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Journal of Molecular and Clinical Medicine  2018, Vol. 1 Issue (2): 99-106    DOI: 10.31083/j.jmcm.2018.02.007
Research article Previous articles | Next articles
Resistance to crizotinib in a cMET gene amplified tumor cell line is associated with impaired sequestration of crizotinib in lysosomes
Nele Van Der Steen1, 2, 3, Richard J Honeywell3, Henk Dekker3, Johan Van Meerloo4, Jeroen Kole5, René Musters5, Rob Ruijtenbeek6, Christian Rolfo7, Patrick Pauwels1, 2, Godefridus J Peters3, Elisa Giovannetti3, 8, *()
1 Center for Oncological Research, University of Antwerp, Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
2 Department of Pathology, Antwerp University Hospital, Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
3 Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands
4 Department of Pediatric Oncology/Hematology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands
5 Department of Physiology, VU University Medical Center, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands
6 Wolvenhoek 10, 5211 HH 's-Hertogenbosch, The Netherlands
7 Phase I - Early Clinical Trials Unit, Oncology Department, Antwerp University Hospital, Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
8 Cancer Pharmacology Lab, AIRC Start-Up Unit, University of Pisa, Cisanello Hospital, via Paradisa 2, 56124 Pisa, Italy
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Several cMET inhibitors have been developed as novel therapeutic candidates and are under investigation in clinical trials. New preclinical models to study mechanisms underlying resistance to these targeted agents are essential, as resistance acquired during treatment may lead to relapse. The squamous non-small-cell lung cancer (NSCLC) cell line EBC-1 harbors a cMET gene amplification and is sensitive to the cMET inhibitor crizotinib. Here, through multiple step selection with gradually increasing concentrations of crizotinib we established a resistant clone of these cells, termed EBC-CR. A tyrosine kinase activity assay did not show increased signaling of a bypassing pathway or renewed activity of cMET after crizotinib treatment. However, the pH-sensitive pHRodo Green AM probe showed increased acidification of the cytoplasm and lysosomes of EBC-CR cells. Live cell fluorescence imaging also showed an increase in lysosomal number after crizotinib treatment, and the intracellular concentration of crizotinib was significantly lower in crizotinib-resistant EBC-CR cells as compared to the drug sensitive parental EBC-1 cells. These findings suggest that the impaired accumulation of crizotinib in EBC-CR cells, together with the increased acidification of the lysosomes, contributes to crizotinib resistance in cMET-amplified NSCLC cells. In conclusion, the present research identified a novel mechanism used by cancer cells to confer resistance to cMET inhibition. These results prompt future studies for the establishment of innovative therapeutic strategies to overcome resistance to cMET kinase inhibitors by modulation of lysosomal acidification.

Key words:  C-MET      Crizotinib      Resistance      Lysosomes     
Submitted:  03 November 2017      Revised:  04 February 2018      Accepted:  09 February 2018      Published:  20 April 2018     
*Corresponding Author(s):  Elisa Giovannetti     E-mail:

Cite this article: 

Nele Van Der Steen, Richard J Honeywell, Henk Dekker, Johan Van Meerloo, Jeroen Kole, René Musters, Rob Ruijtenbeek, Christian Rolfo, Patrick Pauwels, Godefridus J Peters, Elisa Giovannetti. Resistance to crizotinib in a cMET gene amplified tumor cell line is associated with impaired sequestration of crizotinib in lysosomes. Journal of Molecular and Clinical Medicine, 2018, 1(2): 99-106.

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Fig. 1.  FIJI analysis: partial images of the red and green channels are shown. Yellow markings represent regions of interest (ROIs). (A) Lysotracker Red with markings for 6 selected cells for analysis; (B) pHRodo green with 6 selected cells; (C) pHRodo Green with 6 selected cells, containing markings for lysosomes; (D) pHRodo Green with 6 selected cells with deleted intensities for lysosomes; (E) pHRodo Green with 6 selected cells representing only the lysosomes.

Fig. 2.  Growth inhibition by crizotinib: cells were treated with a crizotinib concentration range of 0-10 $\mu $M for 72 h. Values represent mean $\pm $ SEM of triplicates of 3 separate experiments. Horizontal dashed line represents a growth inhibition of 50%. EBC-CR: crizotinib resistant line; par: parental EBC-1 cell line.

Fig. 3.  Tyrosine kinase activity. Cells were treated for 24 h with their respective IC$_{50}$ values of crizotinib (25 nM for EBC-parental (orange) and 7 $\mu $M for the EBC-CR (blue)) or 0.1% DMSO as control. Cells were lysed and tyrosine kinase activity was determined with the pamgene kinase array. (A) Kinase activity of EBC-CR vs parental is represented in this graph. (B) Inhibition of kinase activity after crizotinib treatment compared to the respective basal activity of both cell lines.

Fig. 4.  Intracellular crizotinib concentrations: cells were treated for 24 h with 5 $\mu $M crizotinib. Pellets were snap-frozen and analysis was performed by liquid chromatography coupled to a tandem mass-spectrometer. EBC-par: parental EBC-1 cell line; EBC-CR: crizotinib resistant EBC-1 daughter cell line. Baf: 50 nM Bafilomycin, added 30 min before crizotinib treatment. *: $p <$ 0.05; **: $p <$ 0.01; ***: $p <$ 0.001.

Fig. 5.  Intracellular effect of crizotinib: cells were seeded in a chambered cover glass and treated with drugs for 24 h. Cells were stained with sunitinib and Lysotracker red for 1h and with pHRodo Green for 30 min. Cells were imaged in depth by dividing them in different layers (z-stack). Z-stacks were imaged using a Leica TCS SP8 STED 3$\times$ microscope. (A) pHRodo Green; (B) Lysotracker red; (C) Sunitinib; (D) pHRodo Green $+$ lysotracker red overlay; (E) Bright field; (F) Bright field $+$ lysotracker red overlay; (G) Bright field, lysotracker red and sunitinib overlay; (H) Bright field, lysotracker red and pHRodo Green overlay. Representative images of one z-plane.

Fig. 6.  Subcellular pHRodo Green fluorescence intensity: cells were treated with crizotinib for 24 h and stained with pHRodo Green, lysotracker red and sunitinib as described in Fig. 5. Analysis with FIJI was performed, selecting the lysosomes based on lysotracker red staining and determining the intensity of pHRodo Green in the cytoplasm and the lysosomes respectively for all z-planes of the stack, while taking into account the cell area and number of stacks for each sample. EBC par: parental cell line; EBC CR: crizotinib resistant daughter cell line; ctrl: DMSO treated cells; criz: treated with 5 $\mu $M crizotinib. *: $p<$ 0.05; **: $p<$ 0.01; ***: $p<$ 0.001.

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