Biochemical signatures ofin vitroradiation response in human lung, breast and prostate tumour cells observed with Raman spectroscopy

Abstract

This work applies noninvasive single-cell Raman spectroscopy (RS) and principal component analysis (PCA) to analyze and correlate radiation-induced biochemical changes in a panel of human tumour cell lines that vary by tissue of origin, p53 status and intrinsic radiosensitivity. Six human tumour cell lines, derived from prostate (DU145, PC3 and LNCaP), breast (MDA-MB-231 and MCF7) and lung (H460), were irradiated in vitro with single fractions (15, 30 or 50 Gy) of 6 MV photons. Remaining live cells were harvested for RS analysis at 0, 24, 48 and 72 h post-irradiation, along with unirradiated controls. Single-cell Raman spectra were acquired from 20 cells per sample utilizing a 785 nm excitation laser. All spectra (200 per cell line) were individually post-processed using established methods and the total data set for each cell line was analyzed with PCA using standard algorithms. One radiation-induced PCA component was detected for each cell line by identification of statistically significant changes in the PCA score distributions for irradiated samples, as compared to unirradiated samples, in the first 24-72 h post-irradiation. These RS response signatures arise from radiation-induced changes in cellular concentrations of aromatic amino acids, conformational protein structures and certain nucleic acid and lipid functional groups. Correlation analysis between the radiation-induced PCA components separates the cell lines into three distinct RS response categories: R1 (H460 and MCF7), R2 (MDA-MB-231 and PC3) and R3 (DU145 and LNCaP). These RS categories partially segregate according to radiosensitivity, as the R1 and R2 cell lines are radioresistant (SF(2) > 0.6) and the R3 cell lines are radiosensitive (SF(2) < 0.5). The R1 and R2 cell lines further segregate according to p53 gene status, corroborated by cell cycle analysis post-irradiation. Potential radiation-induced biochemical response mechanisms underlying our RS observations are proposed, such as (1) the regulated synthesis and degradation of structured proteins and (2) the expression of anti-apoptosis factors or other survival signals. This study demonstrates the utility of RS for noninvasive radiobiological analysis of tumour cell radiation response, and indicates the potential for future RS studies designed to investigate, monitor or predict radiation response.

Microdosimetric considerations for radiation response studies using Raman spectroscopy

“…RS has also been used to evaluate radiation response in vitro in mammary epithelial cells, 6 oral cancer cells, 7 prostate tumor cells, 8 and breast and lung tumor cells. 9 The studies cited above involve doses of several Gray, relevant for radiotherapy, however, recent RS applications include investigation of cell response to low radiation doses ( < 1 Gy), relevant for diagnostic radiology or out-of-field radiation in radiotherapy (as well as radiation protection in general). Maguire et al 10 demonstrated that RS is capable of detecting 60 Co radiation response in human lymphocytes for doses as low as 0.05 Gy; DNA damage predicted by changes in Raman spectra were confirmed by comparison with c-H2AX assay results.…”

Vision 20/20: The role of Raman spectroscopy in early stage cancer detection and feasibility for application in radiation therapy response assessment

“…Human prostate cell lines were found to be radiosensitive whereas breast and lung cells seem to be radio resistant. 77 Radiation induced spectral changes were observed in Raman peaks associated with aromatic amino acids, conformation of protein structures, certain nucleic acid, and lipid functional groups. Lakshmi et al 78 noted a small loss of lipid in trabecular regions with no osteoradionecrosis and a complete loss of lipid in osteoradionecrosis bone in oral cancer patients treated with radiation therapy.…”

Raman profile alterations of irradiated human nasopharyngeal cancer cells detected with laser tweezer Raman spectroscopy

“…6,7 The radioresistant lung and breast cells showed radiation-induced RS spectral features as glycogen accumulation at low RT doses, whereas the radiosensitive prostate cells showed some membrane phospholipid-related alterations. 8,9 Roman et al analyzed cytoplasmic and nuclear regions of irradiated prostate cancer cells (PC-3) and suggested various DNA damages located in those two regions. 10 Lasalvia et al observed that DNA was damaged in breast cells (MCF10A) irradiated by protons, in particular the O-P-O stretching mode at about 784 cm À1 .…”