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.

  • Research Article
  • Published:

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes

Nature Biotechnology volume 17, pages 375–378 (1999)Cite this article

Abstract

We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.

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: (A) Schematic diagram of probe activation.
Figure 2: Fluorescence microscopy of LX-1 cells incubated with C-PGC (magnification 250×).
Figure 3: LX-1 tumor implanted into the mammary fat pad of a nude mouse.
Figure 4: NIRF histology of tumor excised from animal shown in Figure 3 (magnification 200×).

Similar content being viewed by others

References

  1. Devita, V., Hellman, S. & Rosenberg, S. Cancer: Principles and Practice of Oncology (Lippincott, Philadelphia, PA, 1997).

    Google Scholar 

  2. Stocchi, L. & Nelson, H. Diagnostic and therapeutic applications of monoclonal antibodies in colorectal cancer. Dis. Colon. Rectum. 41, 232–250 ( 1998).

    Article  CAS  Google Scholar 

  3. Kopans, D.B. Updated results of the trials of screening mammography. Surg. Oncol. Clin. N. Am. 6, 233–263 (1997).

    Article  CAS  Google Scholar 

  4. Baum, R.P. & Brummendorf, T.H. Radioimmunolocalization of primary and metastatic breast cancer. Q. J. Nucl. Med. 42, 33–42 (1998).

    CAS  PubMed  Google Scholar 

  5. Teates, C.D. & Parekh, J.S. New radiopharmaceuticals and new applications in medicine. Curr. Probl. Diagn. Radiol. 22, 229–266 (1993).

    Article  CAS  Google Scholar 

  6. Dessureault, S. et al. Pre-operative assessment of axillary lymph node status in patients with breast adenocarcinoma using intravenous 99mtechnetium mAb-170H.82. Breast Cancer Res. Treat. 45, 29–37 (1997).

    Article  CAS  Google Scholar 

  7. Pasqualini, R., Koivunen, E. & Ruoslahti, E. Alpha v integrins as receptors for tumor targeting by circulating ligands. Nat. Biotechnol. 15, 542–546 (1997).

    Article  CAS  Google Scholar 

  8. Neri, D. et al. Targeting by affinity-matured recombinant antibody fragments on an angiogenesis associated fibronectin isoform. Nat. Biotechnol. 15 , 1271–1275 (1997).

    Article  CAS  Google Scholar 

  9. Buchsbaum, D.J. Experimental tumor targeting with radiolabeled ligands. Cancer 80, 2371–2377 ( 1997).

    Article  CAS  Google Scholar 

  10. Rusckowski, M., Qu, T., Chang, F. & Hnatowich, D.J. Technetium-99m labeled epidermal growth factor-tumor imaging in mice. J. Pept. Res. 50, 393–401 ( 1997).

    Article  CAS  Google Scholar 

  11. Goodwin, D.A. & Meares, C.F. Pretargeting: general principles: October 10-12, 1996. Cancer 80, 2675– 2680 (1997).

    Article  CAS  Google Scholar 

  12. Jain, R. Delivery of molecular medicine to solid tumors. Science 271, 1079–1080 (1996).

    Article  CAS  Google Scholar 

  13. Muldoon, L.L. et al. Comparison of intracerebral inoculation and osmotic blood-brain barrier disruption for delivery of adenovirus, herpesvirus, and iron oxide particles to normal rat brain. Am. J. Pathol. 147, 1840–1851 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Roselli, M. et al. Systemic administration of recombinant interferon alfa in carcinoma patients upregulates the expression of the carcinoma-associated antigens tumor-associated glycoprotein-72 and carcinoembryonic antigen. J. Clin. Oncol. 14, 2031–2042 (1996).

    Article  CAS  Google Scholar 

  15. Leto, G., Gebbia, N., Rausa, L. & Tumminello, F. Cathepsin D in the malignant progression of neoplastic diseases. Anticancer Res. 12, 235–240 ( 1992).

    CAS  PubMed  Google Scholar 

  16. Keppler, D. et al. Tumor progression and angiogenesis: cathepsin B & Co. Biochem. Cell. Biol. 74, 799–810 (1996).

    Article  CAS  Google Scholar 

  17. Kim, J., Yu, W., Kovalski, K. & Ossowski, L. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 94, 353– 362 (1998).

    Article  CAS  Google Scholar 

  18. Callahan, R., Bogdanov, A., Fischman, A., Brady, T. & Weissleder, R. Preclinical evaluation and phase I clinical trial of a 99mTc labeled synthetic polymer used in blood pool imaging. AJR 171, 137–143 (1998).

