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.

Photoacoustic imaging of elevated glutathione in models of lung cancer for companion diagnostic applications

Abstract

Companion diagnostics (CDx) are powerful tests that can provide physicians with crucial biomarker information that can improve treatment outcomes by matching therapies to patients. Here, we report a photoacoustic imaging-based CDx (PACDx) for the selective detection of elevated glutathione (GSH) in a lung cancer model. GSH is abundant in most cells, so we adopted a physical organic chemistry approach to precisely tune the reactivity to distinguish between normal and pathological states. To evaluate the efficacy of PACDx in vivo, we designed a blind study where photoacoustic imaging was used to identify mice bearing lung xenografts. We also employed PACDx in orthotopic lung cancer and liver metastasis models to image GSH. In addition, we designed a matching prodrug, PARx, that uses the same SNAr chemistry to release a chemotherapeutic with an integrated PA readout. Studies demonstrate that PARx can inhibit tumour growth without off-target toxicity in a lung cancer xenograft model.

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

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Application of physical organic chemistry to tune SNAr reactivity for GSH sensing.
Fig. 2: Evaluation of PACDx GSH-responsiveness in vitro and in in cellulo systems.
Fig. 3: Development of PARx, a gemcitabine-based prodrug with matching GSH reactivity.
Fig. 4: Assessment of PARx biodistribution and in vivo activation.
Fig. 5: Determination of PARx efficacy in a murine model of lung cancer.
Fig. 6: Application of PACDx and PARx in unbiased animal studies.

Data availability

All data are available within the Article and its Supplementary Information. Alternatively, data are available upon request from the corresponding author.

References

  1. Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2020. CA Cancer J. Clin. 70, 7–30 (2020).

    Article  PubMed  Google Scholar 

  2. Dracopoli, N. C. & Boguski, M. S. The evolution of oncology companion diagnostics from signal transduction to immuno-oncology. Trends Pharmacol. Sci. 38, 41–54 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Agarwal, A., Ressler, D. & Snyder, G. The current and future state of companion diagnostics. Pharmgenomics Pers. Med. 8, 99–110 (2015).

    PubMed  PubMed Central  Google Scholar 

  4. Wang, L. V. & Hu, S. Photoacoustic tomography: in vivo imaging from organelles to organs. Science 335, 1458–1462 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Heijblom, M. et al. Photoacoustic image patterns of breast carcinoma and comparisons with magnetic resonance imaging and vascular stained histopathology. Sci. Rep. 5, 11778 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yang, M. et al. Photoacoustic/ultrasound dual imaging of human thyroid cancers: an initial clinical study. Biomed. Opt. Express 8, 3449–3457 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Jo, J. et al. A functional study of human inflammatory arthritis using photoacoustic imaging. Sci. Rep. 7, 15026 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Liu, Y., Zhang, L., Li, S., Han, X. & Yuan, Z. Imaging molecular signatures for clinical detection of scleroderma in the hand by multispectral photoacoustic elastic tomography. J. Biophoton. 11, e201700267 (2018).

    Article  Google Scholar 

  9. Reinhardt, C. J. & Chan, J. Development of photoacoustic probes for in vivo molecular imaging. Biochemistry 57, 194–199 (2018).

    Article  CAS  PubMed  Google Scholar 

  10. Knox, H. J. & Chan, J. Acoustogenic probes: a new frontier in photoacoustic imaging. Acc. Chem. Res. 51, 2897–2905 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Li, H., Zhang, P., Smaga, L. P., Hoffman, R. A. & Chan, J. Photoacoustic probes for ratiometric imaging of copper(II). J. Am. Chem. Soc. 137, 15628–15631 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Mishra, A., Jiang, Y., Roberts, S., Ntziachristos, V. & Westmeyer, G. G. Near-infrared photoacoustic imaging probe responsive to calcium. Anal. Chem. 88, 10785–10789 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Roberts, S. et al. Calcium sensor for photoacoustic imaging. J. Am. Chem. Soc. 140, 2718–2721 (2018).

    Article  CAS  PubMed  Google Scholar 

  14. Wang, S. et al. Activatable small-molecule photoacoustic probes that cross the blood–brain barrier for visualization of copper(II) in mice with Alzheimer’s disease. Angew. Chem. Int. Ed. 58, 12415–12419 (2019).

    Article  Google Scholar 

  15. Lucero, M. Y. et al. Activity-based photoacoustic probe for biopsy-free assessment of copper in murine models of Wilson’s disease and liver metastasis. Proc. Natl Acad. Sci. USA 118, e2106943118 (2021).

