Cancer Research with Near-Infrared Fluorescence | Cancer Research using LI-COR Odyssey and Pearl Imagers and NIR Fluorescence Technologies

PI3 Kinase Pathway Studies with Infrared Fluorescence by Odyssey^® Expert, Dr. Geir Bjørkøy

Posted on May 14, 2013 by licor-admin

Dr. Geir Bjørkøy is a Professor at the University College of Sør-Trøndelag and CEMIR Centre of Exellence, NTNU, in Trondheim, Norway. He studies the molecular biology of cancer. Bjørkøy’s major focus uses Western blotting to study phosphorylation in the PI3 Kinase pathway.

Dr. Bjørkøy’s lab uses the Odyssey Imager to examine the differences in phospho-Akt levels in the cross-sections of tumors grown in nude mice and tumor samples from patients. According to Bjørkøy, “Quantifying tumor size reduction will be crucial for future cancer diagnostics and therapies.”

Bjørkøy combines his work on the Odyssey Imager with confocal microscopy by mixing in secondary antibodies that can be detected in the visible light spectra. After scanning the whole tissue section on the Odyssey Imager and gathering data, Bjørkøy uses a confocal microscope to examine particular sections in more detail. He looks at how specific inhibitors affect tumor growth.

Bjørkøy’s innovation using the Odyssey Infrared Imager for immunohistochemistry analysis, and exploring future quantitative cancer diagnostics are just a couple of the reasons we are proud to call him an “Odyssey Expert.”

Read Dr Bjørkøy’s recently published paper utilizing the Odyssey Infrared Imaging System:

Metabolic biomarkers for response to PI3K inhibition in basal-like breast cancer
Moestue SA, Dam CG, Gorad SS, Kristian A, Bofin A, Mælandsmo GM, Engebråten O, Gribbestad, IS, Bjørkøy G.
Breast Cancer Research (Feb 2013) 15: R16. doi:10.1186/bcr3391

Posted in Odyssey Imaging Systems, Proteomics Applications | Tagged cancer research, geir bjorkoy, immunohistochemistry, infrared fluorescent western blots, multiplex detection, odyssey applications, odyssey expert, phospho-akt, phosphorylation, pi3 kinase pathway, quantifying tumor size, tissue section imaging | Leave a comment

MYCC Overexpression and “Oncogene Addiction” in Ovarian Cancer Cells

Posted on April 17, 2013 by licor-admin

Self-sufficiency in growth signals is one of the six hallmarks of cancer cells. In normal cells, proliferation requires externally-produced growth factors. Tumor cells may produce their own growth factors, or become hyper-sensitive to ambient growth factor levels. Other mechanisms of sustained proliferation include deregulation of mitotic signaling, constitutive activation of downstream signaling pathways, and disruption of negative-feedback mechanisms.

The MYCC oncogene (also called MYC or c-MYC) is amplified in many cancers. This study examined MYCC overexpression and “oncogene addiction” in ovarian cancer cells. LI-COR^® products were used to generate some of the data cited in this research paper.

MYCC or MYC paralogs were down-regulated by siRNA in a panel of ovarian cancer cell lines.
Quantitative two-color Westerns and the Odyssey® Imager were used to monitor protein levels.
MYCC inactivation inhibited proliferation and induced senescence in cell lines with amplified MYCC, indicating oncogene addiction.
In growth-arrested cells, expression of the Cdk inhibitor p27^Kip1 was increased. Cyclin A expression and Cdk2 activity were decreased. Arrest could be reversed by RNAi knockdown of p27^Kip1 or Rb.
The authors conclude that MYCC-amplified ovarian cancer cells require MYCC overexpression to prevent senescence by repressing p27Kip1, maintaining Cdk2 activity, and inactivating Rb.

Effect of MYCC knockdown on expression and activity of cell-cycle regulators in MYCC-amplified cells. A) Cyclin A levels were quantified by immunoblotting and normalized to Gapdh levels. B) Top: Cdk2 complexes were immunoprecipitated, and kinase activities assayed using C-terminal Rb as substrate. Bottom: Phospho-Thr160-Cdk2 and Cdk2 were analyzed by immunoblotting. C) Upon MYCC RNAi, p27^Kip1 and p21^Cip1 are up-regulated in MYCC-amplified cells (Hey and Ovca420), but not in MYCC non-amplified cells (A2780 and Caov3). Levels of p27 (left) and p21 (right) were quantified by immunoblotting and normalized to Gapdh. Asterisks indicate significant differences between MYCC siRNA and control scrambled siRNA. Fluorescent Westerns were imaged with Odyssey Classic Infrared Imaging System.

