumors even after vigorous washing. The nanospheres nonspecifically bound to normal mucosa were easily removed through mild washing. These results indicate that the nanospheres combined with colonoscopy, will be a clinically-valuable diagnostic tool for early-stage primary colon carcinoma. Copyright © 2014 John Wiley & Sons, Ltd.
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Specific tumor labeling enhanced by polyethylene glycol linkage of near infrared dyes conjugated to a chimeric anti-carcinoembryonic antigen antibody in a nude mouse model of human pancreatic cancer.
J Biomed Opt
PUBLISHED: 03-16-2014
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Labeling of metastatic tumors can aid in their staging and resection of cancer. Near infrared (NIR) dyes have been used in the clinic for tumor labeling. However, there can be a nonspecific uptake of dye by the liver, lungs, and lymph nodes, which hinders detection of metastasis. In order to overcome these problems, we have used two NIR dyes (DyLight 650 and 750) conjugated to a chimeric anti-carcinoembryonic antigen antibody to evaluate how polyethylene glycol linkage (PEGylation) can improve specific tumor labeling in a nude mouse model of human pancreatic cancer. The conjugated PEGylated and non-PEGylated DyLight 650 and 750 dyes were injected intravenously into non-tumor-bearing nude mice. Serum samples were collected at various time points in order to determine serum concentrations and elimination kinetics. Conjugated PEGylated dyes had significantly higher serum dye concentrations than non-PEGylated dyes (p=0.005 for the 650 dyes and p<0.001 for the 750 dyes). Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with conjugated PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (p<0.001 for the 650 dyes; p=0.01 for 750 dyes). PEGylation of the NIR dyes also decreased their accumulation in lymph nodes, liver, and lung. These results demonstrate enhanced selective tumor labeling by PEGylation of dyes conjugated to a tumor-specific antibody, suggesting their future clinical use in fluorescence-guided surgery.
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Advantages of fluorescence-guided laparoscopic surgery of pancreatic cancer labeled with fluorescent anti-carcinoembryonic antigen antibodies in an orthotopic mouse model.
J. Am. Coll. Surg.
PUBLISHED: 02-24-2014
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Our laboratory has previously developed fluorescence-guided surgery of pancreatic and other cancers in orthotopic mouse models. Laparoscopic surgery is being used more extensively in surgical oncology. This report describes the efficacy of laparoscopic fluorescence-guided surgery of pancreatic cancer in an orthotopic mouse model.
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Osteosarcoma cells enhance angiogenesis visualized by color-coded imaging in the in vivo Gelfoam® assay.
J. Cell. Biochem.
PUBLISHED: 02-24-2014
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We previously described a color-coded imaging model that can quantify the length of nascent blood vessels using Gelfoam® implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice. In ND-GFP mice, nascent blood vessels are labeled with GFP. We report here that osteosarcoma cells promote angiogenesis in the Gelfoam® angiogenesis assay in ND-GFP mice. Gelfoam® was initially transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation of Gelfoam®, skin flaps were made and human 143B osteosarcoma cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in cytoplasm were injected into the transplanted Gelfoam®. The control-group mice had only implanted Gelfoam®. Skin flaps were made at days 14, 21, and 28 after transplantation of the Gelfoam® to allow imaging of vascularization in the Gelfoam® using a variable-magnification small animal imaging system and confocal fluorescence microscopy. ND-GFP expressing nascent blood vessels penetrated and spread into the Gelfoam® in a time-dependent manner in both control and osteosarcoma-implanted mice. ND-GFP expressing blood vessels in the Gelfoam® of the osteosarcoma-implanted mice were associated with the cancer cells and larger and longer than in the Gelfoam®-only implanted mice (P?
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Fluorescence-guided surgery of prostate cancer bone metastasis.
J. Surg. Res.
PUBLISHED: 02-12-2014
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The aim of this study is to investigate the effectiveness of fluorescence-guided surgery (FGS) of prostate cancer experimental skeletal metastasis.
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Fluorescent angiogenesis models using gelfoam® implanted in transgenic mice expressing fluorescent proteins.
Methods Mol. Biol.
PUBLISHED: 02-11-2014
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Fidler's group described an in vivo angiogenesis assay utilizing Gelfoam(®) sponges impregnated with agarose and proangiogenic factors. Vessels were detected by staining with fluorescent antibodies against CD31. We showed that Gelfoam(®) implanted in transgenic mice expressing the nestin promoter-driven green fluorescent protein (ND-GFP mice) was rapidly vascularized with ND-GFP-expressing nascent blood vessels. Angiogenesis in the Gelfoam(®) was quantified by measuring the total length of ND-GFP-expressing nascent blood vessels in a skin flap by in vivo fluorescence microscopy imaging. The ND-GFP-expressing nascent blood vessels formed a network on the surface of the basic fibroblast growth factor (bFGF)-treated Gelfoam(®). We then developed a color-coded imaging model that can visualize the interaction between ?v integrin linked to green fluorescent protein (GFP) in osteosarcoma cells and blood vessels in Gelfoam(®) vascularized after implantation in red fluorescent protein (RFP) transgenic nude mice. The implanted Gelfoam(®) became highly vascularized with RFP-expressing vessels in 14 days. 143B osteosarcoma cells expressing ?v integrin-GFP were injected into the Gelfoam(®) after transplantation of Gelfoam(®). After cancer cell injection, cancer cells interacting with blood vessels were observed in the Gelfoam(®) by color-coded confocal microscopy through the skin flap window. We developed another color-coded Gelfoam(®)-based imaging model that can visualize the anastomosis between blood vessels. RFP-expressing vessels in vascularized Gelfoam(®), previously transplanted into RFP transgenic mice, were re-transplanted into ND-GFP mice. Skin flaps were made and anastomosis between the GFP-expressing nascent blood vessels of ND-GFP transgenic nude mice and RFP blood vessels in the transplanted Gelfoam(®) could be imaged. Our results demonstrate that the Gelfoam(®) in vivo angiogenesis model in combination with fluorescent protein labeling of blood vessels is a powerful system for use in the discovery and evaluation of agents influencing vascularization.
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Fluorescence-guided surgery with a fluorophore-conjugated antibody to carcinoembryonic antigen (CEA), that highlights the tumor, improves surgical resection and increases survival in orthotopic mouse models of human pancreatic cancer.
Ann. Surg. Oncol.
PUBLISHED: 02-06-2014
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We have developed a method of distinguishing normal tissue from pancreatic cancer in vivo using fluorophore-conjugated antibody to carcinoembryonic antigen (CEA). The objective of this study was to evaluate whether fluorescence-guided surgery (FGS) with a fluorophore-conjugated antibody to CEA, to highlight the tumor, can improve surgical resection and increase disease-free survival (DFS) and overall survival (OS) in orthotopic mouse models of human pancreatic cancer.
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Successful fluorescence-guided surgery on human colon cancer patient-derived orthotopic xenograft mouse models using a fluorophore-conjugated anti-CEA antibody and a portable imaging system.
