NanoSIMS combined with fluorescence microscopy as a tool for subcellular imaging of isotopically labeled platinum-based anticancer drugs

Abstract

Multi-elemental, isotope selective nano-scale secondary ion mass spectrometry (NanoSIMS) combined with confocal laser-scanning microscopy was used to characterize the subcellular distribution of ¹⁵N-labeled cisplatin in human colon cancer cells. These analyses indicated predominant cisplatin colocalisation with sulfur-rich structures in both the nucleus and cytoplasm. Furthermore, colocalisation of platinum with phosphorus-rich chromatin regions was observed, which is consistent with its binding affinity to DNA as the generally accepted crucial target of the drug. Application of ¹⁵N-labeled cisplatin and subsequent measurement of the nitrogen isotopic composition and determination of the relative intensities of platinum and nitrogen associated secondary ion signals in different cellular compartments with NanoSIMS suggested partial dissociation of Pt-N bonds during the accumulation process, in particular within nucleoli at elevated cisplatin concentrations. This finding raises the question as to whether the observed intracellular dissociation of the drug has implications for the mechanism of action of cisplatin. Within the cytoplasm, platinum mainly accumulated in acidic organelles, as demonstrated by a direct combination of specific fluorescent staining, confocal laser scanning microscopy and NanoSIMS. Different processing of platinum drugs in acidic organelles might be relevant for their detoxification, as well as for their mode of action.

Fig. 1. Exemplary ¹²C¹⁴N⁻, ³⁴S⁻, ³¹P⁻ and ¹⁹⁴Pt⁻ secondary ion signal intensity distribution maps acquired from semi-thin sections of SW480 cells treated with 25 μM of cisplatin. Regions of interest (ROIs) were manually defined within: (1) the cytoplasm; (2) the nucleus; (3) the nucleolus; and (4) the chromatin. According to the ¹⁹⁴Pt⁻ and ³⁴S⁻ signals, the drug is accumulated in small cytoplasmic, sulfur-rich aggregates (encircled in red), as well as in the nucleoli. Intensities are displayed on a rainbow false-color scale, ranging from dark blue to red for low to high intensities, respectively. The scale bars are 5 μm.

Fig. 2. NanoSIMS ¹⁹⁴Pt⁻ signal intensity distribution images obtained from semi-thin resin sections of SW480 cells treated with CDDP with various concentrations for 24 h. Signal intensities are displayed on a rainbow false-color scale, ranging from 0 counts per pixel (black) to max. 4 and 10 counts per pixel (red) for images from cells exposed to 0 to 10 μM and 25 to 150 μM CDDP, respectively. The primary ion beam was rastered over areas between 32 × 32 to 40 × 40 μm² at a resolution of 512 × 512 pixels for a total dwell time of 200 to 300 ms per pixel. Scale bars = 5 μm.

Fig. 3. Local subcellular ligand vs. central atom accumulation in SW480 cells upon 24 h exposure to ¹⁵N-labeled cisplatin with eight different concentrations ranging from 0 μM (min. values, negative control) to 150 μM (max. values), as inferred from NanoSIMS Pt/N elemental mapping and determination of the local ¹⁵N/¹⁴N isotopic composition. Note that the slope of the curves is proportional to the N/Pt stoichiometry of the accumulated cisplatin. In particular, any flattening of the curves is indicative of cleavage and loss of ammine ligands. Symbols in green, blue, red and cyan refer to values obtained from ROIs defined within the cytoplasm, nucleus (excl. nucleolus), nucleoli and chromatin of individual cells, respectively. The error bars refer to one standard deviation calculated from the compartment specific ROI values. The mean counting error, which is an inverse measure of the analytical precision, was smaller than the dispersion of the individual ROI values (see also Fig. S7 and S8†). Further information about the calculation of the displayed quantities is provided in the text and in more detail in the ESI.

Fig. 4. NanoSIMS secondary ion signal intensity distribution maps and confocal microscopy images of an adherent SW480 cell after 24 h exposure to 25 μM cisplatin, revealing cisplatin colocalisation with acidic organelles in the cell. The screened region is marked with a frame in the differential interference contrast image of the cell (A). The confocal microscopy image of the (fluorine-containing) LysoTracker Red (D) demonstrates a correlation with the signal intensity patterns of both ¹⁹F⁻ originating from LysoTracker Red (B) and ¹⁹⁴Pt⁻ originating from cisplatin (C) detected by NanoSIMS in the same cell. The arrows indicate a few regions of platinum accumulation without fluorescence and ¹⁹F⁻ signal intensity enhancement (2 out of 20 labeled areas). Secondary ion signal intensities are displayed on a rainbow false-color scale ranging from dark blue to red for low to high intensities, respectively. The small misalignment between the fluorescence image and the secondary ion maps may originate from lens aberrations and/or cell shrinkage due to sample preparation. Scale bars = 5 μm.