Article Category: Applications

Visualizing Excised Tumor Samples with IRDye Fluorescent Dyes and the Pearl Trilogy

For cancer surgery to be considered fully successful, a tumor must be completely removed with no diseased tissue left behind. Tumor margin analysis in excised tissue samples is a widely-used assessment of whether a tumor was fully removed. Traditionally, margin analysis has been based on subjective evaluations of tissue differences in white light. In many cases, however, differences in cancerous and non-cancerous tissue are very difficult to discern in white light. For some cancers like head and neck squamous cell carcinoma, “positive margin” rates – rates of cancerous tissue (likely) being left behind – are as high as 40% [1].

Novel methods are needed to reduce positive margin rates and provide better clinical outcomes. Fluorescent dyes conjugated to tumor-specific monoclonal antibodies are emerging as a visual aid for tumor margin analysis and are proving to be particularly useful in cancers where positive margins still dominate clinical outcomes.

Evaluating Tumor Margins in Excised Tissue Samples With IRDye® 800CW-Cetuximab Fluorescent Images

Previous blog posts (Optical Probe Specificity and Dual-Modality Labeling with IRDye Near-Infrared Fluorescent Dyes and The Pivotal Role of Validation in Optical Probe Development) have highlighted in vivo and in situ clinical applications of IRDye 800CW-cetuximab. In an article published Rosenthal et.al. in Clinical Cancer Research, fluorescent contrast agents were shown to also improve visualization of cancer margins in excised tissue samples.

In this first-in-human study, 12 patients scheduled to have squamous-cell carcinoma tumors removed from the head or neck were given an infusion of IRDye 800-cetuximab prior to surgery. Fresh tumor tissue sections were imaged ex vivo with the LI-COR Pearl® Impulse imager to “determine the ability of tumor fluorescence to differentiate tumor from normal tissue and identification of positive margins” [1]. Like the intraoperative in situ results, the histopathological ex vivo results were also promising. The authors noted that “Fluorescence in histologically confirmed tumor tissue was significantly greater (P<0.001) than negative epithelial margins, muscle, and skin for each dose” [1].

Rosenthal and colleagues also utilized the Odyssey® imaging platform (LI-COR Biosciences) to quantify fluorescence in slide-mounted tissue sections after imaging with Pearl Impulse imager within the surgery suite. The fluorescent images taken in the Odyssey imager were correlated with routine H&E (hematoxylin and eosin) stains to compare IRDye 800CW-cetuximab against established pathological standards, corroborating the results of the Pearl Impulse scans.

The authors concluded “Here we demonstrate for the first time that … cetuximab-IRDye 800CW can be safely administered as a tumor-specific contrast agent,” and that “The use of real-time fluorescence imaging during ablative procedures to delineate tumor margins has the potential to reduce morbidity, improve locoregional control and reduce operative time “[1].

Cetuximab-IRDye 800CW in the Clinic Part 2: Enhanced Pathological Assessment with Fluorescent Probes

In a 2016 article published in The Journal of Pathology: Clinical Research, Warram et.al. utilized IRDye 800CW-cetuximab to address the lack of “tools to consistently discriminate tumor and normal tissue in real-time” for pathological assessments of tumor margins in head and neck squamous cell carcinoma (HNSSC) [2]. In this proof-of-principle study, the authors tested fluorescent assessment of diseased tissue margins against standard histological methods. 80 tumor margin assessments were collected from post-resection wound beds of 20 mice with SCC1-luc tumors after administration of IRDye 800CW-cetuximab.

The results were significant: fluorescent images improved pathologist prediction of positive tumor margins from 21/39 (49%) to 33/39 (85%), or a 36% increase in sensitivity in positive tumor margin predictions. The authors noted false negative margin predictions lead to a 90% 5-year post resection mortality rate, demonstrating the magnitude of impact that fluorescent-guided tumor margin analysis may have on patient outcomes.