J Laparoendosc Adv Surg Tech A
PUBLISHED: 02-04-2014
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Fluorescence-guided surgery (FGS) can enable successful cancer surgery where bright-light surgery often cannot. There are three important issues for FGS going forward toward the clinic: (a) proper tumor labeling, (b) a simple portable imaging system for the operating room, and (c) patient-like mouse models in which to develop the technology. The present report addresses all three.
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3-dimensional tissue is formed from cancer cells in vitro on Gelfoam®, but not on Matrigel™.
J. Cell. Biochem.
PUBLISHED: 01-27-2014
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Cell and tissue culture can be performed on different substrates such as on plastic, in Matrigel™, and on Gelfoam(®), a sponge matrix. Each of these substrates consists of a very different surface, ranging from hard and inflexible, a gel, and a sponge-matrix, respectively. Folkman and Moscona found that cell shape was tightly coupled to DNA synthesis and cell growth. Therefore, the flexibility of a substrate is important for cells to maintain their optimal shape. Human osteosarcoma cells, stably expressing a fusion protein of ?(v) integrin and green fluorescent protein (GFP), grew as a simple monolayer without any structure formation on the surface of a plastic dish. When the osteosarcoma cells were cultured within Matrigel™, the cancer cells formed colonies but no other structures. When the cancer cells were seeded on Gelfoam(®), the cells formed three-dimensional tissue-like structures. The behavior of 143B osteosarcoma cells on Gelfoam(®) in culture is remarkably different from those of these cells in monolayer culture or in Matrigel™. Tissue-like structures were observed only in Gelfoam(®) culture. The data in this report suggest a flexible structural substrate such as Gelfoam(®) provides a more in vivo-like culture condition than monolayer culture or Matrigel(TM) and that Matrigel(TM) does not result in actual three-dimensional culture.
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Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging.
Cell Cycle
PUBLISHED: 01-20-2014
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Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G0/G1 cells express a red fluorescent protein and S/G2/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G0/G1 phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G2/M phases. Cancer cells ceased migrating when they entered S/G2/M phases and restarted migrating after cell division when the cells re-entered G0/G1. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G0/G1, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G0/G1 cancer cells should be developed to prevent metastasis.
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Efficacy of Salmonella typhimurium A1-R versus chemotherapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX).
J. Cell. Biochem.
PUBLISHED: 01-13-2014
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The aim of this study is to determine the efficacy of tumor-targeting Salmonella typhimurium A1-R (A1-R) on pancreatic cancer patient-derived orthotopic xenografts (PDOX). The PDOX model was originally established from a pancreatic cancer patient in SCID-NOD mice. The pancreatic cancer PDOX was subsequently transplanted by surgical orthotopic implantation (SOI) in transgenic nude red fluorescent protein (RFP) mice in order that the PDOX stably acquired red fluorescent protein (RFP)-expressing stroma for the purpose of imaging the tumor after passage to non-transgenic nude mice in order to visualize tumor growth and drug efficacy. The nude mice with human pancreatic PDOX were treated with A1-R or standard chemotherapy, including gemcitabine (GEM), which is first-line therapy for pancreatic cancer, for comparison of efficacy. A1-R treatment significantly reduced tumor weight, as well as tumor fluorescence area, compared to untreated control (P?=?0.011), with comparable efficacy of GEM, CDDP, and 5-FU. Histopathological response to treatment was defined according to Evans's criteria and A1-R had increased efficacy compared to standard chemotherapy. The present report is the first to show that A1-R is effective against a very low-passage patient tumor, in this case, pancreatic cancer. The data of the present report suggest A1-1 will have clinical activity in pancreatic cancer, a highly lethal and treatment-resistant disease and may be most effectively used in combination with other agents.
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Comparison of Efficacy and Toxicity of Traditional Chinese Medicine (TCM) Herbal Mixture LQ and Conventional Chemotherapy on Lung Cancer Metastasis and Survival in Mouse Models.
PLoS ONE
PUBLISHED: 01-01-2014
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Unlike Western medicine that generally uses purified compounds and aims to target a single molecule or pathway, traditional Chinese medicine (TCM) compositions usually comprise multiple herbs and components that are necessary for efficacy. Despite the very long-time and wide-spread use of TCM, there are very few direct comparisons of TCM and standard cytotoxic chemotherapy. In the present report, we compared the efficacy of the TCM herbal mixture LQ against lung cancer in mouse models with doxorubicin (DOX) and cyclophosphamide (CTX). LQ inhibited tumor size and weight measured directly as well as by fluorescent-protein imaging in subcutaneous, orthotopic, spontaneous experimental metastasis and angiogenesis mouse models of lung cancer. LQ was efficacious against primary and metastatic lung cancer without weight loss and organ toxicity. In contrast, CTX and DOX, although efficacious in the lung cancer models caused significant weight loss, and organ toxicity. LQ also had anti-angiogenic activity as observed in lung tumors growing in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Survival of tumor-bearing mice was also prolonged by LQ, comparable to DOX. In vitro, lung cancer cells were killed by LQ as observed by time-lapse imaging, comparable to cisplatinum. LQ was more potent to induce cell death on cancer cell lines than normal cell lines unlike cytotoxic chemotherapy. The results indicate that LQ has non-toxic efficacy against metastatic lung cancer.
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Fluorescence-guided surgery in combination with UVC irradiation cures metastatic human pancreatic cancer in orthotopic mouse models.
PLoS ONE
PUBLISHED: 01-01-2014
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The aim of this study is to determine if ultraviolet light (UVC) irradiation in combination with fluorescence-guided surgery (FGS) can eradicate metastatic human pancreatic cancer in orthotopic nude-mouse models. Two weeks after orthotopic implantation of human MiaPaCa-2 pancreatic cancer cells, expressing green fluorescent protein (GFP), in nude mice, bright-light surgery (BLS) was performed on all tumor-bearing mice (n?=?24). After BLS, mice were randomized into 3 treatment groups; BLS-only (n?=?8) or FGS (n?=?8) or FGS-UVC (n?=?8). The residual tumors were resected using a hand-held portable imaging system under fluorescence navigation in mice treated with FGS and FGS-UVC. The surgical resection bed was irradiated with 2700 J/m2 UVC (254 nm) in the mice treated with FGS-UVC. The average residual tumor area after FGS (n?=?16) was significantly smaller than after BLS only (n?=?24) (0.135±0.137 mm2 and 3.338±2.929 mm2, respectively; p?=?0.007). The BLS treated mice had significantly reduced survival compared to FGS- and FGS-UVC-treated mice for both relapse-free survival (RFS) (p<0.001 and p<0.001, respectively) and overall survival (OS) (p<0.001 and p<0.001, respectively). FGS-UVC-treated mice had increased RFS and OS compared to FGS-only treated mice (p?=?0.008 and p?=?0.025, respectively); with RFS lasting at least 150 days indicating the animals were cured. The results of the present study suggest that UVC irradiation in combination with FGS has clinical potential to increase survival.
