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Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering


ABL1 tyrosine-kinase inhibitors (TKI) are front-line therapy for chronic myelogenous leukaemia and are among the best-known examples of targeted cancer therapeutics. However, the dynamic uptake into cells of TKIs of low molecular weight and their intracellular behaviour is unknown because of the difficulty of observing non-fluorescent small molecules at subcellular resolution. Here we report the direct label-free visualization and quantification of two TKI drugs (imatinib and nilotinib) inside living cells using hyperspectral stimulated Raman scattering imaging. Concentrations of both drugs were enriched over 1,000-fold in lysosomes as a result of their lysosomotropic properties. In addition, low solubility appeared to contribute significantly to the surprisingly large accumulation of nilotinib. We further show that the lysosomal trapping of imatinib was reduced more than tenfold when chloroquine is used simultaneously, which suggests that chloroquine may increase the efficacy of TKIs through lysosome-mediated drug–drug interaction in addition to the commonly proposed autophagy-inhibition mechanism.

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Figure 1: Structure and spectral properties of five drug molecules: imatinib, nilotinib, chloroquine, GNF-2 and GNF-5.
Figure 2: hsSRS microscopy reveals enrichment of drugs in living cells: the SRS spectra of the bright spots in drug-treated cells match the SRS spectra of the drug in solution, but differ from that of cytosol.
Figure 3: Accumulations of drugs in lysosomes are confirmed by simultaneous two-photon fluorescence imaging of lysotracker and SRS imaging of drug accumulation.
Figure 4: hsSRS imaging of intracellular uptake of the GNF-2 and GNF-5 drugs shows that only cytoplasm spectra have moderate intensity increases at ~1,600 cm−1 caused by drug accumulation.
Figure 5: Time course of lysosomal drug uptake monitored by hsSRS imaging.
Figure 6: Intracellular interaction of TKI drugs with chloroquine measured by hsSRS imaging.


  1. 1

    Melo, J. V. & Barnes, D. J. Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nature Rev. Cancer 7, 441–453 (2007).

    CAS  Article  Google Scholar 

  2. 2

    Manley, P. W. et al. Structural resemblances and comparisons of the relative pharmacological properties of imatinib and nilotinib. Biorg. Med. Chem. 18, 6977–6986 (2010).

    CAS  Article  Google Scholar 

  3. 3

    Adrian, F. J. et al. Allosteric inhibitors of Bcr-abl-dependent cell proliferation. Nature Chem. Biol. 2, 95–102 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Bellodi, C. et al. Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells. J. Clin. Invest. 119, 1109–1123 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Gupta, A. et al. Autophagy inhibition and antimalarials promote cell death in gastrointestinal stromal tumor (GIST). Proc. Natl Acad. Sci. 107, 14333–14338 (2010).

    CAS  Article  Google Scholar 

  6. 6

    Lee, C. M. & Tannock, I. F. Inhibition of endosomal sequestration of basic anticancer drugs: influence on cytotoxicity and tissue penetration. Br. J. Cancer 94, 863–869 (2006).

    CAS  Article  Google Scholar 

  7. 7

    Rubinsztein, D. C., Codogno, P. & Levine, B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nature Rev. Drug Discov. 11, 709–730 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Verschooten, L. et al. Autophagy inhibitor chloroquine enhanced the cell death inducing effect of the flavonoid luteolin in metastatic squamous cell carcinoma cells. Plos One 7, e48264 (2012).

    CAS  Article  Google Scholar 

  9. 9

    Lamoureux, F. et al. Blocked autophagy using lysosomotropic agents sensitizes resistant prostate tumor cells to the novel Akt inhibitor, AZD5363. Clin. Cancer. Res. 19, 833–844 (2013).

    CAS  Article  Google Scholar 

  10. 10

    Zinn, R. et al. Combination treatment with ABT-737 and chloroquine in preclinical models of small cell lung cancer. Mol. Cancer 12, 16 (2013).

    CAS  Article  Google Scholar 

  11. 11

    Maycotte, P. et al. Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy. Autophagy 8, 200–212 (2012).

    CAS  Article  Google Scholar 

  12. 12

    Satori, C. P. et al. Bioanalysis of eukaryotic organelles. Chem. Rev. 113, 2733–2811 (2013).

    CAS  Article  Google Scholar 

  13. 13

    Baik, J. & Rosania, G. R. Molecular imaging of intracellular drug–membrane aggregate formation. Mol. Pharm. 8, 1742–1749 (2011).

    CAS  Article  Google Scholar 

  14. 14

    Ling, J., Weitman, S. D., Miller, M. A., Moore, R. V. & Bovik, A. C. Direct Raman imaging techniques for study of the subcellular distribution of a drug. Appl. Opt. 41, 6006–6017 (2002).

    CAS  Article  Google Scholar 

  15. 15

    Salehi, H. et al. Label-free detection of anticancer drug paclitaxel in living cells by confocal Raman microscopy. Appl. Phys. Lett. 102, 113701 (2013).

    Article  Google Scholar 

  16. 16

    Matthäus, C. et al. in Confocal Raman Microscopy (eds Dieing, T., Hollricher, O. & Toporski, J.) Ch. 7, 137–163 (Springer Series in Optical Sciences 158, Springer, 2011).

    Google Scholar 

  17. 17

    Harada, Y. et al. Intracellular dynamics of topoisomerase I inhibitor, CPT-11, by slit-scanning confocal Raman microscopy. Histochem. Cell Biol. 132, 39–46 (2009).