Reprinted with permission Prathapam T, et al. p27Kip1 mediates addiction of ovarian cancer cells to MYCC (c-MYC) and their dependence on MYC paralogs. J Biol Chem. 285(42):32529-38 (2010).

Posted in Hallmarks of Cancer, Odyssey Imaging Systems, Proteomics Applications | Tagged cancer hallmarks, cancer research, cell proliferation, hallmarks of cancer, myc overexpression, mycc overexpression, ovarian cancer, self-sufficiency in growth signals, sustained proliferation, sustaining proliferative signaling | Leave a comment

PubAlert: Quantitative Western Blots and Odyssey^® used to Study Phosphorylation in Pancreatic Cancer

Posted on November 16, 2012 by licor-admin

Studies on mechanisms of interferon-gamma action in pancreatic cancer using a data-driven and model-based approach.

F Lange, K Rateitschak, B Fitzner, R Pöhland, O Wolkenhauer and R Jaster
Dept of Systems Biology and Bioinformatics, University of Rostock, 18051 Rostock, Germany
Molecular Cancer 10(1):13 (2011). doi: 1.1186/1476-4598-10-13

Interferon-gamma (IFNγ) has anti-proliferative effects, and may inhibit pancreatic cancer. To examine the molecular action of IFNγ, the authors established a mathematical model of STAT1 activation and combined experimental studies with computer simulations. Using a mouse model of heterotopic pancreatic cancer (PC), the effects of IFNγ on PC growth were examined in vivo and in vitro. IFNγ inhibited growth of both pure DSL-6A/C1 pancreatic tumors and tumors composed of DSL-6A/Ca cells and pancreatic stellate cells (PSC) to a similar extent.

Figure 1. Time course of phospho-STAT1 and STAT1 protein levels in IFNγ-stimulated DSL-6A/C1 cells. A) Cells were treated with IFNγ (100 ng/ml) for up to 720 min. STAT1, pSTAT1, and ERK1/2 were detected with multiplex fluorescence on a single Western blot (Odyssey Imager; LI-COR Biosciences). Samples were loaded in randomized order to exclude gel or transfer irregularities correlated with lane position. B) Quantification of STAT1 activation. Protein levels are expressed as arbitrary units from 4-6 experiments. Experimental data are shown in blue; mathematical models are shown as solid red lines.

Quantitative fluorescent Western blots (Odyssey® Imager) were used to measure changes in STAT1 expression and tyrosine phosphorylation during IFNγ treatment (Fig. 1; above). Immunoblotting of total cell lysates, cytosolic fractions, and nuclear fractions was used to examine subcellular localization of STAT1, and nuclear translocation was confirmed by confocal microscopy. Experimental data and mathematical modeling indicate that DSL-6A/C1 cells require higher doses of IFNγ than PSC for efficient activation of STAT1. The authors suggest that IFNγ may affect not only tumor cells, but also stroma cells in the tumor region.

For more publications using infrared fluorescent technology and Odyssey imagers, see our Odyssey Publication Lists, including Odyssey Publications List Vol 17 Autumn 2012.

Posted in Odyssey Imaging Systems, Proteomics Applications | Tagged fluorescent western blotting, gamma interferon, ifn gamma, ifng, infrared westerns, odyssey clx, odyssey imager, protein phosphorylation, quantitative western blots, signal transduction, stat1 expression, tyrosine phosphorylation | Leave a comment

Enhanced Sensitivity and Low Background with IRDye^® Infrared Dyes

Posted on October 15, 2012 by licor-admin

IRDye Infrared Dyes and near-infrared (NIR) fluorescent imaging deliver enhanced sensitivity due to low background autofluorescence in the near-infrared region and, therefore, higher signal to noise ratios. IRDye fluorescent dyes have absorption and emission wavelengths in the NIR spectrum, between 680 and 800 nm.