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Polyethylene glycol (PEG) linked to near infrared (NIR) dyes conjugated to chimeric anti-carcinoembryonic antigen (CEA) antibody enhances imaging of liver metastases in a nude-mouse model of human colon cancer.
PLoS ONE
PUBLISHED: 01-01-2014
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We report here that polyethylene glycol (PEG) linked to near infrared dyes conjugated to chimeric mouse-human anti-carcinoembryonic antigen (CEA) antibody greatly improves imaging of liver metastases in a nude mouse model of colon-cancer experimental metastases. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to the chimeric anti-CEA antibody. The dyes were initially injected intravenously into nude mice without tumors. Tissue biodistribution was determined by tissue sonication and analyzing tissue dye concentration profiles over time. PEGylated dyes had significantly lower accumulation in the liver (p?=?0.03 for the 650 dyes; p?=?0.002 for the 750 dyes) compared to non-PEGylated dyes. In an experimental liver metastasis model of HT-29 colon cancer, PEGylated dyes conjugated to the anti-CEA antibody showed good labeling of metastatic tumors with high contrast between normal and malignant tissue which was not possible with the non-PEGylated dyes since there was so much non-specific accumulation in the liver. PEGylation of the DyLight 650 and 750 NIR dyes significantly altered tissue biodistribution, allowing brighter tissue labeling, decreased accumulation in normal organs, particularly the liver. This enabled high fidelity and high contrast imaging of liver metastases.
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Parathyroid hormone related-protein promotes epithelial-to-mesenchymal transition in prostate cancer.
PLoS ONE
PUBLISHED: 01-01-2014
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Parathyroid hormone-related protein (PTHrP) possesses a variety of physiological and developmental functions and is also known to facilitate the progression of many common cancers, notably their skeletal invasion, primarily by increasing bone resorption. The purpose of this study was to determine whether PTHrP could promote epithelial-to-mesenchymal transition (EMT), a process implicated in cancer stem cells that is critically involved in cancer invasion and metastasis. EMT was observed in DU 145 prostate cancer cells stably overexpressing either the 1-141 or 1-173 isoform of PTHrP, where there was upregulation of Snail and vimentin and downregulation of E-cadherin relative to parental DU 145. By contrast, the opposite effect was observed in PC-3 prostate cancer cells where high levels of PTHrP were knocked-down via lentiviral siRNA transduction. Increased tumor progression was observed in PTHrP-overexpressing DU 145 cells while decreased progression was observed in PTHrP-knockdown PC-3 cells. PTHrP-overexpressing DU 145 formed larger tumors when implanted orthoptopically into nude mice and in one case resulted in spinal metastasis, an effect not observed among mice injected with parental DU 145 cells. PTHrP-overexpressing DU 145 cells also caused significant bone destruction when injected into the tibiae of nude mice, while parental DU 145 cells caused little to no destruction of bone. Together, these results suggest that PTHrP may work through EMT to promote an aggressive and metastatic phenotype in prostate cancer, a pathway of importance in cancer stem cells. Thus, continued efforts to elucidate the pathways involved in PTHrP-induced EMT as well as to develop ways to specifically target PTHrP signaling may lead to more effective therapies for prostate cancer.
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Nestin-Expressing Stem Cells from the Hair Follicle Can Differentiate Into Motor Neurons and Reduce Muscle Atrophy after Transplantation to Injured Nerves.
Tissue Eng Part A
PUBLISHED: 10-19-2013
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We have previously shown that nestin-expressing hair follicle stem cells from the mouse and human are multipotent and can differentiate into many cell types, including neurons and glial cells. The nestin-expressing hair follicle stem cells can effect nerve and spinal cord repair upon transplantation in mouse models. In the present study, nestin-expressing hair follicle stem cells expressing red fluorescent protein (RFP) were induced by retinoic acid and fetal bovine serum to differentiate and then transplanted together with Matrigel into the transected distal sciatic or tibial nerve stump of transgenic nude mice ubiquitously expressing green fluorescent protein (GFP). Control mice were transplanted with Matrigel only. The transplanted cells appeared neuron like, with large round nuclei and long extensions. Immunofluorescence staining showed that some of the transplanted cells in the distal nerve stump expressed the neuron marker Tuj1 as well as motor neuron markers Isl 1/2 and EN1. These transplanted cells contacted each other as well as host nerve fibers. Two weeks post-transplantation, nerve fibers in the distal sciatic nerve stump of the transplanted mice had greater expression of motor neuron markers and neurotrophic factor-3 than those in the Matrigel-only transplanted mice. Muscle fiber areas in the nestin-expressing stem cell plus Matrigel-transplanted animals were much bigger than that in the Matrigel-only transplanted animals after 4 weeks. The present results suggest that transplanted nestin-expressing hair follicle stem cells can differentiate into motor neurons and reduce muscle atrophy after sciatic nerve transection. This study demonstrates a new and accessible neuron source to reduce muscle atrophy after nerve injury.
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Imaging nuclear - cytoplasm dynamics of cancer cells in the intravascular niche of live mice.
Anticancer Res.
PUBLISHED: 10-15-2013
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We have previously shown that cancer cells can form an intravascular niche where they can proliferate and undergo apoptosis as well as traffic and extravasate. In the present study, green fluorescent protein (GFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and red fluorescent protein (mCherry), linked to histone H2B, was expressed in the nucleus to further investigate intravascular cancer cell nuclear-cytoplasmic dynamics. Nuclear mCherry expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic GFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear deformation. Cancer cells were injected in the epigastric cranialis vein in an abdominal flap of nude mice to enable subcellular in vivo imaging. The cell cycle position of individual living cells was readily-visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Intra- and extra-vascular mitotic cells were visualized by imaging. One hour after cell injection, round and elongated cancer cells were observed in the vessels. Three hours after injection, invadopodia-like structures of the cancer cells were observed. Five hours after injection, dual-color cancer cells began to divide within the vessel. By 10 h, some intravascular cancer cells underwent apoptosis. Deformed new blood vessels in the tumor were observed 10 days later. Extravascular cancer cells were imaged dividing in the tumor at day 14 after injection. The subcellular in vivo imaging approach described in the present report provides new visual targets for trafficking and proliferating intravascular cancer cells as well as extravasating and invading cancer cells.
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A Genetically Engineered Oncolytic Adenovirus Decoys and Lethally Traps Quiescent Cancer Stem-like Cells in S/G2/M Phases.
Clin. Cancer Res.
PUBLISHED: 09-30-2013
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Because chemoradiotherapy selectively targets proliferating cancer cells, quiescent cancer stem-like cells are resistant. Mobilization of the cell cycle in quiescent leukemia stem cells sensitizes them to cell death signals. However, it is unclear that mobilization of the cell cycle can eliminate quiescent cancer stem-like cells in solid cancers. Thus, we explored the use of a genetically-engineered telomerase-specific oncolytic adenovirus, OBP-301, to mobilize the cell cycle and kill quiescent cancer stem-like cells.
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Hand-held high-resolution fluorescence imaging system for fluorescence-guided surgery of patient and cell-line pancreatic tumors growing orthotopically in nude mice.