Figure 1: Fluorescent analysis of primary tumor specimen [2]. Circles represent positive or negative biopsy-confirmed cancer cells, showing the distribution and specificity of IRDye 800CW to diseased cells.
Figure 2: Demonstration of how specificity translates into margin classification in excised tissue samples [2].

The authors ultimately concluded that “The ability of fluorescence assessment to localize diseases in these margins was sensitive and specific with a NPV of 87%, which was superior to both surgical assessment (58%) and pathological assessment (66%)” [2]. The authors also noted that “This report provides evidence that tumor-specific fluorescence can be used by the surgeon or pathologist to guide sampling for frozen sections” [2]. Although the current research does not suggest that fluorescence is a bona fide replacement for current methods, “Fluorescence-guided pathology can … be easily implemented into the clinical care workflow and used in adjunct to fluorescence-guided surgery to help guide the pathologist when assessing margins for both intraoperative assessment and staging” [2].

Conclusion

In certain cancers like head and neck squamous cell carcinoma, even the most effective treatment still has relatively high rates of failure. Novel methods are needed to reduce the failure rate and provide better clinical outcomes.

Fluorescent dyes conjugated to tumor-specific monoclonal antibodies are emerging as a promising visual aid for tumor analysis. Rosenthal et.al. and Warram et.al. showed how fluorescent dye-antibody conjugates can enhance tissue assessments, also demonstrating the versatility of fluorescent probes for both in situ and in vitro assessments.

For more exciting clinical applications of IRDye probes and conjugates, visit the Optical Probe Development and Molecular Activity Measurement web pages.

Do you think IRDye infrared dye-labeled probes could be used in your research? Let us help! Contact LI-COR Custom Services.

References

  1. Rosenthal, E.L., et al. Safety and Tumor-specifity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer. Clin Cancer Res 2015, Aug; 21(16):3658-3666. doi: 10.1158/1078-0432.CCR-14-3284.
  2. Warram, J. M., de Boer, E., van Dam, G. M., Moore, L. S., Bevans, S. L., Walsh, E. M., & Young, E. S., et.al. (2016, March 2). Fluorescence Imaging to Localize Head and Neck Squamous Cell Carcinoma for Enhanced Pathological Assessment. Journal of Pathological Cancer Research, 2(2), 104-112. doi:10.1002/cjp2.40

The Pivotal Role of Validation in Optical Probe Development

Underlying every successful clinical application of fluorescent probes is a rigorous, strategic probe validation process. Previous blog posts have discussed the importance of probe specificity, binding affinity, and distribution, and the validation process is where these and other parameters are determined. Given the time and expenses involved in clinical translation, efficient and accurate probe validation is essential.

A Systematic Approach to Developing and Validating Optical Imaging Contrast Agents

In a foundational 2007 article published in Analytical Biochemistry, Kovar et.al. demonstrated the principal steps involved in developing fluorescent optical probes suitable for human clinical use. The authors began with a comprehensive review of NIR fluorochromes, such as IRDye® 800CW, and targets and ligands for fluorescent optical probes, including monoclonal antibodies, tumor surface proteins, peptides, and small molecules. Steps for development and validation described in the remainder of the article are “applicable to any dye-conjugated optical agent,” demonstrating the versatility of this systematic approach [1].

STEP 1: CONJUGATION

The first step in probe development is the conjugation of target and NIR fluorochrome. In this study, the authors conjugated IRDye 800CW to five commercial epidermal growth factor (EGF) sources in equivalent ratios and evaluated signal intensity via the In-Cell Western™ (ICW) assay method. ICW imaging demonstrated variation between the signal strength of each EGF source. Because “Variations in signal strength measured in this fashion have the potential to predict probe performance in vivo,” choosing the correct target for conjugation is a critical first step [1].

STEP 2: SPECIFICITY AND BINDING AFFINITY VALIDATION IN VITRO

Prior to animal imaging, probe specificity and binding affinity are validated in vitro. Here, the authors again chose the In-Cell Western (cytoblot) method to evaluate IRDye 800CW EGF for binding specificity. Specifically, PC3M-LN4 and 22Rv1 human prostate adenocarcinoma cells were cultured in microtiter plates and were “treated with serial dilutions of labeled EGF to verify a high affinity binding of EGFR-targeted dye” [1]. Specificity was then determined by blocking access of EGF to the EGF receptor with an anti-EGFR monoclonal antibody, and by competition with unlabeled EGF [1]. The authors concluded that “Characterization of the targeting agent in a cell-based assay can simplify probe development,” and that although “success in a cell-based assay format does not guarantee performance in vivo, failure at this step is generally predictive of failure in the animal” [1].

Learn more about the In-Cell Western Method.

STEP 3: SPECIFICITY, DISTRIBUTION, AND CLEARANCE VALIDATION IN VIVO

Next, the authors validated probe specificity and clearance in vivo in living mice. This is the process of determining probe uptake by the target vs surrounding tissues, the rate at which the probe is expelled from the organism, and the probe to background ratio in the body of mice. First, clearance kinetics of unconjugated IRDye 800CW were established. Then, clearance measurements for IRDye 800CW-anti-EGFR antibody conjugates were established in mice bearing PC3M-LN4 subcutaneous or orthotopic tumors to ensure the conjugate did not accumulate non-specifically in the mouse. This interaction between clearance kinetics and specificity can impact in vivo analysis by falsely indicating tumor tissue in pooled optical probe in the liver or kidneys.

STEP 4: EX VIVO VALIDATION IN EXCISED TISSUE SAMPLES

Lastly, the PC3M-LN4 tumors were excised and injected intravenously with IRDye 800CW EGF or pre-injected with C225 anti-EGFR monoclonal antibody prior to dosing with IRDye 800CW EGF for ex vivo analysis. After imaging, the distribution of the IRDye probe was assessed and fluorescence signal area was determined against a control, optical agent only, and C225 competition.

The authors ultimately concluded that “Fluorochrome-labeled molecular probes are valuable tools for non-invasive longitudinal study of tumorigenesis and metastasis, preclinical studies of the effects of therapeutic agents, and pharmacokinetic and pharmacodynamic studies of drug-target interactions” [1]. Since this paper was published over a decade ago, IRDye labeled molecular probes have been featured in more than 20 clinical trials around the world.

EGFR-Specific Optical Probes Improve EGFRvIII-Targeted Molecular Imaging

In a 2014 study published in Cancer Biology & Therapy, Gong et.al. demonstrated an application of structured probe validation. In this investigative study, the authors created and validated the specificity, binding affinity, distribution, and clearance of three EFGRvIII-targeted fluorescent optical probes. An EFGR-specific affibody, the therapeutic antibody panitumumab, and an EGF ligand were conjugated with IRDye 800CW to create three probes: Aff800, Pan800, and EGF800. The experimental target was rat glioma cell line F98, a known over-expresser of EGFR. A control assay contained EGFR expression-devoid F98 parent (F98-p) cells, and two experimental assays contained F98-derived transgenic cells expressing EGFR or EGFR-vIII. Each probe was compared with each cell-based assay and imaged for comparison, creating a total of nine experimental conditions.

Comparison of specificity and binding affinity between the experimental conditions was performed in cell-based assays using the In-Cell Western method. All three probes successfully bound to F98-EGFR, and Pan800 and Aff800 bound to F-98vIII. Signal intensity was also compared in the nine conditions to assess if binding was dose-dependent. The authors concluded “Little signal was detected when Aff800 and Pan800 were incubated with F98-p [the expression-devoid parent cells], indicating that their interactions with F98-EGFR and F98-vIII is highly specific” [2].

Next, probe target specificity to EGFR- and EGFRvIII-expressing tumors and clearance profiles were assessed in vivo. Mice with F98-p, F98-EGFR, and F98-vIII xenograft tumors were injected with the three probes and imaged with the Pearl® Impulse Small Animal Imaging System (LI-COR Biosciences). Fluorescent signal to background ratio for each of the nine probe-tumor conditions were assessed, again revealing highly specific interactions between Aff800 and Pan800 with F98-EGFR and F98-vIII expressing tumors. EGF-800 signal was high in F98-EGFR tumors, corroborating cell based assay results.

