SignificanceAltered 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.AimWe 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.ApproachMCF7 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.ResultsThe 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.ConclusionsThis pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.
Radiation therapy (RT) is a standard treatment for ~50% of cancers worldwide. The major goal in RT is to maximize tumor cell kill while minimizing radiation-induced damage to healthy tissue. However, several fundamental questions remain unanswered related to the radiation-matter interactions, and the ensuing radiobiological responses in cells and tissue. Stimulated Raman scattering (SRS) microscopy is a promising technology for imaging the heterogeneous metabolic responses in cells exposed to ionizing radiation. In this work, we demonstrate SRS imaging of endogenous macromolecules such as lipids and proteins in the C-H stretching region (2800 to 3100 cm-1) in MCF-7 breast cancer cells in vitro. We compare the response of control (unirradiated) cells and cells irradiated at 30 Gy (120 kVp x-rays), at three time points of 24, 48, and 72 hours post exposure to ionizing radiation, and find significant changes between irradiated and control MCF-7 cells over time.
The risk of large-scale radiological/nuclear events has notably increased in recent years. Biodosimetry is considered an essential tool for emergency management following such unplanned exposures to ionizing radiation. For example, by assessing an individual’s received dose to blood, biodosimetry can support medical screening and individual health management. Current biodosimetry techniques, such as the dicentric chromosome assay, are based on the analysis of chromosomal aberrations Although highly accurate, these methods are time-consuming and labour-intensive. We recently developed a new high-throughput approach based on Raman spectroscopy of blood combined with covariate-adjusted multivariate analysis for the detection of irradiated blood. We found that the protein bands in the Raman spectra were the main sources of discrimination between unirradiated (control) and irradiated blood. In this follow up work, we explored the application of Raman spectroscopy and multivariate analysis to blood plasma to avoid dominant hemoglobin contributions. Peripheral blood drawn from a healthy volunteer was irradiated at 0 (control), 5 and 20 Gy using 250 kV X-rays. After a 4 hour incubation time, plasma centrifuged from the blood sample was immediately frozen at -80 deg C. Raman measurements were performed in triplicate on thawed blood plasma samples. Partial least squares-discriminant analysis (PLS-DA) was utilized for multi-class differentiation between Raman spectra of 0, 5 and 20 Gy irradiated plasma. Sparse PLS-DA (sPLS-DA) provided improved dose classification after combining Raman spectral data from different batches. Biomarker information related to radiation-induced changes in blood plasma was also extracted from sPLS-DA. The outcomes of these initial studies highlight the value of Raman spectroscopy to support biodosimetry.
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