J. Surg. Res.
PUBLISHED: 09-15-2013
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In this study, we investigated the advantages of fluorescence-guided surgery (FGS) in mice of a portable hand-sized imaging system compared with a large fluorescence imaging system or a long-working-distance fluorescence microscope.
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High lung-metastatic variant of human osteosarcoma cells, selected by passage of lung metastasis in nude mice, is associated with increased expression of ?(v)?(3) integrin.
Anticancer Res.
PUBLISHED: 09-12-2013
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Altered expression of ?v?3 integrin is associated with tumor progression and metastasis in several types of cancer, including metastatic osteosarcoma. In this study, we demonstrate that in vivo passaging of lung metastasis in nude mice can generate an aggressive variant of human osteosarcoma cells. Experimental metastases were established by injecting 143B human osteosarcoma cells, expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm, in the tail vein of nude mice. Lung metastases were harvested under fluorescence microscopy from nude mice to establish cell lines which were then injected via the tail vein of additional nude mice. This procedure was repeated for four passages in order to isolate highly metastatic variant sublines. When the parental and metastatic variants were transplanted orthotopically into the tibia of nude mice, the 143B-LM4 variant had the highest metastatic rate, approximately 18-fold higher than the parent (p<0.01). ?v?3 integrin expression was increased approximately 5.6-fold in 143B-LM4 compared to parental cells (p<0.05). Thus, serial passage of lung metastases created a highly metastatic variant of human osteosarcoma cells which had increased expression of ?v?3 integrin, suggesting that ?v?3 integrin plays an essential role in osteosarcoma metastasis. With this highly metastatic variant overexpressing ?v?3 integrin, it will now be possible to further investigate the mechanism by which ?v?3 integrin facilitates metastasis.
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Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models.
J Biomed Opt
PUBLISHED: 08-20-2013
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ABSTRACT. The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.
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Comparison of efficacy of Salmonella typhimurium A1-R and chemotherapy on stem-like and non-stem human pancreatic cancer cells.
Cell Cycle
PUBLISHED: 08-06-2013
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The XPA1 human pancreatic cancer cell line is dimorphic, with spindle stem-like cells and round non-stem cells. We report here the in vitro IC 50 values of stem-like and non-stem XPA1 human pancreatic cells cells for: (1) 5-fluorouracil (5-FU), (2) cisplatinum (CDDP), (3) gemcitabine (GEM), and (4) tumor-targeting Salmonella typhimurium A1-R (A1-R). IC 50 values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-FU (P = 0.007) and CDDP (P = 0.012). In contrast, there was no difference between the efficacy of A1-R on stem-like and non-stem XPA1 cells. In vivo, 5-FU and A1-R significantly reduced the tumor weight of non-stem XPA1 cells (5-FU; P = 0.028; A1-R; P = 0.011). In contrast, only A1-R significantly reduced tumor weight of stem-like XPA1 cells (P = 0.012). The combination A1-R with 5-FU improved the antitumor efficacy compared with 5-FU monotherapy on the stem-like cells (P = 0.004). The results of the present report indicate A1-R is a promising therapy for chemo-resistant pancreatic cancer stem-like cells.
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Color-coded imaging of spontaneous vessel anastomosis in vivo.
Anticancer Res.
PUBLISHED: 07-31-2013
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Vessel anastomosis is important in tumor angiogenesis as well as for vascularization therapy for ischemia and other diseases. We report here the development of a color-coded imaging model that can visualize the anastomosis between blood vessels of red fluorescent protein (RFP)-expressing vessels in vascularized Gelfoam® previously transplanted into RFP transgenic mice and then re-transplanted into nestin-driven green fluorescent protein (ND-GFP) mice where nascent blood vessels express GFP. Gelfoam® was initially transplanted subcutaneously in the flank of transgenic RFP nude mice. Skin flaps were made at 14 days after transplantation of Gelfoam® to allow observation of vascularization of the Gelfoam® using confocal fluorescence imaging. The implanted Gelfoam® became highly vascularized with RFP vessels. Fourteen days after transplantation into RFP transgenic nude mice, the Gelfoam® was removed and re-transplanted into the subcutis on the flank of ND-GFP transgenic nude mice in which nascent blood vessels express GFP. Skin flaps were made and anastomosis between the GFP-expressing nascent blood vessels of ND-GFP transgenic nude mice and RFP blood vessels in the Gelfoam® was imaged 14 and 21 days after re-transplantation. The results presented in this report indicate a possible mechanism for tumor angiogenesis and suggest a new paradigm of therapeutic revascularization of ischemic organs requiring new blood vessels and in other diseases.
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Real-time imaging of ?v integrin molecular dynamics in osteosarcoma cells in vitro and in vivo.
Anticancer Res.
PUBLISHED: 07-31-2013
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?v Integrin is involved in various steps of cancer metastasis. In this report, we describe real-time imaging of ?v integrin molecular dynamics in human 143B osteosarcoma cells in vitro and in vivo. We first generated osteosarcoma cells expressing ?v integrin green fluorescent protein (GFP) by transfection of an ?v integrin GFP fusion vector (pCMV6-AC-ITGAV-GFP) into 143B cells. Confocal laser-scanning microscopy demonstrated that ?v integrin immunofluorescence staining co-localized with ?v integrin-GFP fluorescence in 143B cells. When ?v integrin-GFP-expressing 143B osteosarcoma cells were seeded on a dish coated with fibronectin, which is bound by ?v integrin, punctate ?v integrin-GFP was observed by confocal laser-scanning microscopy. When the 143B ?v integrin-GFP cells were seeded onto uncoated plastic, ?v integrin-GFP was diffuse within the cells. When ?v integrin-GFP 143B osteosarcoma cells (1×10(6)) were orthotopically transplanted into the tibia of nude mice, the cells aligned along the collagen fibers within the tumor and had punctuate expression of ?v integrin-GFP. In the orthotopic model, the invading osteosarcoma cells had punctate ?v integrin-GFP in the muscle tissue at the primary tumor margin. These results show that ?v integrin-GFP enables the imaging of the molecular dynamics of ?v integrin in osteosarcoma cells in vitro and in vivo.
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Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.
J Surg Oncol
PUBLISHED: 07-06-2013
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The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer.
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Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force.
J. Cell Biol.
PUBLISHED: 06-27-2013
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Cell migration through 3D tissue depends on a physicochemical balance between cell deformability and physical tissue constraints. Migration rates are further governed by the capacity to degrade ECM by proteolytic enzymes, particularly matrix metalloproteinases (MMPs), and integrin- and actomyosin-mediated mechanocoupling. Yet, how these parameters cooperate when space is confined remains unclear. Using MMP-degradable collagen lattices or nondegradable substrates of varying porosity, we quantitatively identify the limits of cell migration by physical arrest. MMP-independent migration declined as linear function of pore size and with deformation of the nucleus, with arrest reached at 10% of the nuclear cross section (tumor cells, 7 µm²; T cells, 4 µm²; neutrophils, 2 µm²). Residual migration under space restriction strongly depended upon MMP-dependent ECM cleavage by enlarging matrix pore diameters, and integrin- and actomyosin-dependent force generation, which jointly propelled the nucleus. The limits of interstitial cell migration thus depend upon scaffold porosity and deformation of the nucleus, with pericellular collagenolysis and mechanocoupling as modulators.