    Article  CAS  PubMed  Google Scholar 

  16. Knox, H. J. et al. A bioreducible N-oxide-based probe for photoacoustic imaging of hypoxia. Nat. Commun. 8, 1794 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Knox, H. J., Kim, T. W., Zhu, Z. & Chan, J. Photophysical tuning of N-oxide-based probes enables ratiometric photoacoustic imaging of tumor hypoxia. ACS Chem. Biol. 13, 1838–1843 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Chen, M. et al. Simultaneous photoacoustic imaging of intravascular and tissue oxygenation. Opt. Lett. 44, 3773–3776 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhou, E. Y., Knox, H. J., Liu, C., Zhao, W. & Chan, J. A conformationally restricted aza-BODIPY platform for stimulus-responsive probes with enhanced photoacoustic properties. J. Am. Chem. Soc. 141, 17601–17609 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gardner, S. H. et al. A general approach to convert hemicyanine dyes into highly optimized photoacoustic scaffolds for analyte sensing. Angew. Chem. Int. Ed. 60, 18860–18866 (2021).

    Article  CAS  Google Scholar 

  21. Yin, L. et al. Quantitatively visualizing tumor-related protease activity in vivo using a ratiometric photoacoustic probe. J. Am. Chem. Soc. 141, 3265–3273 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Levi, J. et al. Design, synthesis and imaging of an activatable photoacoustic probe. J. Am. Chem. Soc. 132, 11264–11269 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Reinhardt, C. J., Zhou, E. Y., Jorgensen, M. D., Partipilo, G. & Chan, J. A ratiometric acoustogenic probe for in vivo imaging of endogenous nitric oxide. J. Am. Chem. Soc. 140, 1011–1018 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Reinhardt, C. J., Xu, R. & Chan, J. Nitric oxide imaging in cancer enabled by steric relaxation of a photoacoustic probe platform. Chem. Sci. 11, 1587–1592 (2020).

    Article  CAS  Google Scholar 

  25. Lucero, M. Y. et al. Development of NIR-II photoacoustic probes tailored for deep-tissue sensing of nitric oxide. J. Am. Chem. Soc. 143, 7196–7202 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chen, Z. et al. An optical/photoacoustic dual-modality probe: ratiometric in/ex vivo imaging for stimulated H2S upregulation in mice. J. Am. Chem. Soc. 141, 17973–17977 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Forman, H. J., Zhang, H. & Rinna, A. Glutathione: overview of its protective roles, measurement and biosynthesis. Mol. Aspects Med. 30, 1–12 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Balendiran, G. K., Dabur, R. & Fraser, D. The role of glutathione in cancer. Cell Biochem. Funct. 22, 343–352 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Gamcsik, M. P., Kasibhatla, M. S., Teeter, S. D. & Colvin, O. M. Glutathione levels in human tumors. Biomarkers 17, 671–691 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Russo, A., DeGraff, W., Friedman, N. & Mitchell, J. B. Selective modulation of glutathione levels in human normal versus tumor cells and subsequent differential response to chemotherapy drugs. Cancer Res. 46, 2845–2848 (1986).

    CAS  PubMed  Google Scholar 

  31. Giustarini, D. et al. Glutathione, glutathione disulfide and S-glutathionylated proteins in cell cultures. Free Radical Biol. Med. 89, 972–981 (2015).

    Article  CAS  Google Scholar 

  32. Estrela, J. M., Ortega, A. & Obrador, E. Glutathione in cancer biology and therapy. Crit. Rev. Clin. Lab. Sci. 43, 143–181 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Zhang, J. et al. Synthesis and characterization of a series of highly fluorogenic substrates for glutathione transferases, a general strategy. J. Am. Chem. Soc. 133, 14109–14119 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Shibata, A. et al. Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions. Analyst 138, 7326–7330 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. Lee, M. H. et al. Disulfide-cleavage-triggered chemosensors and their biological applications. Chem. Rev. 113, 5071–5109 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Maeda, H. et al. 2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman’s reagent in thiol-quantification enzyme assays. Angew. Chem. Int. Ed. 44, 2922–2925 (2005).

    Article  CAS  Google Scholar 

  37. Hansch, C., Leo, A. & Taft, R. W. A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 91, 165–195 (1991).

    Article  CAS  Google Scholar 

  38. van Iersel, M. L. P. S. et al. Inhibition of glutathione S-transferase activity in human melanoma cells by α,β-unsaturated carbonyl derivatives. Effects of acrolein, cinnamaldehyde, citral, crotonaldehyde, curcumin, ethacrynic acid and trans-2-hexenal. Chem. Biol. Interact. 102, 117–132 (1996).