    Article  CAS  Google Scholar 

  19. Marecos, E., Weissleder, R. & Bogdanov, A., Jr. Antibody-mediated versus nontargeted delivery in a human small cell lung carcinoma model. Bioconjugate Chemistry 9, 184–191 ( 1998).

    Article  CAS  Google Scholar 

  20. Quintart, J., Leroy-Houyet, M.A., Trouet, A. & Baudhuin, P. Endocytosis and chloroquine accumulation during the cell cycle of hepatoma cells in culture. J. Cell Biol. 82, 644– 653 (1979).

    Article  CAS  Google Scholar 

  21. Liaudet, E., Derocq, D., Rochefort, H. & Garcia, M. Transfected cathepsin D stimulates high density cancer cell growth by inactivating secreted growth inhibitors. Cell Growth Differ. 6, 1045–1052 (1995).

    CAS  PubMed  Google Scholar 

  22. Garcia, M. et al. Biological and clinical significance of cathepsin D in breast cancer metastasis. Stem Cells 14, 642–650 (1995).

    Article  Google Scholar 

  23. Alfano, R.R. et al. Time-resolved and nonlinear optical imaging for medical applications. Ann. NY. Acad. Sci. 838, 14– 28 (1998).

    Article  CAS  Google Scholar 

  24. Alfano, R., Demos, S. & SK, G. Advances in optical imaging of biomedical media. Ann. NY Acad. Sci. 820, 248–270 ( 1997).

    Article  CAS  Google Scholar 

  25. Benaron, D.A., Cheong, W.F. & Stevenson, D.K. Tissue optics. Science 276, 2002–2003 (1997).

    Article  CAS  Google Scholar 

  26. Hebden, J., Arridge, S. & Delpy, D. Optical imaging in medicine: I. experimental techniques. Phys. Med. Biol. 42, 825– 840 (1997).

    Article  CAS  Google Scholar 

  27. Hebden, J.C. & Delpy, D.T. Diagnostic imaging with light. Br. J. Radiol. 70, S206–S214 (1997).

    Article  Google Scholar 

  28. Benaron, D.A., Contag, P.R. & Contag, C.H. Imaging brain structure and function, infection and gene expression in the body using light. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 352, 755–761 (1997).

    Article  CAS  Google Scholar 

  29. Villringer, A. Functional neuroimaging. Optical approaches. Adv. Exp. Med. Biol. 413, 1–18 ( 1997).

    Article  CAS  Google Scholar 

  30. Andersson-Engels, S., Klinteberg, C., Svanberg, K. & Svanberg, S. In vivo fluorescence imaging for tissue diagnostics. Phys. Med. Biol. 42, 815–824 ( 1997).

    Article  CAS  Google Scholar 

  31. Manoharan, R. et al. Raman spectroscopy and fluorescence photon migration for breast cancer diagnosis and imaging. Photochem. Photobiol 67, 15–22 (1998).

    Article  CAS  Google Scholar 

  32. Wu, J., Perelman, L., Dasari, R. & Feld, M. Fluorescence tomographic imaging in turbid media using early-arriving photons and Laplace transforms. Proc. Natl. Acad. Sci. USA 94, 8783– 8788 (1997).

    Article  CAS  Google Scholar 

  33. Yavelow, J., Finlay, T.H., Kennedy, A.R. & Troll, W. Bowman-Birk soybean protease inhibitor as an anticarcinogen. Cancer Res. 43, 2454s–2459s ( 1983).

    CAS  PubMed  Google Scholar 

  34. Yu, A.E., Hewitt, R.E., Connor, E.W. & Stetler-Stevenson, W.G. Matrix metalloproteinases. Novel targets for directed cancer therapy. Drugs & Aging 11, 229–244 (1997).

    Article  CAS  Google Scholar 

  35. Wojtowicz-Praga, S.M., Dickson, R.B. & Hawkins, M.J. Matrix metalloproteinase inhibitors. Invest. New Drugs 15, 61–75 ( 1997).

    Article  CAS  Google Scholar 

  36. Tamura, Y. et al. Highly selective and orally active inhibitors of type IV collagenase (MMP-9 and MMP-2): N-sulfonylamino acid derivatives. J. Med. Chem. 41, 640–649 (1998).

    Article  CAS  Google Scholar 

  37. Kennedy, A.R. Cancer prevention by protease inhibitors. Prev. Med. 22, 796–811 (1993).

    Article  CAS  Google Scholar 

  38. Giavazzi, R. et al. Batimastat, a synthetic inhibitor of matrix metalloproteinases, potentiates the antitumor activity of cisplatin in ovarian carcinoma xenografts. Clinical Cancer Research 4, 985–992 (1998).

