Cargando…

Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer

BACKGROUND: Studying the intracellular distribution of pharmacological agents, including anticancer compounds, is of central importance in biomedical research. It constitutes a prerequisite for a better understanding of the molecular mechanisms underlying drug action and resistance development. Hype...

Descripción completa

Detalles Bibliográficos
Autores principales: Englinger, Bernhard, Kallus, Sebastian, Senkiv, Julia, Heilos, Daniela, Gabler, Lisa, van Schoonhoven, Sushilla, Terenzi, Alessio, Moser, Patrick, Pirker, Christine, Timelthaler, Gerald, Jäger, Walter, Kowol, Christian R., Heffeter, Petra, Grusch, Michael, Berger, Walter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590147/
https://www.ncbi.nlm.nih.gov/pubmed/28882160
http://dx.doi.org/10.1186/s13046-017-0592-3
_version_ 1783262477633454080
author Englinger, Bernhard
Kallus, Sebastian
Senkiv, Julia
Heilos, Daniela
Gabler, Lisa
van Schoonhoven, Sushilla
Terenzi, Alessio
Moser, Patrick
Pirker, Christine
Timelthaler, Gerald
Jäger, Walter
Kowol, Christian R.
Heffeter, Petra
Grusch, Michael
Berger, Walter
author_facet Englinger, Bernhard
Kallus, Sebastian
Senkiv, Julia
Heilos, Daniela
Gabler, Lisa
van Schoonhoven, Sushilla
Terenzi, Alessio
Moser, Patrick
Pirker, Christine
Timelthaler, Gerald
Jäger, Walter
Kowol, Christian R.
Heffeter, Petra
Grusch, Michael
Berger, Walter
author_sort Englinger, Bernhard
collection PubMed
description BACKGROUND: Studying the intracellular distribution of pharmacological agents, including anticancer compounds, is of central importance in biomedical research. It constitutes a prerequisite for a better understanding of the molecular mechanisms underlying drug action and resistance development. Hyperactivated fibroblast growth factor receptors (FGFRs) constitute a promising therapy target in several types of malignancies including lung cancer. The clinically approved small-molecule FGFR inhibitor nintedanib exerts strong cytotoxicity in FGFR-driven lung cancer cells. However, subcellular pharmacokinetics of this compound and its impact on therapeutic efficacy remain obscure. METHODS: 3-dimensional fluorescence spectroscopy was conducted to asses cell-free nintedanib fluorescence properties. MTT assay was used to determine the impact of the lysosome-targeting agents bafilomycin A1 and chloroquine combined with nintedanib on lung cancer cell viability. Flow cytometry and live cell as well as confocal microscopy were performed to analyze uptake kinetics as well as subcellular distribution of nintedanib. Western blot was conducted to investigate protein expression. Cryosections of subcutaneous tumor allografts were generated to detect intratumoral nintedanib in mice after oral drug administration. RESULTS: Here, we report for the first time drug-intrinsic fluorescence properties of nintedanib in living and fixed cancer cells as well as in cryosections derived from allograft tumors of orally treated mice. Using this feature in conjunction with flow cytometry and confocal microscopy allowed to determine cellular drug accumulation levels, impact of the ABCB1 efflux pump and to uncover nintedanib trapping into lysosomes. Lysosomal sequestration - resulting in an organelle-specific and pH-dependent nintedanib fluorescence - was identified as an intrinsic resistance mechanism in FGFR-driven lung cancer cells. Accordingly, combination of nintedanib with agents compromising lysosomal acidification (bafilomycin A1, chloroquine) exerted distinctly synergistic growth inhibitory effects. CONCLUSION: Our findings provide a powerful tool to dissect molecular factors impacting organismal and intracellular pharmacokinetics of nintedanib. Regarding clinical application, prevention of lysosomal trapping via lysosome-alkalization might represent a promising strategy to circumvent cancer cell-intrinsic nintedanib resistance. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13046-017-0592-3) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-5590147