Optimized for use with LI-COR^® Infrared Imaging Systems
Can be used with a variety of instruments that offer near-infrared excitation and emission
Designed for antibody, protein, DNA, and peptide labeling (see IRDye Protein and Antibody Labeling Kits)
Suitable for many applications, including quantitative Western blot detection, In-Cell Western™ assays, EMSAs, Northern blots, protein arrays, fluorescence microscopy, tissue sections, and in vivo imaging

Applications vary depending on the IRDye. Please refer to specific dye page. One of the most popular IRDye infrared dyes used is the IRDye 800CW family of products.

IRDye 800CW Products include:

IRDye 800CW and Clinical Translation

LI-COR Clinical Translation seeks to facilitate the use of IRDye 800CW imaging agents in clinical studies for detection of disease and its progression, and for monitoring of treatment and drug efficacy.

Used by leading molecular imaging laboratories that develop translatable targeting agents
Easily conjugated to biomolecules (unlike ICG, which cannot be attached to biomolecules in its clinically approved form)

To discuss commercial development rights using IRDye infrared dyes, please contact Business Development at busdev@licor.com.

Posted in Odyssey Imaging Systems, Pearl Impulse Small Animal Imaging System, Reagents | Tagged clinical translation, infrared fluorescent detection, irdye 800cw labeling kits, irdye 800cw maleimides, irdye 800cw nhs esters, irdye fluorescent dyes | Leave a comment

The Sixth Hallmark of Cancer: Evasion of Apoptosis

Posted on August 10, 2012 by licor-admin

The sixth hallmark of cancer is the ability to evade programmed cell death (apoptosis) which can lead to uncontrolled cell division in normal cell development. This inherent cell suicide program can serve as a safety net because cells with excessive DNA damage are eliminated through apoptosis. Cancer cells, on the other hand, are resistant to apoptosis and continue to grow and divide even after accumulation of mutations.

Caspase-3 Assay on Odyssey^® Infrared Imaging System

There are two major apoptosis signaling pathways: the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. Under most circumstances, activation of either pathway leads to proteolytic cleavage and activation of caspases, a family of cysteine proteases that act as common death effector molecules. In-Cell Western™ Assay is a very helpful research tool for scientists who are quantifying cell signaling.

Below is an example of how Bond et al. (2008) used LI-COR Biosciences’ Odyssey^® system for this purpose. S2 cells were treated with actinomycin D to induce apoptosis. Figure A (green) shows active caspase-3 in the wells. Figure B (red), shows f-actin staining of the same wells as a control for cell number normalization. The results are expressed quantitatively in real-time. The graph shows a time course of caspase-3 activation after treatment with actinomycin D.

Posted in Hallmarks of Cancer, Proteomics Applications | Tagged apoptosis, caspase 3, caspase assay, evasion of apoptosis, in-cell elisas, in-cell wese, infrared fluorescent detection, near-infrared fluorescence, signal transduction | Leave a comment

Quantitative Protein Analysis in Cancer Research Webinar

Posted on July 16, 2012 by licor-admin

Quantitative protein detection contributes to a deeper understanding of cancer. See how you can use applications such as Infrared fluorescent Western blots, In-Cell Western™ cell-based assays, zymography and protein arrays for your cancer research.

Handout for Cancer Webinar 1: Quantitative Protein Detection in Cancer Research

Posted in Odyssey Imaging Systems, Proteomics Applications | Tagged immunofluorescent assays, in-cell elisas, infrared fluorescent detection, near-infrared fluorescence, two-color fluorescent detection, western blots, western blotting | Leave a comment

Publication Alert: Hallmarks of Cancer

Posted on June 28, 2012 by licor-admin

Hanahan and Weinberg^1-2 have proposed six “hallmarks of cancer” – acquired traits that distinguish cancer cells from normal cells. The publication reviewed below addresses one of these “hallmarks”. Future blogs will have additional publication reviews addressing other cancer hallmarks.

1. D Hanahan and RA Weinberg. Cell 100(1):57-70 (2000) 2. D Hanahan and RA Weinberg. Cell 144(5):646-74 (2011)

Hallmark: Resisting Cell Death
Vimentin is a novel anti-cancer therapeutic target; insights from in vitro and in vivo mice xenograft studies.