Lastly, tumor-containing organs were dissected and imaged ex vivo, validating the previously-measured fluorescence signals and assessing probe distribution in targets. This last step in validation was consistent with in vitro scans, again demonstrating Aff800 and Pan800 affinity to F98-EGFR and F98-vIII tumors. Based on these results, Aff800 and Pan800 may be valuable in “imaging of heterogenous tumors containing both versions of receptors” (EGFR, EGFRvIII) [2]. Alternatively, due to optimal clearance kinetics, Aff800 EGF800 is preferable in scenarios where imaging must be performed within a short time after probe administration” [2].

Conclusion

This example from Gong et.al. demonstrates how different optical probes may be used to assess different tumor properties. Additionally, the authors showed how structured approach to optical probe validation successively builds proof of probe parameters and provides several spots for go/no-go decision-making. Proof of probe parameters are critical for clinical application, and clear decision points provide efficiency and allow for early determination if a probe is worth exploring further.

Do you think IRDye dye-labeled probes could be used in your research? LI-COR Custom Services include chemistry and probe conjugation, biological assay services, translational services, and manufacturing, including cGMP manufacturing. Request a free project evaluation today.

REFERENCES

  1. Kovar, J. L., Simpson, M. A., Geschwender, A., & Olive, D. M. (2007, August 1). A Systematic Approach to the Development of Fluorescent Contrast Agents for Optical Imaging of Mouse Cancer Models. Analytical Biochemistry, 367(1), 1-12. doi:10.1016/j.ab.2007.04.011
  2. Gong, H., Kovar, J. L., Cheung, L., Rosenthal, E. L., & Olive, D. M. (2014, February). A Comparative Study of Affibody, Panitumumab, and EGF for Near-Infrared Fluorescence Imaging of EGFR- and EGFRvIII-expressing Tumors. Cancer Biology & Therapy, 15(2), 185-193. doi:10.4161/cbt.26719

No-Hassle Near-Infrared Fluorescent and Bioluminescent Optical Imaging

Pearl Trilogy Workstation
The Pearl® Trilogy Small Animal Imaging System is a simple, economical way for your lab to begin in vivo optical imaging. Starting at $65,000 (US List), the Pearl Trilogy offers affordable small animal near-infrared fluorescent and bioluminescent optical imaging.

Combine the Pearl Trilogy with the Odyssey® CLx Imager and create a complete workstation that allows you to go from in vitro to in vivo to ex vivo using the same trusted technology and reagents.

The revolutionary FieldBrite™ Xi2 technology approach to imaging in the Pearl Trilogy Imager allows you to detect smaller and deeper targets accurately in a single acquisition. You don’t need to worry about saturation or having to adjust images.

FieldBrite Xi2 is specifically optimized for small animal imaging, ensuring that you get the highest quality data possible. FieldBrite Xi2 technology offers:

  • Uniform Illumination
  • Excellent Sensitivity
  • Wide Dynamic Range

Bioluminescent Optical Imaging

Figure 1. Bioluminescent detection of subcutaneous 4175 (LM2) luc + human triple negative breast cancer cell line in athymic NCR nu/nu mouse.

Image courtesy of Michael Chiorazzo, Elizabeth Browning and Jim Delikatny, Small Animal Imaging Facility, University of Pennsylvania.

Ask for a quote today. Get your lab up and running quickly – it’s easy to use so little training will be needed for even novices. And, you can count on the reliable data for your in vivo imaging research from the Pearl Trilogy Small Animal Imaging System.

