Temporal imaging of drug dynamics in live cells using stimulated Raman scattering microscopy and a perfusion cell culture system

Stimulated Raman scattering (SRS) microscopy is a powerful technique for visualising the cellular uptake and distribution of drugs and small molecules in live cells under biocompatible imaging conditions. The use of bio-orthogonal groups within the drug molecule, including alkynes and nitriles, has enabled the direct detection of a plethora of bioactive molecules in a minimally perturbative fashion. Limited progress has been made towards real-time detection of drug uptake and distribution into live cells under physiological conditions, despite the accordant potential it presents for preclinical drug development. SRS microscopy has been applied to the study of cellular dynamics of the drug 7RH, which is a potent inhibitor of dicoidin domain receptor 1 (DDR1) and prevents cellular adhesion, proliferation and migration in vitro. The uptake of 7RH into a variety of mammalian cell models was shown to be independent of DDR1 expression. Using a perfusion chamber, the recurrent treatment of live cancer cells was achieved, enabling 7RH uptake to be visualised in real-time using SRS microscopy, after which the viability of the same cellular population was assessed using commercially available fluorescent markers in a multimodal imaging experiment. The effect of 7RH treatment in combination with the chemotherapeutic, cisplatin was investigated using sequential perfusion and time-lapse imaging in the same live cell population, to demonstrate the application of the approach. SRS microscopy also identified potent inhibition of cellular adhesion and migration in breast cancer cell models with increasing 7RH treatment concentrations, thus representing a novel read-out methodology for phenotypic assays of this kind. The direct assessment of drug–cell interactions under physiological conditions offers significant potential for the preclinical drug development process.


Fluorescence imaging
A549 cells were treated with 7RH (500 nM, 2 h) before treating with lysotracker red (75 nM, 30 min). The cells were mounted in PBS containing lysotracker red (75 nM) and affixed onto a microscope slide as performed for live cell SRS imaging. Fluorescence images were acquired using a Leica Microsystems SP8 confocal microscope equipped with a 40× NA 1.10 water immersion objective lens. Lysotracker red signal was detected using λex = 561 nm; λem = 565-650 nm ( Figure S2). Pearson correlation coefficient ( Figure S6) determined using Coloc2 tool on ImageJ.

Figure S1
Investigating the uptake of 7RH into MCF-7 cells. A MCF-7 cells were treated with 7RH at a concentration of 5 μM for the indicated timepoints. Cells were imaged live at the following frequencies: 2930 cm -1 (CH3 symmetric stretch), 2851 cm -1 (CH2 symmetric stretch) and 2217 cm -1 (C≡C, 7RH). An off-resonance image was acquired at 2117 cm -1 which was subtracted from the 2217 cm -1 image. SRS images were acquired across 512×512 pixels, 24 μs/pixel and with false colours applied to detection wavenumbers. Scale bars: 10 μm.  Figure S2 Investigating the uptake of 7RH into A549 cells. A A549 cells were treated DMSO (control) or 7RH at 500 nM for the indicated timepoints. Cells were imaged live at the following frequencies: 2930 cm -1 (CH3 symmetric stretch), 2851 cm -1 (CH2 symmetric stretch) and 2217 cm -1 (C≡C, 7RH). An off-resonance image was acquired at 2117 cm -1 which was subtracted from the 2217 cm -1 image. SRS images were acquired across 512×512 pixels, 24 μs/pixel and with false colours applied to detection wavenumbers. Scale bars: 10 μm. B Quantification of the mean 2217 cm -1 signal per cell in A. The mean 2217 cm -1 intensity per cell is quantified from n>15 cells from three replicate samples. A one-way ANOVA analysis with Tukey post-hoc analysis was performed; ***P≤0.005. C A549 cells were treated with 7RH (500 nM, 2h) before treating with lysotracker red (75 nM, 30 min) and were imaged live. Scale bars 10 μm.

Figure S3
The perfusion chamber set-up and imaging system. A Multimodal SRS microscope at the University of Strathclyde used for live cell imaging studies. B Perfusion chambers used in this study. The chambers are adhesive and are attached to a coverslip. The channel depth of the chambers used in this study was 0.1 mm which contains around 80 μL media, or alternatively, where long-term imaging was performed, a channel depth of 0.6 mm (400 μL media) was used. Product details are available online: https://gracebio.com/products/imagingmicroscopy/coverwell-perfusionchambers-imaging/; accessed 30th May 2022. Only one channel of the 3-channel systems was used for imaging.

Figure S4
Perfusion experiment using multimodal imaging and 7RH. A Schematic workflow describing cell culture under media exchange conditions. B SRS images were acquired at 2930 cm -1 (CH3 symmetric stretch), 2851 cm -1 (CH2 symmetric stretch), 2117 cm -1 (cell-silent region) and 2217 cm -1 (C≡C, 7RH) prior to treatment with 7RH. The cells were then treated with 7RH in RPMI media (1 μM) and SRS images were acquired at 2217 cm -1 every 2 minutes for 40 minutes. Representative images are provided at the indicated timepoints. The cells were then washed with PBS prior to imaging at 2930 cm -1 and staining with a solution of Calcein AM (λex = 488 nm; λem = 495-525 nm, live cells) and ethidium homodimer-1 (EthD-1, λex = 514 nm; λem = 535-650 nm, dead cells) in PBS. The cells were then washed with PBS before permeabilization with Triton-X and subsequent staining with Calcein AM and EthD-1. Scale bars: 20 μm.

Time
Pearson correlation coefficient: 0.72