Utilizing Stimulated Raman Scattering Microscopy To Study Intracellular Distribution of Label-Free Ponatinib in Live Cells

Abstract

Stimulated Raman scattering (SRS) microscopy represents a powerful method for imaging label-free drug distribution with high resolution. SRS was applied to image label-free ponatinib with high sensitivity and specificity in live human chronic myeloid leukemia (CML) cell lines. This was achieved at biologically relevant, nanomolar concentrations, allowing determination of ponatinib uptake and sequestration into lysosomes during the development of acquired drug resistance and an improved understanding of target engagement.

Figure 1

(a) Chemical structure of ponatinib; (b) Raman spectrum of solid ponatinib. The following peak has been annotated: 2221 cm^–1 (C≡C, ponatinib). Raman spectra were acquired at λ_ex = 532 nm for 10 s using a 50× objective. (c) Energy level diagram showing the working principle of SRS microscopy.

Figure 2

(a) Expression of CYP3A4 in lysates from KCL22 and KCL22^Pon-Res cells. β-actin was used as a loading control. (b) Ponatinib and ponatinib metabolites identified by LC-MS. Cells were treated with ponatinib for 1 h prior to analysis. Mean values from five biological repeats expressed relative to ponatinib.

Figure 3

(a–c) Imaging ponatinib uptake in KCL22^Pon-Res cells. KCL22^Pon-Res cells were treated with DMSO (0.0003%, v/v) or ponatinib (500 nM) for 1, 6, 24, or 48 h (left to right). SRS images acquired at (a) 2940 cm^–1 (CH₃, proteins); (b) 2221 cm^–1 (C≡C, ponatinib); (c) 2257 cm^–1 (off-resonance). Images acquired at 1024 × 1024 pixels, 20 μs pixel dwell time, laser power p300, gain 2 with false colors applied to different detection wavenumbers. Scale bars: 10 μm. (d) Mean ponatinib intensity per cell quantified from 2221 cm^–1 in n = 30 cells, three biological repeats. The Mann–Whitney test was used to compare ponatinib Raman intensity values against the DMSO control.

Figure 4

Multimodal imaging and quantitative assessment of ponatinib uptake in KCL22 and KCL22^Pon-Res cell lines. KCL22 cells were treated with (a) DMSO (0.0003%, v/v) or (b) ponatinib (5 μM, 1 h). KCL22 ^Pon-Res cells were treated with (c) ponatinib (5 μM ponatinib, 1 h). SRS images acquired at (from left to right) 2940 cm^–1 (CH₃, proteins), 2221 cm^–1 (C≡C, ponatinib), 2257 cm^–1 (off-resonance), TPF image acquired at 861 nm (Lysotracker Green), overlay of ponatinib and TPF. (d) Mean ponatinib Raman intensity. (e) Maximum ponatinib Raman intensity inside the vesicles of each individual cell quantified for KCL22 and KCL22^Pon-Res cells that were treated with 5 μM ponatinib for 1 h, n = 30 cells, three biological repeats. (f) Mean ponatinib Raman intensity quantified outside of the vesicles of individual cells, n = 10, three biological repeats. Images acquired at 1024 × 1024 pixels, 20 μs pixel dwell time, laser power p200 gain 1 with false colors applied to different detection wavenumbers. Scale bars: 10 μm. The Mann–Whitney test was used to compare ponatinib Raman intensity values, ***p < 0.0001.

Figure 5

Multimodal imaging and quantitative assessment of the effect of chloroquine treatment on the vesicular uptake of ponatinib. KCL22 and KCL22^Pon-Res cells were treated with (a) ponatinib (5 μM, 1 h), (b) chloroquine (20 μM, 2 h) followed by combination treatment of ponatinib (5 μM, 1 h) and chloroquine (20 μM, 1 h). Images shown (left to right) 2221 cm^–1 (C≡C, ponatinib), overlay TPF image at 861 nm (Lysotracker Green) merged with 2221 cm^–1. (c,d) Mean ponatinib Raman intensity inside the vesicles of each individual cell quantified in (c) KCL22 and (d) KCL22^Pon-Res cell line, n = 10 cells, three biological repeats. Images acquired at 1024 × 1024 pixels, 20 μs pixel dwell time with false colors applied to different detection wavenumbers. Scale bars: 10 μm. (e,f) p-CRKL level quantification from Western blots where KCL22 and KCL22^Pon-Res cells were treated with (left to right) either DMSO (0.0003%, v/v), ponatinib (10, 100, 500 nM, 1 h), or a combination of chloroquine (20 μM, 2 h) pretreatment and ponatinib (10, 100, or 500 nM, 1 h). p-CRKL level was quantified against α-tubulin control and normalized to DMSO using Image Lab Software. One-Way ANOVA (Tukey’s multiple comparisons test) was used to compare ponatinib (10 nM) alone vs CQ combination treatment.