Use Near-Infrared Fluorescent Probes for Pharmacokinetics and Biodistribution Studies

In Vivo Imaging with NIR Fluorescent ProbesNon-invasive preclinical imaging methods are critical for development of imaging agents and targeted therapeutics. Pharmacokinetics is the study of what the body does to a drug with respect to biodistribution and clearance. Traditionally-used radiolabeled probes have limitations such as cost, access, and safety. Near-infrared (NIR) fluorescence imaging offers a powerful alternative to radiolabeled probes for pharmacokinetics and biodistribution studies. NIR fluorescent optical imaging agents can be used to image the whole animal over time. And, more than one agent can be tracked in the same animal if each agent is labeled with a spectrally-distinct fluorophore.

In this webinar, Dr Amy Geschwender examines several case studies from the literature, and discusses:

  • Why NIR fluorescent probes are widely used for in vivo imaging
  • How fluorescence imaging of excised tissues and tissue sections is used to examine biodistribution in more detail
  • How to measure serum half-life and % injected dose per gram with NIR fluorescent probes


This webinar features data from the Pearl® Small Animal Imaging System, which was recently honored by Frost & Sullivan, in addition to advancements in NIR technology. Click here to learn more about this award.

Visit our website to learn more about BrightSite™ Optical Imaging Agents and IRDye® infrared dyes that can be used for your pharmacokinetic and biodistribution studies.

Use NEW! VRDye™ Secondary Antibodies to Correlate Near-Infrared Application Data with Microscopy and Flow Cytometry Data

VRDye Secondary Antibody IconsLI-COR is expanding its portfolio of reagents by offering VRDye™ 490, VRDye 549, and IRDye® 650 dye-labeled secondary antibodies and protein labeling kits. These new secondaries can be used for for a variety of applications, including immunofluorescence microscopy and flow cytometry. Just like our IRDye dye-labeled secondary antibodies, these new visible fluorescence antibodies are highly cross-adsorbed. The dyes are conjugated to the same antibodies as the existing IRDye secondary antibodies, which are used for Western blotting and In-Cell Western™ Assay applications. This gives researchers the ability to correlate microscopy and flow data with Western blot and cell-based assay data. The VRDye secondary antibodies are suitable for multiplex experiments when combined with other secondary antibodies labeled with proper fluorescent dyes and using instrumentation with appropriate excitation and detection capabilities.

Immunofluorescence staining of tubulin protein in HeLa cells.

Figure 1. Immunofluorescence staining of tubulin protein in HeLa cells. Cells were cultured on cover slips. After fixation and permeabilization, cells were incubated with rabbit anti-tubulin mAb (CST), followed by VRDye™ 490 Goat anti-Rabbit IgG (LI-COR P/N 926-49020). Nuclei were stained with DAPI. Image acquired with Olympus IX81 microscope.

Immunohistochemistry staining of EGFR protein on F98-EGFR tumor slides.

Figure 2. Immunohistochemistry staining of EGFR protein on F98-EGFR tumor slides. F98-EGFR tumors were snap-frozen in O.C.T. ™ compound and sectioned at 4-µm thickness. After fixation and permeabilization, cells were incubated with rabbit anti-EGFR mAb (CST), followed by detection with VRDye™ 549 Goat anti-Rabbit IgG (LI-COR P/N 926-54020). DAPI was used to stain the nuclei. Image acquired on Olympus IX81 microscope.

In addition, many researchers use labeled primary antibodies for flow cytometry. LI-COR now offers visible fluorescent dye protein labeling kits that are ideal for customers who need to label custom monoclonal antibodies for this application.

Visit our website for more information on these new visible fluorescence antibodies and protein labeling kits or to order them for your research.

Rethinking the Traditional Western Blot

Traditional Western blotting is a labor-intensive process that includes gel electrophoresis, protein transfer to a blotting membrane, incubation with primary and secondary antibodies, and chemiluminescent or fluorescent detection of target proteins. (View a typical Western blotting workflow.) Day-to-day reproducibility is poor, because small variations in lysate preparation, gel loading, electrophoresis, transfer, and detection are unavoidable sources of technical variability.

