Article Category: Cancer Research

Optical Surgical Navigation Clinical Trials with IRDye Infrared Dyes

Recent blog posts have highlighted some of the most exciting clinical developments of IRDye® near-infrared fluorescent dyes as surgical aides. Beyond these examples, IRDye infrared dye products are being used in more than 20 clinical trials around the globe, many of which involve the deadliest and most common cancers.

Brain and Pancreatic Cancer

Glioblastoma and glioma brain and pancreatic adenocarcinoma tumors are particularly aggressive forms of cancer that are difficult to treat. IRDye dye-conjugated optical probes are currently being investigated as an alternative to traditional surgical treatments for these cancers.

Very recently in April 2018, Deling Li and colleagues announced the results their first-in-human study a novel 68Ga-IRDye 800CW-BBN positron emission tomography (PET) and near-infrared fluorescent (NIRF) dual modality optical probe in patients with glioblastoma multiforme (GBM) [1, 2]. The authors used preoperative PET and intraoperative fluorescence-guided surgery methods, demonstrating that the “novel dual model imaging technique is feasible for integrated pre- and intra-operative targeted imaging via the same molecular receptor improved intraoperative GBM visualization and maximum safe resection” [1]. GBM is the deadliest and most aggressive glioma type, and novel GBM therapies have the potential to impact many lives.

Learn More About Dual Imaging Modalities with IRDye Optical Probes.


Figure 1. Intraoperative images of glioblastoma multiforme resection with IRDye 800CW-BBN fluorescent dyes [1].

The Dartmouth-Hitchcock Medical Center is sponsoring an investigatory study utilizing IRDye 800CW for ABY-029 in patients with recurrent glioma to determine if proper tumor/background ratio is present for surgical resection [3]. This study is expected to conclude in September 2019. A similar study is being conducted by Eben Rosenthal (Stanford University) to evaluate the effectiveness of IRDye 800CW-panitumumab in glioma surgery for distinguishing tumor cells from other central nervous system tissue [5]. Rosenthal’s study is set to conclude in 2022. Rosenthal has also studied Cetuximab-IRDye 800CW intraoperatively in patients with malignant glioma [4].

Pancreatic cancer has one of the highest mortality rates of all cancers. G.M. van Dam at the University Medical Center Groningen is currently evaluating dosing of IRDye 800CW-bevacuzimab conjugates for pancreatic adenocarcinoma [7]. Similar studies set to conclude soon by Eben Rosenthal are also evaluating the intraoperative potential of IRDye conjugates in pancreatic cancer [6].

Breast and Colorectal Cancer

Breast and colon cancers are some of the most common cancers around the globe with millions of cases diagnosed each year. Two very recent clinical trials by G.M. van Dam at University Medical Center Groningen in collaboration with Martini Hospital Groningen and UMC Utrecht have evaluated the anti-vascular endothelial growth factor antibody-IRDye 800CW-bevacizumab conjugate in breast cancer study [8, 9]. van Dam’s studies are currently assessing dosing, uptake, quantification, and localization of the optical probe, as well as determining if the conjugate is appropriate for intraoperative breast cancer surgery [8, 9].

Learn More About Optical Probe Validation and Parameters.

Colorectal cancer is also a very common diagnosis around the world. Two recent clinical trials by W.B. Nagengast of the University Medical Center Groningen evaluated IRDye 800CW-bevacizumab for colorectal cancer diagnosis [10, 11]. Nagengast noted “there is a need for better visualization of polyps during surveillance endoscopy in patients with hereditary colon cancer syndromes like Familial Adenomatous Polyposis (FAP) and Lynch Syndrome (LS), to improve adenoma detection rate,” also stating that “optical molecular imaging of adenoma associated biomarkers is a promising technique to accommodate this need” [10]. In addition to detection, IRDye 800CW-bevacizumab is also being investigated as an aid for narrowing down specific colon cancer management surgeries and therapies [11].

Other Clinical Applications

While the focus of this blog post series has been on pre-clinical and clinical applications of IRDye conjugates for cancer, these are not the only applications. Currently, IRDye fluorophores are being evaluated in several trials for clinical use in non-cancer surgeries, like abdominal and urological. Ureters, the path for urine between the kidneys and bladder, and the urethra, often present difficulties in abdominal and urological surgery. By illuminating these pathways with fluorescent dyes, the anatomy is bright and clear, which may allow surgeons to more precisely navigate around them during surgery.