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Stromal-cell and cancer-cell exosomes leading the metastatic exodus for the promised niche.
Breast Cancer Res.
PUBLISHED: 06-18-2013
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Exosomes are thought to play an important role in metastasis. Luga and colleagues have described the production of exosomes by stromal cells such as cancer-associated fibroblasts that are taken up by breast cancer cells and are then loaded with Wnt 11, which is associated with stimulation of the invasiveness and metastasis of the breast cancer cells. Previous studies have shown that exosomes produced by breast cancer cells are taken up by stromal fibroblasts and other stromal cells, suggesting that exosomes are agents of cross-talk between cancer and stromal cells to stimulate metastasis. Imaging of exosomes by labeling with fluorescent proteins will enlighten the process by which exosomes enhance metastasis, including premetastatic niche formation.
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Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling.
Cell
PUBLISHED: 06-04-2013
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Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from premalignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.
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Imaging UVC-induced DNA damage response in models of minimal cancer.
J. Cell. Biochem.
PUBLISHED: 05-09-2013
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We have previously demonstrated that the ultraviolet (UV) light is effective against a variety of cancer cells in vivo as well as in vitro. In the present report, we imaged the DNA damage repair response of minimal cancer after UVC irradiation. DNA-damage repair response to UV irradiation was imaged on tumors growing in 3D culture and in superficial tumors grown in vivo. UV-induced DNA damage repair was imaged with GFP fused to the DNA damage response (DDR)-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells. Three-dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP nuclear foci to be observed within 1?h after UVC irradiation, indicating the onset of DNA damage repair response. A clonogenic assay showed that UVC inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, while UVA and UVB showed little effect on cell proliferation. Induction of UV-induced 53BP1-GFP focus formation was limited up to a depth of 40?µm in 3D-culture of MiaPaCa-2 cells. The MiaPaCa-2 cells irradiated by UVC light in a skin-flap mouse model had a significant decrease of tumor growth compared to untreated controls. Our results also demonstrate that 53BP1-GFP is an imageable marker of UV-induced DNA damage repair response of minimal cancer and that UVC is a useful tool for the treatment of residual cancer since UVC can kill superficial cancer cells without damage to deep tissue.
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A dual-color genetically engineered mouse model for multispectral imaging of the pancreatic microenvironment.
Pancreas
PUBLISHED: 05-08-2013
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To develop a mouse model for multispectral fluorescence imaging of the pancreas and pancreatic microenvironment.
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Salmonella typhimurium A1-R tumor targeting in immunocompetent mice is enhanced by a traditional Chinese medicine herbal mixture.
Anticancer Res.
PUBLISHED: 05-07-2013
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We have developed a bacterial cancer therapy strategy using the genetically-engineered strain Salmonella typhimurium A1-R (A1-R). A1-R is auxotrophic for leu and arg which attenuates bacterial growth in normal tissue but allows high tumor virulence. A1-R is effective against metastatic human and murine cancer cell lines in clinically-relevant nude-mouse models. However, A1-R treatment of tumors in immunocompetent mouse models with high doses is limited by toxicity. The current study evaluated a traditional Chinese medicine (TCM) herbal mixture in combination with A1-R therapy in a syngeneic metastatic immunocompetent mouse model of highly aggressive lung cancer. In a model of Lewis lung carcinoma, the combination of a TCM herbal mixture and S. typhimurium A1-R enabled bacteria to be safely administered at the large dose of 2 × 10(7) colony forming units once a week i.v. with increased treatment efficacy and reduced toxicity compared to monotherapy with A1-R. The herbal mixture prevented body weight loss, spleen weight gain and liver infection by A1-R, as well as hemorrhagic lesions on the skin, liver, and spleen, all observed with A1-R monotherapy. The results of the present study suggest that the combination of A1-R and TCM has important potential for therapy of highly aggressive types of cancer, including those which are resistant to standard therapy.
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Paclitaxel nanosuspensions for targeted chemotherapy - nanosuspension preparation, characterization, and use.
Pharm Dev Technol
PUBLISHED: 04-25-2013
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Abstract Objective: The purpose of this work was to prepare a stable paclitaxel nanosuspension and test it for potential use as a targeted chemotherapeutic. Different particle coatings were employed to assess their impact on cellular uptake in vitro. In vivo work was then performed to demonstrate efficacy in tumor-bearing mouse models. Materials and method: Paclitaxel nanosuspensions were prepared using a homogenization process and coated with excipients. Surface charge was measured by zeta potential, potency by high-performance liquid chromatography, and solubility using an in-line UV probe. Cellular uptake studies were performed via flow cytometry. In vivo experiments were performed to determine residence time, maximum tolerated dose, and the efficacy of paclitaxel nanosuspensions (Paclitaxel-NS). Results: A stable paclitaxel nanosuspension was prepared and coated with various excipients. Studies in mice showed that the nanosuspension was well-tolerated and at least as effective as the IV Taxol control in prolonging mouse survival in a head and neck cancer model as well as an ovarian cancer model with a lower overall drug dose than the traditional IV administration route. Conclusions: The paclitaxel nanosuspension is suitable for cellular uptake. The nanosuspension was effective in prolonging life in two separate xenograft orthotopic murine cancer models through two separate routes of administration.
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Single cell time-lapse imaging of focus formation by the DNA damage-response protein 53BP1 after UVC irradiation of human pancreatic cancer cells.
Anticancer Res.
PUBLISHED: 04-09-2013
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We have previously demonstrated that ultraviolet (UV) light treatment is effective against various types of cancer cells expressing fluorescent proteins. In order to further understand the efficacy of UV treatment of cancer cells, we determined the kinetics of focus formation by imaging of a DNA damage-response (DDR) protein after UVC irradiation of human pancreatic cancer cells. A fusion protein consisting of the DDR protein 53BP1 and green fluorescent protein (GFP) (GFP-53BP1) was used as a live-cell imaging marker for cellular response after UVC irradiation. GFP-53BP1 foci were observed after UVC irradiation of MiaPaCa-2 human pancreatic cancer cells. During live-cell imaging, GFP-53BP1 foci were observed in the cells within 15 min after UVC irradiation, and some of the foci remained stable for at least three hours. GFP-53BP1 focus formation was observed in the pancreatic-cancer cells irradiated by 25-200 J/m(2) UVC. Our results indicate that an early response to DNA damage caused by UVC irradiation can be visualized by increased GFP-53BP1 focus formation by pancreatic cancer cells.
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Dynamic subcellular imaging of cancer cell mitosis in the brain of live mice.
Anticancer Res.