Snapshot of In-Cell Western Assay MethodThe In-Cell Western™ (ICW) Assay, a quantitative immunofluorescent method, is an alternative to traditional Western blots that increases both reproducibility and sample throughput. (View a typical ICW workflow.)

We recently hosted a webinar called “Rethinking the Traditional Western Blot”, during which John Lyssand, PhD, from LI-COR Biosciences, discussed the In-Cell Western Assay and an example of its use in neuroscience research, in this case, Alzheimer’s Disease. The In-Cell Western Assay enables screening and analysis of many more samples in each experiment, eliminates error-prone protocol steps, and delivers higher reproducibility for biological and technical replicates.

ICW Use: Tau Protein Accumulation and InhibitionThe data presented demonstrated how ICW assays were used in Alzheimer’s Disease research to screen HSP90 inhibitors for their effectiveness in reducing tau activity levels. Dr Lyssand discussed how and why the In-Cell Western Assay is superior to traditional methods for screening of cell samples.

If you didn’t have a chance to join us in September for “Rethinking the Traditional Western blot”, you can view this webinar online and on-demand. Check out the information on In-Cell Western assays on our website. You can also read Professor Dickey’s white paper as cited above that outlines how he and his group used higher throughput method to study Alzheimer’s Disease.

New Cell Stain Increases Ease of Use for In-Cell Western™ Normalization

CellTag 700 Stain ICW Kits for Quantitative Cell Signaling AnalysisHave you ever wanted to try an in-cell ELISA but you just weren’t sure how to get started? With the new LI-COR® CellTag™ 700 Stain, a near-infrared fluorescent, non-specific cell stain that provides accurate normalization to cell number, you have a easier — and more affordable — way to try this powerful application. CellTag 700 Stain accumulates in both the nucleus and cytoplasm of permeabilized cells, and provides linear fluorescent signal across a wide range of cell types and cell numbers (see Figure 1 below). CellTag 700 Stain is applied to the cells during incubation with IRDye® 800CW secondary antibody, and enables accurate measurement of target protein levels with much higher throughput than Western blotting.

CellTag 700 Stain - Linear Relationship between Fluorescence and Cell Number.

Figure 1. Linear Relationship between Fluorescence and Cell Number. Two-fold serial dilutions of A431 and NIH/3T3 cells were plated in 96-well plate, then fixed, permeabilized, stained with CellTag 700 Stain, and detected with Odyssey Classic (Resolution: 169um; Quality: medium; Focus offset: 4.0mm; Intensity: 5). The Trim Signals were used to generate the graphs.

CellTag 700 Stain ICW Kits offer a convenient way to try cell-based In-Cell Western Assays. Each kit includes blocking buffer, IRDye® 800CW secondary antibody for detection of a specific protein target in the 800 nm channel, and CellTag 700 Stain to normalize well-to-well variations in cell number. This cost-effective normalization method makes quantification of the target protein more precise.
In-Cell Western Normalization with CellTag 700 Stain in EGF-stimulated A431 Cells.Figure 2. In-Cell Western Assay with CellTag 700 Stain in EGF-stimulated A431 Cells. (Go to the CellTag 700 Stain Overview page for more details on this data).

Try one of our new In-Cell Western Assay Kits with CellTag 700 Stain today and find out just how easy it is to perform fast, cost-effective cell-based Western assays.

Journal Articles Citing Use of Odyssey® or Pearl® Imaging Systems and Near-Infrared Fluorescence

The following are 4 journal references citing the use of either Odyssey or Pearl Imaging Systems.

Affibody-DyLight Conjugates for in vivoAssessment of HER2 Expression by Near-Infrared Optical Imaging.