A study published in 2017 by T.G. Barnes et.al. in Techniques in Coloproctology demonstrated urethra illumination in cadavers with IRDye 800BK as a method for enhancing low rectal surgical navigation [15]. The authors concluding that “IRDye 800BK is a promising alternative to ICG [indocyanine green] in visualizing the urethra,” and that “Its greater depth of penetration may allow earlier detection of the urethra during surgery and prevent wrong plane surgery sooner” [15].


Figure 2: Intraoperative images of low rectal surgery demonstrating urethral fluorescence at various depths of incision. The first row of images shows how IRDye 800BK illuminates the urethra through layers of tissue to better guide further incisions [15].

LI-COR Biosciences is currently sponsoring a clinical trial being conducted at the University of Alabama-Birmingham evaluating the dose response and safety/toxicity of IRDye 800BK for ureter delineation, which is expected to conclude soon [12]. A similar study is being conducted at the University of Oxford, sponsored by the Oxford University Hospitals NHS Trust, and is evaluating the signal/background ratio of IRDye 800BK in the ureter [13]. This trial will likely conclude later this year.


Figure 3: Intraoperative laproscopic images showing ureter delineation with IRDye 800BK. In minimally invasive surgery (such as laproscopy) ureters may be difficult to see in white light without fluorescent illumination [16].

One final application of IRDye fluorescent dyes is in endometriosis surgery. G.M. van Dam at the University Medical Groningen is investigating the feasibility of IRDye 800CW-bevacizumab for endometriosis surgery [14]. van Dam noted that “incomplete resection of endometriosis lesions often results in recurrence of symptoms and the need for repeated surgery, with considerable associated morbidity” [14]. This is the first feasibility study for IRDye 800CW and endometriosis, and is expected to conclude in early 2019.

Conclusion

This blog series on optical surgery navigation has illuminated the potential of IRDye fluorescent dyes as surgical aids. From the deadliest cancers to routine, minimally-invasive gynecological surgeries, IRDye fluorophores present a valuable opportunity for visualizing, understanding, and ultimately treating various diseases.

Could your probe be our next clinical study? For questions regarding probe development services, contact LI-COR Custom Services.

More information about the studies mentioned can be found at ClinicalTrials.gov at the locations mentioned below or on our Optical Probe Development and Molecular Activity Measurement web pages.

IRDye fluorophores are only used for investigative purposes in clinical trials.

References

  1. Li, D., Zhang, J., Chi, C., Xiao, X., Wang, J., Lang, L., & Ali, I. (2018, April 3). First-In-Human Study of PET and Optical Dual-Modality Image-Guided Surgery in Glioblastoma Using 68Ga-IRDye800CW-BBN. Theranostics, 8(9), 2508-2520. doi:10.7150/thno.25599
  2. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02901925, A Microdose Evaluation Study in Recurrent Glioma (ABY-029); 2016 December. Available from: https://clinicaltrials.gov/ct2/show/NCT02901925.
  3. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02910804, IRDye800CW-BBN PET-NIRF Imaging Guiding Surgery in Patients With Glioblastoma; 2015 November. Available from: https://clinicaltrials.gov/ct2/show/NCT02910804.
  4. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02855086, Cetuximab-IRDye 800CW in Detecting Tumors in Patients With Malignant Glioma Undergoing Surgery; 2016 October. Available from: https://clinicaltrials.gov/ct2/show/NCT02855086.
  5. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT03510208, Panitumumab-IRDye800 in Diagnosing Participants With Malignant Glioma Undergoing Surgery; 2018 May 14. Available from: https://clinicaltrials.gov/ct2/show/NCT03510208.
  6. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02736578, Cetuximab-IRDye800CW and Intraoperative Imaging in Finding Pancreatic Cancer in Patients Undergoing Surgery; 2016 July. Available from: https://clinicaltrials.gov/ct2/show/NCT02736578.
  7. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02743975, Near-Infrared Image Guided Surgery in Pancreatic Adenocarcinoma (PENGUIN); 2016 September. Available from: https://clinicaltrials.gov/ct2/show/NCT02743975.
  8. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02583568, Fluorescence Guided Surgery in Breast Cancer (MARGIN); 2015 October. Available from: https:/clinicaltrials.gov/ct2/show/NCT02583568.
  9. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT01508572, VEGF-Targeted Fluorescent Tracer Imaging in Breast Cancer; 2011 October. Available from: https://clinicaltrials.gov/ct2/show/NCT01508572.
  10. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02113202, Molecular Fluorescence Endoscopy in Patients With Familial Adenomatous Polyposis, Using Bevacizumab-IRDye800CW (FLUOFAP); 2014 March. Available from: https://clinicaltrials.gov/ct2/show/NCT02113202.
  11. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT01972373, Visualization of Rectal Cancer During Endoscopy, Using a Fluorescent Tracer (RAPIDO-TRACT);2013 October. Available from: https://clinicaltrials.gov/ct2/show/NCT01972373.
  12. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT03106038, Dose-Escalation Study of a Constrant Agent for Delineation of Urological Anatomy in Minimally Invasive Surgery; 2017 May 4. Available from: https://clinicaltrials.gov/ct2/show/NCT03106038.
  13. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT03387410, Ureter Identification with IRDye 800BK; 2018 April 6. Available from: https://clinicaltrials.gov/ct2/show/NCT03387410.
  14. ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US). Identifier NCT02975219, Feasibility Study of Using Molecular Fluorescence Guided Surgery in Endometriosis (Endo-Light); 2017 May 1. Available from: https://clinicaltrials.gov/ct2/show/NCT02975219.
  15. Barnes, T. G., Volpi, D., Cunningham, C., Vonjovic, B., & Hompes, R. (2018, February 19). Improved Urethral Fluorescence During Low Rectal Surgery: A New Dye and a New Method. Techniques in Coloproctology, 22, 115-119. doi:10.1007/s1051-018-1757-6
  16. Al-Taher, M., van den Bos, J., Schols, R. M., Kubat, B., Bouvy, N. D., & Stassen, L. S. (2018, February 2). Evaluation of a Novel Dye for Near-Infrared Fluorescence Delineation of the Ureters During Laparoscopy. British Journal of Surgery BJS Open. doi:10.1002/bjs5.59