PUBLISHED: 04-09-2013
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The ability to visualize cancer cell mitosis and apoptosis in the brain in real time would be of great utility in testing novel therapies. In order to achieve this goal, the cancer cells were labeled with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm, such that mitosis and apoptosis could be clearly imaged. A craniotomy open window was made in athymic nude mice for real-time fluorescence imaging of implanted cancer cells growing in the brain. The craniotomy window was reversibly closed with a skin flap. Mitosis of the individual cancer cells were imaged dynamically in real time through the craniotomy-open window. This model can be used to evaluate brain metastasis and brain cancer at the subcellular level.
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A color-coded imaging model of the interaction of ?v integrin-GFP expressed in osteosarcoma cells and RFP expressing blood vessels in Gelfoam® vascularized in vivo.
Anticancer Res.
PUBLISHED: 04-09-2013
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The integrin family of proteins has been shown to be involved in the malignant behavior of cells. We report here development of a color-coded imaging model that can visualize the interaction between ?v integrin linked to green fluorescent protein (GFP) in osteosarcoma cells and blood vessels in Gelfoam® vascularized after implantation in red fluorescent protein (RFP) transgenic nude mice. Human 143B osteosarcoma cells expressing ?v integrin-GFP were generated by transfection with an ?v integrin-GFP vector. Gelfoam® (5×5 mm) was transplanted subcutaneously in transgenic RFP nude mice. The implanted Gelfoam® became highly vascularized with RFP vessels within 14 days. Skin flaps were made at days 7, 14, 21, 28 after transplantation of Gelfoam® for observing vascularization of the Gelfoam® using fluorescence imaging. Gelfoam® is a useful tool to observe angiogenesis in vivo. 143B cells (5 × 10(5)) expressing ?v integrin-GFP were injected into the Gelfoam® seven days after transplantation of Gelfoam®. Seven days after cancer-cell injection, cancer cells and blood vessels were observed in the Gelfoam® by color-coded confocal microscopy via the skin flap. The 143B cells expressing ?v integrin-GFP proliferated into the Gelfoam®, which contained RFP-expressing blood vessels. Strong expression of ?v integrin-GFP in 143B cells was observed near RFP vessels in the Gelfoam®. The observation of the behavior of ?v integrin-GFP and blood vessels will allow further understanding of the role of ?v integrin in cancer cells.
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Dynamic color-coded fluorescence imaging of the cell-cycle phase, mitosis, and apoptosis demonstrates how caffeine modulates cisplatinum efficacy.
J. Cell. Biochem.
PUBLISHED: 03-21-2013
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Caffeine enhances the effect of certain anticancer drugs, but the mechanism of modulation is poorly understood. In this study, modulation of cisplatinum efficacy induced by caffeine was visualized at the subcellular level by real-time fluorescent-protein imaging. Mitotic and apoptotic changes were observed by imaging 143B human osteosarcoma dual-color cells, in which GFP is expressed in the nucleus and RFP is expressed in the cytoplasm. Modulation of the cell cycle was imaged using time-lapse imaging of HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (FUCCI) in the nucleus. Clonogenic assays showed that caffeine increased the inhibition by cisplatinum on cell proliferation. Subcellular imaging demonstrated that cisplatinum decreased mitosis and induced apoptosis in 143B cells. The combination of cisplatinum and caffeine enhanced mitosis and subsequently increased apoptosis. Time-lapse imaging showed that cisplatinum strongly induced cell-cycle arrest in the S/G2 phase in HeLa-FUCCI cells. Caffeine overcame the cell-cycle arrest induced by cisplatinum, thereby increasing its efficacy, since cisplatinum is ineffective against quiescent cells. The data in this report indicate that caffeine modulates the cell cycle in cancer cells, thereby enhancing efficacy of cell-cycle-dependent anticancer drugs such as cisplatinum.
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Progression-free survival is accurately predicted in patients treated with chemotherapy for epithelial ovarian cancer by the histoculture drug response assay in a prospective correlative clinical trial at a single institution.
Anticancer Res.
PUBLISHED: 03-14-2013
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This study aimed to prospectively correlate clinical outcomes of advanced epithelial ovarian cancer (AEOC), with the results of in vitro chemosensitivity testing of taxol and carboplatin using the in vitro histoculture drug response assay (HDRA). A total of 104 patients with AEOC were treated with combination chemotherapy of taxol and carboplatin after primary cytoreductive surgery between 2007 and 2012 at the Asan Medical Center, Seoul, Korea. To compare chemosensitivity in the HDRA with clinical response, all patients were first categorized into two groups as either sensitive to both taxol and carboplatin (SS), or not sensitive to one or both drugs (R) based on HDRA results. The recurrence rate was much lower in the SS group compared to the R group; 29.2% vs 69.8%, respectively (p=0.02). The SS group had a significantly longer progression-free survival compared to the R group, 34.0 months vs 16.0 months, respectively (p=0.025). These results demonstrate that the HDRA prospectively correlates to clinical outcome from chemotherapy and that treatment regimens can be individualized based on the HDRA.
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In Vivo Fluorescence Imaging of Gastrointestinal Stromal Tumors Using Fluorophore-Conjugated Anti-KIT Antibody.
Ann. Surg. Oncol.
PUBLISHED: 03-08-2013
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Gastrointestinal stromal tumors (GISTs) are frequently characterized by KIT overexpression. Tumor-free margins and complete cytoreduction of disease are mainstays of treatment. We hypothesized that fluorescently labeled anti-KIT antibodies can label GIST in vivo.
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Fluorescence-guided surgery and fluorescence laparoscopy for gastrointestinal cancers in clinically-relevant mouse models.
Gastroenterol Res Pract
PUBLISHED: 02-19-2013
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There are many challenges that face surgeons when attempting curative resection for gastrointestinal cancers. The ability to properly delineate tumor margins for complete resection is of utmost importance in achieving cure and giving the patient the best chance of prolonged survival. Targeted tumor imaging techniques have gained significant interest in recent years to enable better identification of tumor lesions to improve diagnosis and treatment of cancer from preoperative staging modalities to optimizing the surgeons ability to visualize tumor margins at the initial operation. Using unique characteristics of the tumor to fluorescently label the tissue can delineate tumor margins from normal surrounding tissue, allowing improved precision of surgical resection. In this paper, different methods of fluorescently labeling native tumor are discussed as well as the development of fluorescence laparoscopy and the potential role for fluorescence-guided surgery in the treatment of gastrointestinal cancers.
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Efficacy comparison of traditional Chinese medicine LQ versus gemcitabine in a mouse model of pancreatic cancer.