Zielinski R, M Hassan, I Lyakhov, D Needle, V Chernomordik, A Garcia-Glaessner, Y Ardeshirpour, J Capala and A Gandjbakhche
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
PLoS ONE 7(7): e41016 (2012). doi:10.1371/journal.pone.0041016

The HER2/neu gene is overexpressed in ~20% of invasive breast carcinomas. in vivo assessment of HER2 levels would aid development of HER2-targeted therapies and perhaps assist in selection of appropriate treatment strategies. This study describes HER2-specific probes for in vivo monitoring of receptor levels by near-infrared (NIR) optical imaging. Affibody molecules were labeled with DyLight750 dye, and affinity and specificity were confirmed in vitro. in vivo, Affibody-DyLight probes accumulated in HER2-positive breast cancer xenografts, but not in HER2-negative xenografts.

Fluorescent images were acquired at different time intervals after probe injection.
Fluorescent images were acquired at different time intervals after probe injection. Mouse bearing BT-474 xenograft tumor was injected with 10 µg HER2-Affibody-DyLight750 conjugate. Images were acquired every second for 1 minute with Pearl Impulse Imager (LI-COR Biosciences). doi:10.1371/journal.pone.0041016.s004

Animals were imaged with a custom NIR fluorescence-lifetime imaging system. The Pearl® Impulse Imager (LI-COR Biosciences) was used to monitor real-time accumulation of the Affibody probe in HER2-positive tumors during very early time points. Probe was injected during image acquisition, and images were captured every second for 1 minute. Probe accumulation in the kidney first, followed by tumor accumulation. Tumor fluorescence could still be detected 5 days after probe injection. This Affibody conjugate is useful for preclinical monitoring of HER2 status, and may have clinical utility.


Disruption of Kv1.3 Channel Forward Vesicular Trafficking by Hypoxia in Human T Lymphocytes

AA Chimote, Z Kuras, and L Conforti
Departments of Internal Medicine and Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
Journal of Biological Chemistry 287(3): 2055-67 (2012) DOI 10.1074/jbc.M111.274209

In solid tumors, hypoxia decreases immune surveillance. Kv1.3 channels on T lymphocytes are down-regulated by an unknown mechanism, inhibiting T cell function. The authors hypothesize that changes in membrane trafficking cause reduced expression of Kv1.3 at the cell surface. On-Cell Western cell based assays (Odyssey® Imager, LI-COR Biosciences) were extensively used to measure cell surface expression of Kv1.3.

Chronic hypoxia decreased cell surface expression of Kv1.3 in Jurkat cells. Inhibition of protein synthesis, degradation, or endocytosis did not block this effect. However, inhibition of forward trafficking in the trans-Golgi with brefeldin A (BFA) prevented hypoxia-induced reduction of Kv1.3 cell surface expression. Confocal microscopy confirmed retention of Kv1.3 in the trans-Golgi. Quantitative fluorescent Westerns (Odyssey Imager) demonstrated that expression of AP-1, which is required for clathrin-coated vesicle formation, is downregulated by hypoxia. These data indicate that chronic hypoxia disrupts clathrin-mediated forward trafficking of Kv1.3, thereby reducing immune surveillance by T cells.


Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

M Lee, A Ye, A Gardino, A Hheijink, P Sorger, G MacBeath, and M Yaffe
Dept of Biology, David H. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA.
Cell 149:780-794 (2012). doi: 10.1016/j.cell.2012.03.031

Historically, standard treatments for human malignancies have been single drug therapies that cause DNA damage. Systems-based approaches and network analysis are now being used to examine how signaling can be re-wired by drug treatments that target dynamic network states. This study suggests that the timing and order of administration of certain drug combinations increases treatment effectiveness. Lee et al. pre-treated cells with epidermal growth factor receptor (EGFR) inhibitors, prior to DNA-damaging chemotherapy drugs.

Pre-treatment with erlotinib (an EGFR inhibitor) sensitized triple-negative breast cancers (TNBCs) to the DNA damage agent doxorubicin, and cell death increased by nearly 500%. Sensitization occurred only if the drugs were given sequentially. Transcriptional, proteomic, and computational analysis of signaling networks showed that dynamic network re-wiring was responsible for sensitization. Quantitative Westerns (Odyssey Imager; high-density, 48-sample blots) were used to monitor systems-level signaling dynamics. Erlotinib treatment made cells more susceptible to DNA damage by reactivating an apoptotic pathway that had been suppressed.