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

Your Commitment to Producing Reproducible Research is Critical

Reproducibility is becoming a highly discussed issue in all research sciences. The ability for major research findings to be independently replicated after an initial experiment is essential to building upon foundational discoveries. When experiments are not conducted thoroughly or published articles lack sufficient details for replication, we lose the ability to move ahead with accurate science. This is a major problem for researchers today.

NIH QuoteThis problem will only begin to be addressed if institutions, universities, industry, and others alike take on the responsibility of producing scientific experiments and reporting scientific methods that can be replicated at a later date. Thus, the conventions of reproducible science are paramount to the future of biomedical research findings in particular.

Several areas are being scrutinized in the discussion on biomedical reproducibility. Including:

  • Thoroughness of experimental details in journal articles
  • Review of studies submitted to journals
  • Scientific fraud
  • Utilization of highly reproducible techniques

reproducibility initiative logo smallThoroughness in research is important, because without knowing all the details of a foundational experiment future scientists are unable to efficiently build upon that research. To increase thoroughness, the Reproducibility Initiative, headed by Elizabeth Iorns, is advising full disclosure of experimental procedures in published papers. The initiative aims to identify and reward high quality, published research that can be successfully reproduced by independent validation labs. The first step in this process is pinpointing a pool of research that is true and accurate —a task The Reproducibility Initiative has begun by investigating 50 of the most impactful cancer biology studies from 2010 – 2012.

In light of the growing concern regarding scientific reproducibility, the review processes for scientific journal submissions are seeing stringent changes as well. The plans to increase the reproducibility of published papers laid out by the National Institutes of Health (NIH) at the beginning of the year are just one example. In their plan the NIH instituted a training module for enhancing the transparency of cited methods, provided a checklist for routine evaluation of grant applications, and began to urge scientific journals to revise their current review practices. Since then, high-impact journals like Nature and Science have implemented precautionary statistical checklists intended to qualify submitted research papers before publishing them in their magazines.

Unfortunately, though, there are times reported science is proved to be inaccurate, and fraudulent papers claiming breakthrough research are retracted. These retractions can severely affect scientists who have based their careers on such published inaccuracies.

ireland flag smallIn response, Ireland has taken precautions against fraudulent publication. By the end of the year The Science Foundation Ireland will be funding auditors at leading universities. The auditors will look into best practices related to research, procedures “for reporting and investigating misconduct; whether management has followed those procedures in real cases; and whether any investigations have been carried out to a satisfactory standard.” The purpose of these audits is to encourage researchers to take protocols seriously and to put standards in place that will decrease the likelihood of scientific fraud occurring.

