Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes

Nature Protocols volume 11, pages 184–191 (2016)Cite this article

Subjects

Abstract

Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4–8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of BMV109 and general protocol for tissue labeling using a quenched activity-based probe.
Figure 2: Topical staining of fresh-frozen human biopsy tissues with the optical cathepsin probe BMV109.
Figure 3: Inhibition of probe signal by the addition of the cathepsin inhibitor GB111-NH2 on fresh-frozen human biopsy tissues that have been labeled with the optical cathepsin probe BMV109.
Figure 4: Pictorial representation of protocol steps.

Similar content being viewed by others

References

  1. McMahon, J. et al. Influence of condition of surgical margins on local recurrence and disease-specific survival in oral and oropharyngeal cancer. Br. J. Oral Maxillofac. Surg. 41, 224–231 (2003).

    Article  CAS  Google Scholar 

  2. Ravasz, L.A., Slootweg, P.J., Hordijk, G.J., Smit, F. & van der Tweel, I. The status of the resection margin as a prognostic factor in the treatment of head and neck carcinoma. J. Craniomaxillofac. Surg. 19, 314–318 (1991).

    Article  CAS  Google Scholar 

  3. Haque, R., Contreras, R., McNicoll, M.P., Eckberg, E.C. & Petitti, D.B. Surgical margins and survival after head and neck cancer surgery. BMC Ear Nose Throat Disord. 6, 2 (2006).

    Article  Google Scholar 

  4. Nagtegaal, I.D. & Quirke, P. What is the role for the circumferential margin in the modern treatment of rectal cancer? J. Clin. Oncol. 26, 303–312 (2008).

    Article  Google Scholar 

  5. Singletary, S.E. Surgical margins in patients with early-stage breast cancer treated with breast conservation therapy. Am. J. Surg. 184, 383–393 (2002).

    Article  Google Scholar 

  6. Snijder, R.J., Brutel de la Riviere, A., Elbers, H.J. & van den Bosch, J.M. Survival in resected stage I lung cancer with residual tumor at the bronchial resection margin. Ann. Thorac. Surg. 65, 212–216 (1998).

    Article  CAS  Google Scholar 

  7. Kunos, C. et al. Breast conservation surgery achieving ≥2 mm tumor-free margins results in decreased local-regional recurrence rates. Breast J. 12, 28–36 (2006).

    Article  Google Scholar 

  8. Kong, C.S. & Jensen, K.C. Specimen processing. in Practical Breast Pathology 15–23 (Elsevier Saunders, Expert Consult, 2013).

  9. Bydlon, T.M. et al. Performance metrics of an optical spectral imaging system for intraoperative assessment of breast tumor margins. Opt. Express 18, 8058–8076 (2010).

    Article  CAS  Google Scholar 

  10. Jacobs, L. Positive margins: the challenge continues for breast surgeons. Ann. Surg. Oncol. 15, 1271–1272 (2008).

    Article  Google Scholar 

  11. Abraham, S.C., Fox, K., Fraker, D., Solin, L. & Reynolds, C. Sampling of grossly benign breast reexcisions: a multidisciplinary approach to assessing adequacy. Am. J. Surg. Pathol. 23, 316–322 (1999).

    Article  CAS  Google Scholar 

  12. Nguyen, Q.T. & Tsien, R.Y. Fluorescence-guided surgery with live molecular navigation—a new cutting edge. Nat. Rev. Cancer 13, 653–662 (2013).

    Article  CAS  Google Scholar 

  13. Stummer, W. et al. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 7, 392–401 (2006).

    Article  CAS  Google Scholar 

  14. Blum, G. et al. Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nat. Chem. Biol. 1, 203–209 (2005).

    Article  CAS  Google Scholar 

  15. Cutter, J.L. et al. Topical application of activity-based probes for visualization of brain tumor tissue. PLoS ONE 7, e33060 (2012).

    Article  CAS  Google Scholar 

  16. Funovics, M., Weissleder, R. & Tung, C.H. Protease sensors for bioimaging. Anal. Bioanal. Chem. 377, 956–963 (2003).

    Article  CAS  Google Scholar 

  17. Segal, E. et al. Detection of intestinal cancer by local, topical application of a quenched fluorescence probe for cysteine cathepsins. Chem. Biol. 22, 148–158 (2015).

    Article  CAS  Google Scholar 

  18. Verdoes, M. et al. Improved quenched fluorescent probe for imaging of cysteine cathepsin activity. J. Am. Chem. Soc. 135, 14726–14730 (2013).

    Article  CAS  Google Scholar 

  19. Kos, J., Werle, B., Lah, T. & Brunner, N. Cysteine proteinases and their inhibitors in extracellular fluids: markers for diagnosis and prognosis in cancer. Int. J. Biol. Markers 15, 84–89 (2000).

    Article  CAS  Google Scholar 

  20. Fonovic, M. & Turk, B. Cysteine cathepsins and extracellular matrix degradation. Biochim. Biophys. Acta 1840, 2560–2570 (2014).

    Article  CAS  Google Scholar 

  21. Repnik, U., Stoka, V., Turk, V. & Turk, B. Lysosomes and lysosomal cathepsins in cell death. Biochim. Biophys. Acta 1824, 22–33 (2012).

    Article  CAS  Google Scholar 

  22. Zheng, T. et al. Role of cathepsin S-dependent epithelial cell apoptosis in IFN-γ–induced alveolar remodeling and pulmonary emphysema. J. Immunol. 174, 8106–8115 (2005).