    Article  CAS  PubMed  Google Scholar 

  39. Rahman, I., Kode, A. & Biswas, S. K. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 1, 3159–3165 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Manegold, C. Gemcitabine (Gemzar®) in non-small cell lung cancer. Expert Rev. Anticancer Therapy 4, 345–360 (2004).

    Article  CAS  Google Scholar 

  41. Hayashi, H., Kurata, T. & Nakagawa, K. Gemcitabine: efficacy in the treatment of advanced stage nonsquamous non-small cell lung cancer. Clin. Med. Insights Oncol. 5, 177–184 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Toschi, L., Finocchiaro, G., Bartolini, S., Gioia, V. & Cappuzzo, F. Role of gemcitabine in cancer therapy. Future Oncol. 1, 7–17 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. He, M., Xuehong, C. & Yepeng, L. Small molecular gemcitabine prodrugs for cancer therapy. Curr. Med. Chem. 26, 5562–5582 (2019).

    Google Scholar 

  44. Ullman-Cullere, M. H. & Foltz, C. J. Body condition scoring: a rapid and accurate method for assessing health status in mice. Lab. Anim. Sci. 49, 319–323 (1999).

    CAS  PubMed  Google Scholar 

  45. Milovanovic, I. S., Stjepanovic, M. & Mitrovic, D. Distribution patterns of the metastases of the lung carcinoma in relation to histological type of the primary tumor: an autopsy study. Ann. Thorac. Med. 12, 191–198 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Glutathione (GSH) Probes (Ursa Bioscience, 2013); https://ursabioscience.com/technology/gsh-probes

  47. Jiang, X. et al. Quantitative real-time imaging of glutathione. Nat. Commun. 8, 16087 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhou, H. et al. Intracellular endogenous glutathione detection and imaging by a simple and sensitive spectroscopic off–on probe. Analyst 143, 2390–2396 (2018).

    Article  CAS  PubMed  Google Scholar 

  49. Guo, R. et al. GSH activated biotin-tagged near-infrared probe for efficient cancer imaging. Theranostics 9, 3515–3525 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zou, Y. et al. Bioimaging of glutathione with a two-photon fluorescent probe and its potential application for surgery guide in laryngeal cancer. ACS Sens. 5, 242–249 (2020).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health (R35GM133581). M.Y.L acknowledges the Alfred P. Sloan Foundation for financial support. Major funding for the 500-MHz Bruker CryoProbeTM was provided by the Roy J. Carver Charitable Trust (Muscatine, Iowa; grant no. 15-4521) to the School of Chemical Sciences NMR Lab. The Q-TOF Ultima mass spectrometer was purchased in part with a grant from the National Science Foundation, Division of Biological Infrastructure (DBI-0100085). We also acknowledge the Core Facilities at the Carl R. Woese Institute for Genomic Biology for access to the Zeiss LSM 700 confocal microscope and corresponding software. We also acknowledge I. Dobrucka and the Molecular Imaging Laboratory at the Beckman Institute for use of the IVIS imaging system. M.Y.L. thanks H. Knox for initial animal training and assistance with the NanoZoomer. We thank T. Bearrood and C. Anorma for assistance with initial confocal imaging experiments, L. Akin for aid with mass spectrometry experiments, S. Anakk and A. Dean for help with interpreting results from H&E staining experiments, N. Herndon and J. Xu for help with generating the orthotopic lung cancer and liver metastasis models, S. Gardner for providing 4T1 tumour models, J. Sarol and A. Kaur from Biostatistics, Epidemiology, & Research Design at the Interdisciplinary Health Sciences Institute (UIUC) for aid in statistical analysis, and A. Bennet for helpful discussions.

Author information

Authors and Affiliations

Authors

Contributions

M.Y.L. performed all experiments in this study that include chemical synthesis, in vitro characterization, cellular studies, tumour model studies, in vivo imaging and sample preparation for ex vivo analysis. J.C. assisted with the blinded animal study. M.Y.L. and J.C. analysed the data and prepared the manuscript. J.C. conceived the project, with intellectual contributions from M.Y.L.

Corresponding author

Correspondence to Jefferson Chan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Chemistry thanks Junjie Yao, Deju Ye and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–34, Tables 1 and 2 and NMR spectra 2–21.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lucero, M.Y., Chan, J. Photoacoustic imaging of elevated glutathione in models of lung cancer for companion diagnostic applications. Nat. Chem. 13, 1248–1256 (2021). https://doi.org/10.1038/s41557-021-00804-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-021-00804-0

This article is cited by

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