Another area of the reproducibility discussion highlights the need for highly consistent research techniques and instrumentation. The nature of complex research and varying protocols between labs can cause inherent fluctuating results from experiment to experiment. To help combat the variability, there is a need for improved and consistent training of researchers using Western blotting and other scientific techniques in their research, just as there is a need for the instruments researchers use to be of the highest quality and to generate reproducible results. Putting more emphasis on training researchers and utilizing the highest quality instruments will help to improve the reproducibility of the studies research labs are currently conducting.

Only time will tell if the scientific community will really begin to take the issues and repercussions of reproducible science seriously. While science is shifting it is important you stay ahead of the curve and close the gaps in your research confidently. Your commitment to producing reproducible research is critical to redressing the reputation of the scientific method from beginning research stages to the published piece.

Are your findings reproducible? Read more about how reproducibility is affecting the life sciences and where the future of Western blotting may be headed.

If you’d like to learn more about reliable instrumentation, check out LI-COR Imaging Systems, which offer a digital imaging solution that ensures reproducible results. See how LI-COR can help you improve your research.

Listen to the Reproducibility in Science Webinar Series:

Studying Colon Cancer? Use the C-DiGit® Scanner for Western Blots.

Cortactin (CTTN) is a substrate of Src tyrosine kinase involved in actin dynamics, and is overexpressed in several cancers. Phosphorylated cortactin (pTyr421) is required for cancer cell motility and invasion. This study demonstrates elevated expression of pTyr421-CTTN in primary colorectal tumors, with no change in mRNA levels. Curcumin (a natural compound derived from the spice turmeric) reduced association of CTTN with plasma membrane fractions in surface biotinylation, mass spectrometry, and Western blot experiments. Curcumin also decreased pTyr421-CTTN levels in certain cell lines.

Western blot analysis of cortactin, actin and GAPDH proteins

Figure 1. Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p<0.05 DMSO vs. curcumin; Student’s T-test. All images are representative of three independent experiments.

Quantitative chemiluminescent Westerns (using the LI-COR® C-DiGit Blot Scanner and SuperSignal® West Pico substrate) showed that curcumin treatment reduced CTTN levels in cytoskeletal fractions, and increased cytoplasmic localization. In Western blotting and immunofluorescent microscopy studies, curcumin induced dephosphorylation of cortactin by activation of the PTPN1 protein tyrosine phosphatase. Western blotting demonstrated that biotinylated curcumin directly binds to PTPN1, and that curcumin blocks the interaction between CTTN and p120 catenin. Curcumin inhibits cell migration in colon cancer cells overexpressing CTTN, and it holds promise as a colon cancer therapeutic.

Reference:

pTyr421 cortactin is overexpressed in colon cancer and is dephosphorylated by curcumin: involvement of non-receptor type 1 protein tyrosine phosphatase (PTPN1)
VM Radhakrishnan, P Kojs, G Young, R Ramalingam, B Jagadish, EA Mash, JD Martinez, FK Ghishan, PR Kiela
University of Arizona Health Sciences Center, Tucson, Arizona; Arizona Cancer Center, Tucson, AZ, USA
PLoS ONE 9(1): e85796 (2014). 10.1371/journal.pone.0085796

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:

In-Cell Western™ Assay Webinar – Applications Review

In-Cell Western Assays - Fluorescent ImmunoassaysFor those of you that like to watch videos and listen to information, here is a great webinar on In-Cell Western Assays.

In this In-Cell Western webinar, the basics of ICW assays are covered and these applications:

For more information on these plate-based fluorescent immunoassays, go to the In-Cell Western assay application page. There are also several sample protocols and information on how to set up, optimize, and analyze ICW assays.

In-Cell Western™ Assay Application: Response of COS-7 Cells to Hydroxyurea

Application: Detecting phospho-p53 in COS cells in response to Hydroxyurea


Example of In-Cell Western Assay: Effects of Hydroxyurea on phospho-p53 on COS-7 cells

In this In-Cell Western assay application, the response of COS-7 cells to increasing doses of hydroxyurea was measured by a specific antibody (Anti-phospho-p53 from Cell Signaling Technology, P/N 9286) that detects phosphorylated-p53 (Ser16). Total ERK1 was used for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 nm channels detecting phosphorylated-p53 (Ser16) and total ERK1, respectively. Background wells were incubated with secondary antibody but no primary antibody. IRDye® 680RD secondary antibodies were used for detection in the 700nm channel and IRDye 800CW secondary antibodies were usd for detection in the 800nm channel.