    Article  CAS  Google Scholar 

  23. Blum, G., von Degenfeld, G., Merchant, M.J., Blau, H.M. & Bogyo, M. Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes. Nat. Chem. Biol. 3, 668–677 (2007).

    Article  CAS  Google Scholar 

  24. Withana, N.P. et al. Cathepsin B inhibition limits bone metastasis in breast cancer. Cancer Res. 72, 1199–1209 (2012).

    Article  CAS  Google Scholar 

  25. Sanman, L.E. & Bogyo, M. Activity-based profiling of proteases. Annu. Rev. Biochem. 83, 249–273 (2014).

    Article  CAS  Google Scholar 

  26. Lavy, R. et al. A comparative study on two different pathological methods to retrieve lymph nodes following gastrectomy. Int. J. Surg. 12, 725–728 (2014).

    Article  Google Scholar 

  27. Shi, S.R. et al. Evaluation of the value of frozen tissue section used as “gold standard” for immunohistochemistry. Am. J. Clin. Pathol. 129, 358–366 (2008).

    Article  CAS  Google Scholar 

  28. Fischer, A.H., Jacobson, K.A., Rose, J. & Zeller, R. Fixation and permeabilization of cells and tissues. CSH Protoc. 2008 pdb.top36 (2008).

  29. Randall, H.W., Bogdanffy, M.S. & Morgan, K.T. Enzyme histochemistry of the rat nasal mucosa embedded in cold glycol methacrylate. Am. J. Anat. 179, 10–17 (1987).

    Article  CAS  Google Scholar 

  30. Verdoes, M. et al. A nonpeptidic cathepsin S activity-based probe for noninvasive optical imaging of tumor-associated macrophages. Chem. Biol. 19, 619–628 (2012).

    Article  CAS  Google Scholar 

  31. Greenbaum, D. et al. Chemical approaches for functionally probing the proteome. Mol. Cell. Proteomics 1, 60–68 (2002).

    Article  CAS  Google Scholar 

  32. Edgington, L.E. et al. Functional imaging of legumain in cancer using a new quenched activity-based probe. J. Am. Chem. Soc. 135, 174–182 (2013).

    Article  CAS  Google Scholar 

  33. Puri, A.W., Broz, P., Shen, A., Monack, D.M. & Bogyo, M. Caspase-1 activity is required to bypass macrophage apoptosis upon Salmonella infection. Nat. Chem. Biol. 8, 745–747 (2012).

    Article  CAS  Google Scholar 

  34. Edgington, L.E. et al. An optimized activity-based probe for the study of caspase-6 activation. Chem. Biol. 19, 340–352 (2012).

    Article  CAS  Google Scholar 

  35. Edgington, L.E. et al. Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes. Nat. Med. 15, 967–973 (2009).

    Article  CAS  Google Scholar 

  36. Ben-Nun, Y. et al. Photodynamic quenched cathepsin activity based probes for cancer detection and macrophage targeted therapy. Theranostics 5, 847–862 (2015).

    Article  CAS  Google Scholar 

  37. Berkers, C.R. et al. Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib. Nat. Methods 2, 357–362 (2005).

    Article  CAS  Google Scholar 

  38. Patricelli, M.P., Giang, D.K., Stamp, L.M. & Burbaum, J.J. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes. Proteomics 1, 1067–1071 (2001).

    Article  CAS  Google Scholar 

  39. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 96, 14694–14699 (1999).

    Article  CAS  Google Scholar 

  40. Bogyo, M. et al. Applications for chemical probes of proteolytic activity. Curr. Protoc. Protein Sci. 36, 21.17.1–21.17.35 (2004).

    Article  Google Scholar 

Download references

Acknowledgements

We thank P. Chu from the Department of Pathology at Stanford University for assistance with processing the histology samples. We thank C.S. Kong and R. West from the Department of Pathology at Stanford University for intellectual input and pathological analysis of tissue samples. This work was funded by US National Institutes of Health grants R01 HL116307 and R01 EB005011 (to M.B.), by the Howard Hughes Medical Institute Medical Research Fellows program (to M.G.), and by the Stanford Medical Scientist Training Program (to M.G.).

Author information

Author notes

  1. Martijn Verdoes

    Present address: Present address: Radboud University Medical Centre, Nijmegen, the Netherlands.,

  2. Nimali P Withana and Megan Garland: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, Stanford, California, USA

    Nimali P Withana, Megan Garland, Martijn Verdoes, Leslie O Ofori, Ehud Segal & Matthew Bogyo

  2. Department of Chemical Systems and Biology, Stanford University School of Medicine, Stanford, California, USA

    Matthew Bogyo

  3. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

    Matthew Bogyo

Authors
  1. Nimali P Withana
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Megan Garland
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martijn Verdoes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Leslie O Ofori
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ehud Segal
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Matthew Bogyo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.P.W. and M.G. designed and performed all experiments and drafted the manuscript. M.V. and L.O.O. designed and synthesized BMV109 and GB111-NH2 used in experiments. E.S. helped establish the tile scans and obtain tissue samples for analysis. M.B. developed and coordinated the project, analyzed the data and prepared and edited the manuscript.

Corresponding author

Correspondence to Matthew Bogyo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Withana, N., Garland, M., Verdoes, M. et al. Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes. Nat Protoc 11, 184–191 (2016). https://doi.org/10.1038/nprot.2016.004

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.004

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer