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

Abstract

ABL1 tyrosine-kinase inhibitors (TKI) are a front-line therapy for chronic myelogenous leukemia and represent the best known examples of targeted cancer therapeutics. However, the dynamic uptake of low molecular weight TKIs into cells and their intracellular behavior is largely unknown due to 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. Both drugs were enriched over 1000-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 by more than 10-fold when using chloroquine simultaneously, suggesting that chloroquine may increase the efficacy of TKIs through lysosome mediated drug-drug interaction besides the commonly proposed autophagy inhibition mechanism.

Lysosomal Sequestration Determines Intracellular Imatinib Levels

“…In line with our observations, it has been reported that drugs that sequester to lysosomes can reach final intracellular concentrations that are much higher than the drug concentrations in the surrounding medium (Kornhuber et al, 2010;Fu et al, 2014).…”

“…It has been suggested that drug transfer directly from the cytosol into the luminal space of lysosomes may proceed via three different pathways: passive diffusion, autophagocytosis, and/ or transporter-mediated accumulation (Kaufmann and Krise, 2007). Since passive diffusion has the highest accumulation capacity and is most efficient, we and others (Fu et al, 2014) speculate that the uptake into lysosomes likely proceeds by simple diffusion, although other mechanisms cannot be excluded. Within the lysosome, imatinib will be effectively protonated and become membrane-impermeable (Szakacs et al, 2005).…”

“…Instead, our findings demonstrate that the IUR of imatinib is primarily determined by lysosomal sequestration. Accumulation of imatinib in lysosomes was described before by Chapuy et al (2009) and, more recently, by Fu et al (2014).…”

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

“…Being a nonlinear optical microscopy, it offers 3D sectioning capability with a diffraction-limited spatial resolution. SRS microscopy has been extensively applied to image biomolecules in cells and tissues (9)(10)(11)(12)(13)(14)(15).…”

Vibrational spectroscopy as a tool for studying drug-cell interaction: Could high throughput vibrational spectroscopic screening improve drug development?

“…In this review, the potential for SRS as the latest development for in vitro drug screening is explored, pointing towards SRS as the future for spectroscopic HTS. In a particularly exciting study, Fu et al [97] used hyperspectral SRS to study the uptake and distribution of two tyrosine-kinase inhibitors (TKI), imatinib and nilotinib, in murine cell line BaF3 cells. Hyperspectral SRS images a number of Raman frequencies, providing additional biochemical information regarding the drug distribution inside cells by allowing both the drug and cellular signals to be recorded.…”