Dose response graph of % induction of p53 phosphorylation with hydroxyurea in COS-7 cells

The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16). Plate-based assays such as this can be imaged on the Odyssey® CLx or Odyssey Sa Infrared Imaging System.

For more uses of In-Cell Westerns Assays, visit our website.

Are You Studying Phosphorylation or Quantitative Cell Signaling Analysis? How About for IC50 Determinations?

In-Cell Western Assays - Fluorescent Immunoassays

In a previous post, I talked about how In-Cell Western™ assays could be used when studying apoptosis. So, you may be asking yourself, for what other applications can quantitative cell signaling analysis be used? GREAT QUESTION!!

Well, In-Cell ELISAs (as these immunofluorescent assays are also called) have been used successfully in studying protein phosphorylation. Whether you are looking at the effects of drug compounds on signaling pathways, or the timing/kinetics of signal transduction, or trying to determine the IC50 of compounds, In-Cell Western assays are a valuable tool.

Here are two examples of data from IC50 and EC50 determination experiments.
Use of labeling for In-Cell Western Assay normalization.
Figure 1. Use of cell labeling for In-Cell Western normalization. A) HeLa cells were treated with increasing amounts of rapamycin in a 384-well format. Fixed cells were stained with phospho-rpS6 antibody and NHS-ester reactive dye (for cell number). Dose dependent inhibition of phospho-rpS6-staining yielded an IC50 of 224 pM (n=4). B) Raw microplate image. For details, see Hoffman, GR et al. Assay Drug Dev Tech 8(2):186-99 (2010).

Dose titration of Wnt3a treatment of mouse L-cells.  An In-Cell Western Assay Application.
Figure 2. Dose titration of Wnt3a treatment of mouse L-cells. Half-maximal activation (EC50) of cellular beta-catenin levels occurs at 33 ng/ml ligand. Hannoush, RN. PLoS One. 3(10):e3498 (2008). Creative Commons license 2.5.

To help you get started in designing your experiment, here is a complete sample protocol for measuring IC50 of the inhibitor PD168393 in A431 cells responding to epidermal growth factor (EGF).

Check here for future blog posts on other applications of quantitative cell signaling analysis!

Seeding Cells in Microplates for In-Cell Western™ Assays – Hints & Tips

In-Cell Western Assays - Fluorescent Immunoassays
One of the first steps in an In-Cell Western Assay experiment is to seed cells into the wells of a tissue culture microplate. Cell density is more important for some cell lines than others. In particular, cells that depend more on extracellular activity for proliferation (such as epithelial cells) are affected to a greater extent by initial growth conditions. There are three factors to consider when seeding cells:

  1. Plates: For most adherent cells that stick to wells tightly (e.g. A431, HeLa, HEK293, CHO), we recommend regular tissue culture microplates with low auto-fluorescence, such as Nunc P/N 167008. For adherent cells that could detach from wells during In-Cell Western assay wash steps (e.g. NIH/3T3), we recommend Poly-D-lysine coated 96-well microplates.
  2. Cell seeding density: Typically, 15,000 to 40,000 cells are seeded per well. Two to three days are usually required for cells to reach the appropriate confluency, depending on growth rate. Seeding with low cell numbers is recommended if you plan to culture for several days before use. Plates seeded with higher cell numbers will be ready to use earlier.
  3. Confluence: To obtain maximal fluorescent signals, complete or near complete confluency is recommended for cells that stick to wells tightly. For cells that adhere loosely to wells, such as NIH3T3, 70% confluency should be used. Please note that cell type and experimental conditions may affect the acceptable level of growth confluency.

The example below illustrates the importance of cell seeding density for A431 cells. As shown in the corresponding graph, cell growth is greatly inhibited when there are too few neighboring cells.

cell seed plate

Graph showing why Seeding Plates for ICW Assays is Important

Use Quantitative Cell Signaling to Study Apoptosis

AGAIN with the quantitative cell signaling! YES! because it is so versatile!! I am sure you will find that this will become a valuable technique to use in your research.

This quantitative immunofluorescent assay – the one that we call an In-Cell Western™ (ICW) Assay – can be used to study a variety of mechanisms. Here is an example of an ICW used to study apoptosis.

As you may already know, 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. The In-Cell Western Assay is a very helpful research tool for scientists who are quantifying cell signaling.

Time Course of Caspase-3 Activation in SP2 Cells Performed using an In-Cell Western Assay

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).

Here is our complete apoptosis assay example protocol of the HeLa cellular response to anisomycin treatment (detailing the seeding, induction, and detection).