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-55901472017-09-14 Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer Englinger, Bernhard Kallus, Sebastian Senkiv, Julia Heilos, Daniela Gabler, Lisa van Schoonhoven, Sushilla Terenzi, Alessio Moser, Patrick Pirker, Christine Timelthaler, Gerald Jäger, Walter Kowol, Christian R. Heffeter, Petra Grusch, Michael Berger, Walter J Exp Clin Cancer Res Research BACKGROUND: Studying the intracellular distribution of pharmacological agents, including anticancer compounds, is of central importance in biomedical research. It constitutes a prerequisite for a better understanding of the molecular mechanisms underlying drug action and resistance development. Hyperactivated fibroblast growth factor receptors (FGFRs) constitute a promising therapy target in several types of malignancies including lung cancer. The clinically approved small-molecule FGFR inhibitor nintedanib exerts strong cytotoxicity in FGFR-driven lung cancer cells. However, subcellular pharmacokinetics of this compound and its impact on therapeutic efficacy remain obscure. METHODS: 3-dimensional fluorescence spectroscopy was conducted to asses cell-free nintedanib fluorescence properties. MTT assay was used to determine the impact of the lysosome-targeting agents bafilomycin A1 and chloroquine combined with nintedanib on lung cancer cell viability. Flow cytometry and live cell as well as confocal microscopy were performed to analyze uptake kinetics as well as subcellular distribution of nintedanib. Western blot was conducted to investigate protein expression. Cryosections of subcutaneous tumor allografts were generated to detect intratumoral nintedanib in mice after oral drug administration. RESULTS: Here, we report for the first time drug-intrinsic fluorescence properties of nintedanib in living and fixed cancer cells as well as in cryosections derived from allograft tumors of orally treated mice. Using this feature in conjunction with flow cytometry and confocal microscopy allowed to determine cellular drug accumulation levels, impact of the ABCB1 efflux pump and to uncover nintedanib trapping into lysosomes. Lysosomal sequestration - resulting in an organelle-specific and pH-dependent nintedanib fluorescence - was identified as an intrinsic resistance mechanism in FGFR-driven lung cancer cells. Accordingly, combination of nintedanib with agents compromising lysosomal acidification (bafilomycin A1, chloroquine) exerted distinctly synergistic growth inhibitory effects. CONCLUSION: Our findings provide a powerful tool to dissect molecular factors impacting organismal and intracellular pharmacokinetics of nintedanib. Regarding clinical application, prevention of lysosomal trapping via lysosome-alkalization might represent a promising strategy to circumvent cancer cell-intrinsic nintedanib resistance. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13046-017-0592-3) contains supplementary material, which is available to authorized users. BioMed Central 2017-09-07 /pmc/articles/PMC5590147/ /pubmed/28882160 http://dx.doi.org/10.1186/s13046-017-0592-3 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Englinger, Bernhard
Kallus, Sebastian
Senkiv, Julia
Heilos, Daniela
Gabler, Lisa
van Schoonhoven, Sushilla
Terenzi, Alessio
Moser, Patrick
Pirker, Christine
Timelthaler, Gerald
Jäger, Walter
Kowol, Christian R.
Heffeter, Petra
Grusch, Michael
Berger, Walter
Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer
title Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer
title_full Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer
title_fullStr Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer
title_full_unstemmed Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer
title_short Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer
title_sort intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in fgfr-driven lung cancer
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590147/
https://www.ncbi.nlm.nih.gov/pubmed/28882160
http://dx.doi.org/10.1186/s13046-017-0592-3
work_keys_str_mv AT englingerbernhard intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT kallussebastian intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT senkivjulia intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT heilosdaniela intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT gablerlisa intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT vanschoonhovensushilla intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT terenzialessio intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT moserpatrick intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT pirkerchristine intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT timelthalergerald intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT jagerwalter intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT kowolchristianr intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT heffeterpetra intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT gruschmichael intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer
AT bergerwalter intrinsicfluorescenceoftheclinicallyapprovedmultikinaseinhibitornintedanibrevealslysosomalsequestrationasresistancemechanisminfgfrdrivenlungcancer