J. Cell. Biochem.
PUBLISHED: 02-14-2013
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Pancreatic cancer is highly treatment-resistant and has one of the highest fatality rates of all cancers and is the fourth highest cancer killer worldwide. Novel, more effective strategies are needed to treat this disease. We report here on the use of patient-like orthotopic nude-mouse models of human metastatic pancreatic cancer to compare the traditional Chinese medicine (TCM) herbal mixture LQ to gemcitabine, which is first-line therapy for this disease, for anti-metastatic and anti-tumor activity as well as safety. The human pancreatic cancer cell line, MiaPaCa-2, labeled with red fluorescent protein (RFP), was used for the orthotopic model. LQ (gavage, 600?mg/kg/day) significantly inhibited pancreatic cancer tumor growth and metastasis, as measured by imaging, with no overt toxicity. Survival of tumor-bearing mice was also prolonged by LQ. The therapeutic efficacy of LQ is comparable with gemcitabine but with less toxicity, as indicated by a lack of body-weight loss with LQ, but not gemcitabine. The results indicate that TCM can have non-toxic efficacy against metastatic pancreatic cancer comparable to gemcitabine in a clinically-relevant orthotopic mouse model.
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Intraoperative imaging of metastatic lymph nodes using a fluorophore-conjugated antibody in a HER2/neu-expressing orthotopic breast cancer mouse model.
Anticancer Res.
PUBLISHED: 02-09-2013
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We investigated in this study whether fluorescence imaging with a fluorophore-conjugated anti-human epidermal growth factor receptor 2 (HER2)/neu probe could be used to differentiate metastatic lymph nodes (LN) from normal LN to guide surgical resection. A fluorescent probe for detecting HER2/neu-expressing cells was generated by conjugation of the humanized anti-HER2/neu antibody trastuzumab with rhodamine. The green fluorescent protein-expressing breast cancer cell line 4T1-GFP was used for in vitro binding analysis and for the establishment of a model of HER2/neu expressing LN-metastatic breast cancer. All tumor-bearing mice were given a single intravenous injection of either rhodamine-conjugated anti-HER2/neu probe, or conjugated control IgG antibody, when LN metastasis developed. Each animal was imaged with both green and red fluorescence to assess in vivo binding of the rhodamine-conjugated anti-HER2/neu probe to the tumors and LN metastases. Hematoxylin and eosin (H&E) staining and immunohistochemistry was performed to confirm the presence of tumor and metastasis as well as HER2/neu expression. The imaging probe was able to bind to HER2/neu-expressing 4T1-GFP tumor cells in vitro. The primary tumors and LN metastases from the animals which were treated with rhodamine-conjugated anti-HER2/neu probe exhibited a visible red fluorescence signal. The fluorescent axillary LN metastasis was easily distinguishable from the surrounding normal tissue and normal LN. Sensitivity of 78% (14 out of 18) and specificity of 100% (20 out of 20) could be achieved with the rhodamine-conjugated anti-HER2/neu probe for the detection of LN metastasis. These data support further investigation of the fluorophore-conjugated anti-HER2/neu antibody to detect LN metastasis in the surgical setting.
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VEGF-C ShRNA inhibits pancreatic cancer growth and lymphangiogenesis in an orthotopic fluorescent nude mouse model.
Anticancer Res.
PUBLISHED: 02-09-2013
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The aim of this study was to assess the inhibitory efficacy of short hairpin RNA (ShRNA) targeting vascular endothelial growth factor C (VEGF-C) in an orthotopic pancreatic cancer mouse model. BxPC-3 human pancreatic cancer cells expressing green fluorescent protein (GFP) were orthotopically implanted onto the pancreas of nude mice. All mice were randomly divided into four groups when the average tumor size had reached 100 mm(3) and were treated with either vehicle or gemcitabine at 150 mg/kg; or intravenous VEGF-C ShRNA at 150 mg/kg; or intratumoral VEGF-C ShRNA at 150 ?g/kg. In vivo fluorescence imaging was performed to monitor tumor growth and metastasis during the study. Real-time quantitative polymerase chain reaction (RT-qPCR) and an enzyme-linked immunosorbent assay (ELISA) were performed to determine the mRNA and protein level of VEGF-C in tumor tissues. Lymphatic vessel marker D2-40, blood vessel marker CD31 and proliferation marker Ki67 expression of the tumor tissues were analyzed by immunohistochemistry staining. Intravenous and intratumoral VEGF-C ShRNA treatment significantly inhibited tumor growth, downregulated the expression of VEGF-C mRNA, reduced tumor microlymphatic vessel density (MLVD), and inhibited cancer cell proliferation. Gemcitabine, as the standard treatment for pancreatic cancer, demonstrated a stronger inhibitory effect on tumor growth, with less inhibition of MLVD and more inhibition of microvessel density (MVD) and proliferation than VEGF-C ShRNA. These results indicate that different mechanisms are associated with the efficacy of gemcitabine and VEGF-C ShRNA.
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Specific route mapping visualized with GFP of single-file streaming contralateral and systemic metastasis of Lewis lung carcinoma cells beginning within hours of orthotopic implantation [correction of implantion].
J. Cell. Biochem.
PUBLISHED: 01-30-2013
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In this study, we visualized the origin of Lewis lung carcinoma metastasis after transducing tumor cells with green fluorescent protein (GFP) and transplanting them orthotopically in the middle lobe of the right lung of nude mice. Metastasis was visualized in live tissue at single cell resolution by GFP-expression as early as 18 h post-tumor transplant. At this time, single-file streaming lung carcinoma cells already had invaded inferiorly via a tubular lymphatic structure crossing the lower lobes of the lung to the ipsilateral diaphragmatic surface. By post-implantation day 2, the ipsilateral lower lobes of the lung were involved with metastatic cells. By post-implantation day 3, the ipsilateral lower lobes of the lung and the ipsilateral diaphragmatic surface were highly involved with streaming metastatic cells trafficking in single file. By day 4 post-implantation, cancer cells invaded across the diaphragm to the contralateral diaphragmatic surface. Metastatic cells then invaded superiorly through a lymphatic vessel to involve the contralateral mediastinal lymph nodes. In this model of lung cancer, the origin of metastasis was an inferior invasion from the implanted tumor via a lymphatic duct to the ipsilateral diaphragmatic surface. The cancer cells from this site invaded on the surface of the diaphragm to the contralateral diaphragmatic surface and proceeded superiorly through a lymphatic duct to contralateral lymph nodes. Other organs such as the kidneys and the adrenal glands later became involved with metastasis with the contralateral mediastinal lymph nodes as the source. The use of GFP and the highly metastatic orthotopic lung cancer model allowed the visualization of the origin of metastasis at the single-cell level and demonstrated the critical role of lymphatic ducts and the diaphragmatic surface as the path to the contralateral side.
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The role of hair follicle nestin-expressing stem cells during whisker sensory-nerve growth in long-term 3D culture.
J. Cell. Biochem.
PUBLISHED: 01-16-2013
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We have previously reported that nestin-expressing hair follicle stem cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, vibrissa hair follicles, including their sensory nerve stump, were excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice), and were placed in 3D histoculture supported by Gelfoam®. ?-III tubulin-positive fibers, consisting of ND-GFP-expressing cells, extended up to 500 µm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F-actin. These findings indicate that ?-III tubulin-positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND-GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results of the present report suggest a major function of the nestin-expressing stem cells in the hair follicle is for growth of the follicle sensory nerve.
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In vivo serial selection of human pancreatic cancer cells in orthotopic mouse models produces high metastatic variants irrespective of Kras status.