    CAS  PubMed  Google Scholar 

  39. Denis, L.J. & Verweij, J. Matrix metalloproteinase inhibitors: present achievements and future prospects. Invest. New Drugs 15, 175–185 (1997).

    Article  CAS  Google Scholar 

  40. Bogdanov, A.A. Jr., Martin, C., Bogdanova, A.V., Brady, T.J. & Weissleder, R. An adduct of cis-diamminedichloroplatinum(II) and poly(ethylene glycol)poly(L-lysine)-succinate: synthesis and cytotoxic properties. Bioconj. Chem. 7, 144–149 (1996).

    Article  CAS  Google Scholar 

  41. Weissleder, R., Cheng, H., Marecos, E., Kwong, K. & Bogdanov, A. Non-invasive in vivo mapping of tumor vascular and interstitial volume fractions. Eur. J. Cancer 34, 1448 –1454 (1998).

    Article  CAS  Google Scholar 

  42. Mitch, W. & Goldberg, A. Mechanism of muscle wasting. N. Engl. J. Med. 335, 1897–1905 (1996).

    Article  CAS  Google Scholar 

  43. Tsurugi, K. & Ogata, K. Presence of a thiol protease in regenerating rat-liver nuclei. Partial purification and some properties. Eur. J. Biochem. 109, 9–15 ( 1980).

    Article  CAS  Google Scholar 

  44. Hashida, S., Towatari, T., Kominami, E. & Katunuma, N. Inhibitions by E-64 derivatives of rat liver cathepsin B and cathepsin L in vitro and in vivo. J. Biochem. (Tokyo) 88, 1805–1811 (1980).

    Article  CAS  Google Scholar 

  45. Barrett, A.J., Kembhavi, A.A. & Hanada, K. E-64 [L-trans-epoxysuccinyl-leucyl-amido(4-guanidino)butane] and related epoxides as inhibitors of cysteine proteinases. Acta Biol Med Ger 40, 1513–1517 (1981).

    CAS  PubMed  Google Scholar 

  46. Shau, H. & Dawson, J.R. Regulation of human natural killing by lysosomotropic and thiol- reactive agents. Immunology 55, 647–654 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Harper, J.W., Hemmi, K. & Powers, J.C. Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor. Biochemistry 24, 1831 –1841 (1985).

    Article  CAS  Google Scholar 

  48. Hudig, D., Allison, N.J., Kam, C.M. & Powers, J.C. Selective isocoumarin serine protease inhibitors block RNK-16 lymphocyte granule-mediated cytolysis. Mol. Immunol. 26, 793– 798 (1989).

    Article  CAS  Google Scholar 

  49. Powers, J.C. et al. Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design. J. Cell Biochem. 39, 33–46 (1989).

    Article  CAS  Google Scholar 

  50. Tearney, G. et al. In vivo endoscopic optical biopsy with optical coherence tomography. Science 276, 2037–2039 (1997).

    Article  CAS  Google Scholar 

  51. el-Shirbiny, A.M. Prostatic specific antigen. Adv. Clin. Chem. 31, 99–133 (1994).

    Article  CAS  Google Scholar 

  52. Himelstein, B.P., Canete-Soler, R., Bernhard, E.J., Dilks, D.W. & Muschel, R.J. Metalloproteinases in tumor progression: the contribution of MMP-9. Invasion Metastasis 14, 246–258 (1994).

    CAS  PubMed  Google Scholar 

  53. Rha, S.Y. et al. Different expression patterns of MMP-2 and MMP-9 in breast cancer. Oncology Reports 5, 875–879 (1998).

    CAS  PubMed  Google Scholar 

  54. Berquin, I.M. & Sloane, B.F. Cathepsin B expression in human tumors. Adv. Exp. Med. Biol. 389, 281– 294 (1996).

    Article  CAS  Google Scholar 

  55. Bogdanov, A., Weissleder, R. & Brady, T. Long-circulating blood pool imaging agents. Advanced Drug Delivery Reviews 16, 335– 348 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Anna Moore for providing all cell cultures and Lee Josephson and Michael Feld for valuable discussion. This study was supported, in part, by RO1 CA74424-01.

Author information

Author notes

  1. Ching-Hsuan Tung and Umar Mahmood: These authors contributed equally to this project.

Authors and Affiliations

  1. Center of Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA

    Ralph Weissleder, Ching-Hsuan Tung, Umar Mahmood & Alexei Bogdanov Jr.

Authors
  1. Ralph Weissleder
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ching-Hsuan Tung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Umar Mahmood
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexei Bogdanov Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ralph Weissleder.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Weissleder, R., Tung, CH., Mahmood, U. et al. In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat Biotechnol 17, 375–378 (1999). https://doi.org/10.1038/7933

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/7933

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing