Investigating ionizing radiation-induced changes in breast cancer cells using stimulated Raman scattering microscopy

Abstract

Significance: Altered lipid metabolism of cancer cells has been implicated in increased radiation resistance. A better understanding of this phenomenon may lead to improved radiation treatment planning. Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of cellular lipids but has never been applied in this domain.

Aim: We sought to investigate the radiobiological response in human breast cancer MCF7 cells using SRS microscopy, focusing on how radiation affects lipid droplet (LD) distribution and cellular morphology.

Approach: MCF7 breast cancer cells were exposed to either 0 or 30 Gy (X-ray) ionizing radiation and imaged using a spectrally focused SRS microscope every 24 hrs over a 72-hr time period. Images were analyzed to quantify changes in LD area per cell, lipid and protein content per cell, and cellular morphology. Cell viability and confluency were measured using a live cell imaging system while radiation-induced lipid peroxidation was assessed using BODIPY C11 staining and flow cytometry.

Results: The LD area per cell and total lipid and protein intensities per cell were found to increase significantly for irradiated cells compared to control cells from 48 to 72 hrs post irradiation. Increased cell size, vacuole formation, and multinucleation were observed as well. No significant cell death was observed due to irradiation, but lipid peroxidation was found to be greater in the irradiated cells than control cells at 72 hrs.

Conclusions: This pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.

Keywords: ionizing radiation; lipids imaging; stimulated Raman scattering microscopy.

Fig. 1

(a) Confluency (%) and (b) cell death (%) as a function of time for irradiated and control MCF7 cells. These results are the mean values of three trials with error bars (± 1 SD) and were measured using the Incucyte S3 live cell imaging system.

Fig. 2

Representative SRS images of MCF7 cells taken at $2850{\mathrm{cm}}^{-1}$ (a,c) and $2926{\mathrm{cm}}^{-1}$ (b,d) for unirradiated (a,b) and irradiated (c,d). The corresponding spectrally unmixed lipid-rich (e,g) and protein-rich (f,h) images after applying MCR-ALS. The threshold masks (i,k) from the above lipid images and composite lipid-protein images (j,l).

Fig. 3

(a) Average LD area per cell, (b) total lipid intensity per cell, and (c) total protein intensity per cell over 72 hrs. LD Area/cell is the average LD area per cell for an imaged region ($n=10$). (ns = no significance, *$\mathrm{p}<0.05$, **$\mathrm{p}<0.01$, ***$\mathrm{p}<0.001$, and ****$\mathrm{p}<0.0001$).

Fig. 4

Representative cell images at $2926{\mathrm{cm}}^{-1}$ for (a) 0 Gy and (b) 30 Gy at 72 hrs. (c) Mean cell area at each time point where the cell area was determined from protein images ($n=10$). (**$\mathrm{p}<0.01$, ***$\mathrm{p}<0.001$, and ****$\mathrm{p}<0.0001$).

Fig. 5

Some regions of MCF7 cells contained multi-nucleated (red circles) cells (a,b) and even more regions (c,d) contained cells containing vacuoles (arrows).

Fig. 6

Flow cytometry data for MCF7 cells over a 3-day recovery period at (a) 24, (b) 48, and (c) 72 hrs for a single representative trial. Histograms show profiles for unstained (0 Gy;orange), control (0 Gy with BODIPY;red), and irradiated cells (30 Gy irradiated with BODIPY;blue). The $Y$-axis represents the normalized cell count as a percentage of the Max, and the $X$-axis represents the fluorescent intensity of the portion of C11-BODIPY dye that was oxidized after interaction with peroxyl radicals, emitting in the green region (490 to 510 nm) of the visible spectrum.