Revealing the molecular and biological functions of a clinically approved drug through light microscopy

Session

Poster Session

Authors

Domink Kirchhofer (1), Gerald Timelthaler (1), Elena Mosca (1), Christine Pirker (1), Andy Sombke (2), Walter Berger (1)

Affiliations

1. Center for Cancer Research, Comprehensive Cancer Center, Medical University of Vienna
2. Center of Anatomy and Cell Biology Medical University of Vienna

Keywords

Cancer Research, Olympus Super Resolution, Drug, Lysosomes, Live Cell Microscopy, Cells

Abstract text

Revealing the molecular and biological functions of a clinically approved drug through light microscopy

Dominik Kirchhofer¹, Gerald Timelthaler¹, Elena Mosca¹, Christine Pirker¹, Andy Sombke², Walter Berger¹

¹Center for Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8A, 1090 Vienna, Austria

¹Center of Anatomy and Cell Biology Medical University of Vienna Schwarzspanierstrasse 17, 1090 Vienna, Austria

Nintedanib (BIBF 1120) is a small molecule inhibitor (SMI) clinically approved for the treatment of non-small cell lung cancer (NSCLC) and pulmonary fibrosis. In US, this drug is commercially available under the names Ofev or Vargatef. Due to its strong anti-tyrosine kinase activity, nintedanib efficiently inhibits the molecular pathways following activation of vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor (PDGF). Nevertheless, nintedanib treatment also correlates with several severe side effects - such as nausea, stomach pain and diarrhea – which might even require treatment discontinuation. In addition, acquired resistance represents a commonly unwanted outcome of nintedanib administration to NSCLC patients. Finally, even though nintedanib is a well-known and widely adopted tyrosine kinase inhibitor, mechanisms influencing pharmacodynamics and acquired resistance in nintedanib-treated cancer patients seem to be complex and need further investigation.

In our group we use light microscopy to uncover the exact molecular behavior of this SMI for a better understanding of the drug mode-of-action and improvement of nintedanib-based therapy.

Interestingly, nintedanib-treated cells emit a strong green fluorescent signal following 488 nm excitation as published by Englinger et al., Journal of Experimental & Clinical Cancer Research (2017). Already in basic epifluorescence, a distinct localization of green fluorescent vesicles can be observed within nintedanib-treated cells. In fluorescence localization experiments, cells co-incubated with the lysosomal fluorescent marker Lysotracker-red and nintedanib revealed co-localization of fluorescence signals. This indicates altered fluorescence properties of the drug specifically in the intralysosomal environment. 

With Olympus-Super Resolution spinning disk confocal microscopy and deep learning algorithms, we were able to demonstrate significant lysosomal swelling in nintedanib-treated cells. Considering the acidic pH typically found in lysosomes, we performed fluorescence measurements of nintedanib crystals derived from cell-free buffer solutions at different pH conditions. We identified a strong pH-dependency of nintedanib crystal fluorescence. Interestingly, nintedanib crystals grown from cell-free acidic solution resulted in crystalline structures exerting fluorescence properties similar to those of lysosomes of nintedanib-treated cells. 

Subsequently, laser scanning confocal microscopy revealed different fluorescent compartments of drug-filled lysosomes. This discovery initiated the hypothesis of discrete pH compartments within the drug-filled lysosomes. Adopting the spinning disk confocal microscope, we could distinctly identify membranous structures within nintedanib-treated lysosomes emitting characteristic fluorescence signals. Further analyses of nintedanib-treated cells with transmission electron microscopy suggested induction of irregular phospholipidosis and lamellar body formation. 

Another aspect of nintedanib`s opto-physical properties concerns its photosensitivity. The strong and almost immediate photobleaching of the drug rendered experimental analyses at light microscopy highly challenging. Additionally, any disruption of the fragile system of the functional lysosomes, like treatment with vATPse inhibitors or chemical fixation, eliminated the nintedanib fluorescence signals. 

The exposure of nintedanib-treated cells to light resulted in immediate shrinkage of lysosomes. Following photobleaching during our live cell epifluorescence microscopy studies, we also observed strong phototoxicity in nintedanib-treated cells upon exposure to violet/blue light. 

Summarizing, we could conclude that nintedanib forms, in acidic intracellular vesicles, crystalline-like material sensitive to light exposure, able to induce phototoxicity, and to affect morphology and functionality of treated lysosomes. Indeed, birefringence information obtained using polarization microscopy strengthened our assumption. In conclusion, this research study, conducted at the Center for Cancer Research, proves that, by adopting different light microscopy modalities and techniques, we can improve our understanding concerning the mode-of-action of clinically used drugs and uncover drug pharmacokinetic/pharmacodynamic impacts.