Lahat G, Zhu Q-S, Huang K-L, Wang S, Bolshakov S, Liu J, Torres K, Langley RR, Lazar AJ, Hung MC, Lev D
Sarcoma Research Center, Dept of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA

PLoS ONE 5(4): e10105 (2010). doi:10.1371/journal.pone.0010105

Vimentin, a mesenchymal intermediate filament, provides structural support to cells and plays a role in adhesion, migration, cell signaling, and survival. Withaferin-A (WFA) may target vimentin, and this study evaluated the impact of WFA on tumor growth.

Fluorescent Western blots (Odyssey^® Imager; LI-COR Biosciences) were used to monitor protein expression and cleavage. WFA strongly elicited apoptosis and vimentin degradation in tumor cells that express vimentin, with a lesser response in normal mesenchymal cells. Vimentin knockdown or caspase inhibition abolished the proapoptotic response. In xenograft models, WFA blocked soft tissue sarcoma growth, recurrence, and metastasis. Apoptosis and decreased angiogenesis were observed. WFA and other anti-vimentin therapeutics may have clinical relevance.

Figure 1. WFA sensitivity of cancer cells with epithelial origin is enhanced in cells exhibiting epithelial to mesenchymal transition (EMT). A) WFA-induced growth inhibition corresponds to vimentin expression level in epithelial origin cancer cells. C) Western blots (Odyssey Imager) show vimentin degradation in SKLMS1 and MDA231 cells, in conjunction with enhanced cleaved caspase-3 and activated PARP expression levels. Minimal or no expression of cleaved caspase-3 or activated PARP is seen in vimentin-negative cells (MCF7 and HT29). Graph shows the average of three experiments ±SD. (Vim FL = full length vimentin; VDP = vimentin degradation products.) doi:10.1371/journal.pone.0010105.g007

See publications related to: Cancer
See publications related to: Odyssey Infrared Imager

Posted in Hallmarks of Cancer, Odyssey Imaging Systems, Proteomics Applications | Tagged angiogenesis, apoptosis, caspase 3, fluorescent western blotting, infrared fluorescent detection, infrared westerns, monitoring protein expression, odyssey applications, PARP, programmed cell death | Leave a comment

The Fourth Hallmark of Cancer: Uncontrolled Cell Proliferation

Posted on June 7, 2012 by licor-admin

The fourth hallmark of cancer cells is the ability to escape normal regulatory mechanisms that control how many times a cell can divide. This uncontrolled proliferative capacity essentially produces the same result as avoiding anti-growth signals or generating positive growth signals. The limits to cell division are set in large part by the ends of chromosomes called telomeres. In normal somatic (body) cells, each round of cell division shrinks telomeres and cell division stops when these telomeres become too short. Cancer cells, in contrast, can lengthen their telomeres, which allows them to divide an indefinite number of times.

In a study conducted by Vidal-Cardenas and Greider (2010) at Johns Hopkins University, School of Medicine, the Odyssey^® Imaging System was used to quantify expression of the reverse transcriptase component TERT, which is the catalytic protein subunit that carries out telomere repeat additions. TR is the essential telomerase RNA component needed for both enzymatic activity and to provide the template for the telomeric repeats that are synthesized. Camptothecin, a cytotoxic quinoline alkaloid, induces a similar DNA damage response.

Reprinted with permission from NARESE- Nucleic Acids Research, 38(1); 60-71, 2010 (Figure 2)

Posted in Hallmarks of Cancer, Odyssey Imaging Systems, Proteomics Applications | Tagged cancer control, growth signals in cancer, infrared fluorescent detection, journal citations, li-cor western blotting, near-infrared fluorescence, uses of the odyssey imagers, western blots, western blotting | Leave a comment

Advance Your Research with Quantitative Cell Signaling Analysis

Posted on May 11, 2012 by licor-admin

Quantitative cell signaling analysis has the ability to improve your research and find answers that you may not have found with other methods. LI-COR provides the complete package for your research needs, including imagers, software, and infrared reagents that are rigorously validated and tested for every application we recommend, so you can have the confidence to get started right away. Follow the links below to learn more about a few quantitative cell signaling assays that have been incredibly successful with LI-COR^® reagents and imaging systems.