    CAS  Article  Google Scholar 

  18. 18

    Freudiger, C. W. et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 322, 1857–1861 (2008).

    CAS  Article  Google Scholar 

  19. 19

    Wang, M. C., Min, W., Freudiger, C. W., Ruvkun, G. & Xie, X. S. RNAi screening for fat regulatory genes with SRS microscopy. Nature Methods 8, 135–138 (2011).

    CAS  Article  Google Scholar 

  20. 20

    Freudiger, C. W. et al. Multicolored stain-free histopathology with coherent Raman imaging. Lab. Invest. 92, 1492–1502 (2012).

    CAS  Article  Google Scholar 

  21. 21

    Zhang, X. et al. Label-free live-cell imaging of nucleic acids using stimulated Raman scattering microscopy. Chemphyschem 13, 1054–1059 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Fu, D. et al. Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy. J. Am. Chem. Soc. 134, 3623–3626 (2012).

    CAS  Article  Google Scholar 

  23. 23

    Ozeki, Y. et al. High-speed molecular spectral imaging of tissue with stimulated Raman scattering. Nature Photon. 6, 845–851 (2012).

    CAS  Article  Google Scholar 

  24. 24

    Fu, D., Holtom, G., Freudiger, C., Zhang, X. & Xie, X. S. Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers. J. Phys. Chem. B 117, 4634–4640 (2013).

    CAS  Article  Google Scholar 

  25. 25

    Zhang, D. et al. Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis. Anal. Chem. 85, 98–106 (2013).

    CAS  Article  Google Scholar 

  26. 26

    Colthup, N. B., Daly, L. H. & Wiberley, S. E. Introduction to Infrared and Raman Spectroscopy (Academic Press, 1990).

    Google Scholar 

  27. 27

    Cînt[acaron]-Pînzaru, S. et al. FT-Raman and NIR-SERS characterization of the antimalarial drugs chloroquine and mefloquine and their interaction with hematin. J. Raman Spectrosc. 37, 326–334 (2006).

    Article  Google Scholar 

  28. 28

    De Duve, C. et al. Lysosomotropic agents. Biochem. Pharmacol. 23, 2495–2531 (1974).

    CAS  Article  Google Scholar 

  29. 29

    Trapp, S., Rosania, G. R., Horobin, R. W. & Kornhuber, J. Quantitative modeling of selective lysosomal targeting for drug design. Eur. Biophys. J. 37, 1317–1328 (2008).

    CAS  Article  Google Scholar 

  30. 30

    Codogno, P. & Meijer, A. J. Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ. 12, 1509–1518 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Kimura, T., Takabatake, Y., Takahashi, A. & Isaka, Y. Chloroquine in cancer therapy: a double-edged sword of autophagy. Cancer Res. 73, 3–7 (2013).

    CAS  Article  Google Scholar 

  32. 32

    Poole, B. & Ohkuma, S. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J. Cell Biol. 90, 665–669 (1981).

    CAS  Article  Google Scholar 

  33. 33

    Zheng, N., Zhang, X. & Rosania, G. R. Effect of phospholipidosis on the cellular pharmacokinetics of chloroquine. J. Pharmacol. Exp. Ther. 336, 661–671 (2011).

    CAS  Article  Google Scholar 

  34. 34

    Larson, R. A. et al. Imatinib pharmacokinetics and its correlation with response and safety in chronic-phase chronic myeloid leukemia: a subanalysis of the IRIS study. Blood 111, 4022–4028 (2008).

    CAS  Article  Google Scholar 

  35. 35

    Deininger, M. W. Nilotinib . Clin. Cancer. Res. 14, 4027–4031 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Yamakoshi, H. et al. Alkyne-tag Raman imaging for visualization of mobile small molecules in live cells. J. Am. Chem. Soc. 134, 20681–20689 (2012).

    CAS  Article  Google Scholar 

  37. 37

    Palacios, R. & Steinmetz, M. IL3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo. Cell 41, 727–734 (1985).

    CAS  Article  Google Scholar 

  38. 38

    Daley, G. Q. & Baltimore, D. Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein. Proc. Natl Acad. Sci. 85, 9312–9316 (1988).

    CAS  Article  Google Scholar 

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We thank S. Martin, J. Hastewell, M. Ji, F-K. Lu, C. Freudiger and W. Yang for helpful discussions. We also thank S. Moss for his help with the Raman peak assignments. This work was supported by the National Institute of Health's T-R01 (1R01EB010244-01) awarded to X.S.X.

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D.F., J.Z., Y.K.W. and X.S.X conceived the study. D.F. and J.Z. designed the study. D.F., J.Z., Y.K.W., X.S.X., and P.W.M participated extensively in the scientific discussion about the study. D.F. performed the hsSRS imaging study and analysed the imaging data. W.S.Z. performed the cell proliferation and phospho-STAT5 assay, with supervision from A.W., and J.Z., W.S.Z., A.W. and T.H. prepared the drug and cell samples. D.F., J.Z., P.M. and X.S.X. wrote the manuscript with contributions from W.S.Z., A.W. and T.H.

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Correspondence to X. Sunney Xie.

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The authors declare no competing financial interests.

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Fu, D., Zhou, J., Zhu, W. et al. Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering. Nature Chem 6, 614–622 (2014).

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