Investigation of Ovarian Cancer Associated Sialylation Changes in N-linked Glycopeptides by Quantitative Proteomics

V Shetty, J Hafner, P Shah, Z Nickens, and R Philip
Immunotope, Inc., Doylestown, Pennsylvania, USA
Clinical Proteomics 9:10 (2012) doi:10.1186/1559-0275-9-10.

CA125 is currently used as a biomarker for ovarian cancer, but is ineffective for detection of early stage disease. Previous research indicates that the level of sialic acid in total serum of ovarian cancer patients is elevated. Based on that idea, the authors suggest using N-linked sialyated glycopeptides as potential targets for early stage ovarian cancer biomarker discovery.

Shetty et al. used Lectin-directed Tandem Lableing (LTL) and iTRAQ quantitative proteomics to investigate N-linked sialyated glycopeptides, and identified 10 that were up-regulated in serum from ovarian cancer patients. Quantitative Western blot analysis of lectin-enriched glycoproteins (Odyssey Imager) was used to confirm the proteomic analysis. In ovarian cancer, increased sialylation of haptoglobin, PON1, and Zinc-alpha-2-glycoprotein was observed. Cancer-specific sialylation of glycopeptides may be a target for biomarker discovery.


Check out some of our Publications Lists for:

Analyze Glycoproteins with Sensitive, Quantitative Infrared Fluorescent Techniques

O-Linked Glycan StructureGlycosylation is one of the most common and important events in post-translational modification. Over half of all proteins are believed to be glycosylated, and the resulting glycoconjugates play an important role in many biological processes. They have been connected to instances of cancer development, retrovirus infection, and other diseases. In an effort to understand these diseases, glycoprotein analysis has become a growing area of research. (See examples of typical glycan structures.)

Analysis of glycoproteins requires sensitive and quantitative applications. LI-COR offers a single, optimized solution using the Odyssey® Systems and IRDye® labeled conjugates to analyze glycoproteins. This solution provides sensitive and quantitative results using two-color near-infrared detection at 700 nm and 800 nm wavelengths. Operating at this wavelength produces lower background from biological materials, buffer components, and standard membranes used in Western blotting and lectin binding applications and, thus, superior data.

Outlined below are a variety of applications for several one-color, visible glycoprotein applications that have been adapted to near-infrared fluorescence detection on an Odyssey Imaging System:

Read Glycoprotein Detection with the Odyssey Infrared Imaging System for more indepth information on using your Odyssey Infrared Imaging System for glycobiology research.

Create a Complete Molecular Imaging Workstation

pearltrilogybuildsystemCombining the Odyssey® CLx Infrared Imaging System with the Pearl® Small Animal Imaging System creates a versatile molecular imaging workstation for in vivo and in vitro imaging.

BrightSite™ Optical Imaging Agents or probes developed using IRDye® infrared dyes can be used for in vitro, in vivo, and tissue imaging. This technology offers researchers the ability to take research from the cell to the animal, all within one lab.

Odyssey CLx Infrared Imaging System Capabilities:

  • Cell-based assays (binding capacity, specificity, competition, etc.) for optical agent development
  • Histology and whole organ imaging for studying clearance and specificity
  • Simultaneous two-color detection for two targets or one target with sample normalization

Pearl Small Animal Imaging Capabilities:

Validation Workflow and Molecular Imaging WorkstationFigure 1. Validation and Use of an IRDye Fluorescent Probe. After probe labeling, in vitro cellular assays and microscopy are used to confirm specificity. The desired target is then imaged in animals. Excised organs and tissues> can be examined for more detailed localization of the probe. Animal image captured with Pearl Imaging System. A more comprehensive discussion of approaches for the development of fluorescent contrast agents has also been published. Reference: Kovar, et al. Anal Biochem 367(2007) 1-12.

Molecular imaging – achieved with near-infrared fluorescent technology from LI-COR!