J. Surg. Res.
PUBLISHED: 01-04-2013
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Kras mutations have been thought to play an important role in pancreatic cancer progression. In this study, we evaluated how serially passaging primary pancreatic tumors with and without Kras mutations, in nude mice, can generate more aggressive variants of human pancreatic cancer.
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Development of a clinically-precise mouse model of rectal cancer.
PLoS ONE
PUBLISHED: 01-01-2013
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Currently-used rodent tumor models, including transgenic tumor models, or subcutaneously growing tumors in mice, do not sufficiently represent clinical cancer. We report here development of methods to obtain a highly clinically-accurate rectal cancer model. This model was established by intrarectal transplantation of mouse rectal cancer cells, stably expressing green fluorescent protein (GFP), followed by disrupting the epithelial cell layer of the rectal mucosa by instilling an acetic acid solution. Early-stage tumor was detected in the rectal mucosa by 6 days after transplantation. The tumor then became invasive into the submucosal tissue. The tumor incidence was 100% and mean volume (±SD) was 1232.4 ± 994.7 mm(3) at 4 weeks after transplantation detected by fluorescence imaging. Spontaneous lymph node metastasis and lung metastasis were also found approximately 4 weeks after transplantation in over 90% of mice. This rectal tumor model precisely mimics the natural history of rectal cancer and can be used to study early tumor development, metastasis, and discovery and evaluation of novel therapeutics for this treatment-resistant disease.
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In vivo fluorescence imaging reveals the promotion of mammary tumorigenesis by mesenchymal stromal cells.
PLoS ONE
PUBLISHED: 01-01-2013
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Mesenchymal stromal cells (MSCs) are multipotent adult stem cells which are recruited to the tumor microenvironment (TME) and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.
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Nestin-Expressing Stem Cells Promote Nerve Growth in Long-Term 3-Dimensional Gelfoam®-Supported Histoculture.
PLoS ONE
PUBLISHED: 01-01-2013
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We have previously reported that hair follicles contain multipotent stem cells which express nestin. The nestin-expressing cells form the hair follicle sensory nerve. In vitro, the nestin-expressing hair follicle cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, the sciatic nerve was excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP mice). The ND-GFP cells of the sciatic nerve were also found to be multipotent as the ND-GFP cells in the hair follicle. When the ND-GFP cells in the mouse sciatic nerve cultured on Gelfoam® and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker ?-III tubulin, the immature Schwann-cell marker p75(NTR) and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The ?-III tubulin-positive fibers had growth cones on their tips expressing F-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co-cultured on Gelfoam® with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo. Nestin-expressing cells were also observed in posterior nerves but not in the spinal cord itself, when placed in 3-D Gelfoam® culture. The results of the present report suggest a critical function of nestin-expressing cells in peripheral nerve growth and regeneration.
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Ovarian tumor attachment, invasion, and vascularization reflect unique microenvironments in the peritoneum: insights from xenograft and mathematical models.
Front Oncol
PUBLISHED: 01-01-2013
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Ovarian cancer relapse is often characterized by metastatic spread throughout the peritoneal cavity with tumors attached to multiple organs. In this study, interaction of ovarian cancer cells with the peritoneal tumor microenvironment was evaluated in a xenograft model based on intraperitoneal injection of fluorescent SKOV3.ip1 ovarian cancer cells. Intra-vital microscopy of mixed GFP-red fluorescent protein (RFP) cell populations injected into the peritoneum demonstrated that cancer cells aggregate and attach as mixed spheroids, emphasizing the importance of homotypic adhesion in tumor formation. Electron microscopy provided high resolution structural information about local attachment sites. Experimental measurements from the mouse model were used to build a three-dimensional cellular Potts ovarian tumor model (OvTM) that examines ovarian cancer cell attachment, chemotaxis, growth, and vascularization. OvTM simulations provide insight into the relative influence of cancer cell-cell adhesion, oxygen availability, and local architecture on tumor growth and morphology. Notably, tumors on the mesentery, omentum, or spleen readily invade the "open" architecture, while tumors attached to the gut encounter barriers that restrict invasion and instead rapidly expand into the peritoneal space. Simulations suggest that rapid neovascularization of SKOV3.ip1 tumors is triggered by constitutive release of angiogenic factors in the absence of hypoxia. This research highlights the importance of cellular adhesion and tumor microenvironment in the seeding of secondary ovarian tumors on diverse organs within the peritoneal cavity. Results of the OvTM simulations indicate that invasion is strongly influenced by features underlying the mesothelial lining at different sites, but is also affected by local production of chemotactic factors. The integrated in vivo mouse model and computer simulations provide a unique platform for evaluating targeted therapies for ovarian cancer relapse.
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The preclinical discovery of bacterial therapy for the treatment of metastatic cancer with unique advantages.
Expert Opin Drug Discov
PUBLISHED: 12-22-2011
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The potential of bacteria as therapeutics for cancer has a long history, dating at least as far back as the early 19(th) Century. Bacteria have a large genome that can be manipulated in order to target and eradicate tumors. Many types of bacteria have been shown to target tumors but most are obligate anaerobes whose growth is confined to the necrotic parts of tumors, thereby limiting their efficacy. Salmonella, on the other hand, are facultative aerobes that can grow aerobically or anaerobically and, therefore, grow on viable tumor tissue as well as necrotic tissue.
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Glowing tumors make for better detection and resection.
Sci Transl Med
PUBLISHED: 11-26-2011
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Tumor-specific fluorescent probes that can be administered topically make tumors glow selectively and thus have great potential for improving cancer detection and removal.
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Comparison of cancer-cell seeding, viability and deformation in the lung, muscle and liver, visualized by subcellular real-time imaging in the live mouse.
Anticancer Res.
PUBLISHED: 11-24-2011
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The comparison of cancer cell seeding, deformation and viability in the lung, muscle and liver of nude mice in real-time is reported here. The mice were intubated to support ventilation with positive end-respiratory pressure (PEEP) for imaging on the lung. Human fibrosarcoma cells with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (dual-color HT-1080 cells) were injected into the tail vein for lung imaging, the portal vein for liver imaging or the abdominal aorta for muscle imaging which was performed with an Olympus OV100 Small Animal Imaging System. The length of the cytoplasm and nuclei in 20 seeded cancer cells were measured. A large number of cells initially arrested in the lung capillaries and many cells formed aggregates. The cell number decreased rapidly at 6 and 24 h. There was no significant difference in cancer cell survival when immunocompetent C57BL/6 mice were used in place of the nude mice, suggesting that T cell reaction is not very important in the first 24 h after seeding of cancer cells in the lung. In the lung and liver, little cancer cell deformation occurred. In contrast in the muscle, the cytoplasm and nuclei of the seeded cells were highly deformed and many fragmented cells were observed. The rate of cancer cell death was highest in the lung and lowest in the muscle. In each organ, single disseminated cells tended to die earlier than aggregated cells. The results of this study suggest that the early steps of metastasis are different in the lung, liver and muscle.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.