Figure 1. Time course of caspase-3 activation in S2 cells. (A-C) In-Cell Western analysis of S2 cells treated with Actinomycin D (Act D) to induce apoptosis. Each time point was measured in triplicate and stained for anti-active-caspase-3 (A; green) and f-actin (B; red, stained with near-infrared fluorescent phalloidin). Panel C shows merged pseudocolor images. (D) Active-caspase-3 protein levels from (A) were quantified and normalized to f-actin levels in (B) for each time point. The active caspase-3:f-actin ratio at 0min Actinomycin D exposure was designated as 1, and all other ratios are shown relative to this value. Error bars represent the standard error of each independent measurement. Exposure of S2 cells to Actinomycin D increased the relative levels of active caspase-3 over time.

Reprinted with permission from Bond, D.et al. Biol Proced Online. 10(1):20-28(2008).

Other Resources:
Cancer Research Publications Using Odyssey Imaging Systems

Posted in Odyssey Imaging Systems, Proteomics Applications | Tagged cell proliferation assay, cell signaling assays, in-cell western assays, in-cell westerns, infrared fluorescent detection, odyssey applications, p53 activation assays, protein phosphorylation, western blots, western blotting, westerns | Leave a comment

Infrared Fluorescence Imaging for Tumor Detection Using Compounds Labeled with IRDye^® 800CW

Posted on April 27, 2012 by licor-admin

Intraoperative near-infrared fluorescence tumor imaging with vascular endothelial growth factor and human epidermal growth factor receptor 2 targeting antibodies

Anton G.T. Terwisscha van Scheltinga, Gooitzen M. van Dam, Wouter B. Nagengast, Vasilis Ntziachristos, Harry Hollema, Jennifer L. Herek, Carolien P. Schröder, Jos G.W. Kosterink, Marjolijn N. Lub-de Hoog, and Elisabeth G.E. de Vries

Dept of Medical Oncology, Dept of Hospital and Clinical Pharmacy, Dept of Surgery, Dept of Pathology, and Dept of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Groningen, The Netherlands; Institute for Biological and Medical Imaging, Technical University of Munich and Helmholtz Center Munich, Munich, Germany; Optical Sciences Group, MESA1 Institute for Nanotechnology, University of Twente, Enschede, The Netherlands

J Nucl Med 52:1778-85 (2011) doi: 10.2967/jnumed.111.092833

Fluorescence imaging is of great interest for intraoperative tumor detection, but many fluorescent tracers lack tumor specificity. To increase specificity, this study combined monoclonal-antibody-based tumor detection with intraoperative optical imaging, in preclinical mouse models. Bevacizumab (anti-VEGF) and Trastuzumab (anti-HER2) therapeutic antibodies were labeled with the near-infrared (NIR) fluorescent dye IRDye® 800CW (LI-COR Biosciences). Tumor uptake of the tracers (and 89Zr-labeled radioactive counterparts for PET) was assessed in athymic mice with human xenograft tumors, followed by ex vivo biodistribution and pathology studies. Excellent selective tumor uptake was observed. Real-time intraoperative imaging detected tumor lesions, even at the submillimeter level. The authors conclude that NIR fluorescence-labeled antibodies against VEGF or HER2 can be used for highly specific, sensitive detection of tumors in vivo. They are preparing for clinical trials with Bevacizumab-IRDye 800CW.

Other Resources:
In Vivo Imaging Publication List – scientific journal references citing the use of BrightSite™ Optical Imaging Agents or IRDye Infrared Dyes

LI-COR Cancer Web Site

Posted in In vivo Imaging Applications, Pearl Impulse Small Animal Imaging System, Reagents | Tagged Bevacizumab, biodistribution, fluorescent dyes, infrared fluorescent detection, intraoperative optical imaging, irdye 800cw, irdye800cw, near-infrared fluorescence, NIR fluorescent dye, peer-reviewed journal references, Trastuzumab, tumor imaging | Leave a comment

Reprinted with permission from Bond, D.et al. Biol Proced Online. 10(1):20-28(2008).

Anton G.T. Terwisscha van Scheltinga, Gooitzen M. van Dam, Wouter B. Nagengast, Vasilis Ntziachristos, Harry Hollema, Jennifer L. Herek, Carolien P. Schröder, Jos G.W. Kosterink, Marjolijn N. Lub-de Hoog, and Elisabeth G.E. de Vries

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