Near-infrared Raman microspectroscopy and Fluorescence-Activated cell sorting (FACS) were used to investigate the effect of etoposide on C6 glioma cells. Cells were incubated at different drug concentrations (0, 0.2, 2, and 20 µM) over 1, 2, and 3 days. After drug treatment, cells were harvested, and two cell pellets prepared: one pellet was analysed by FACS to investigate the effect of etoposide on cell cycle. The other was analysed by Raman microspectroscopy to identify spectroscopic markers linked to changes during the cell cycle. To correlate these changes with the cell cycle, Raman spectra were measured at G0G1, G2-M, and S phases obtained after fetal bovine serum starvation, nocodazol incubation, and thymidine exposure, respectively. These treatments lead to a maximum of 94% cells in G0G1 phase, 97% in G2-M and 78% in S phase. Multivariate statistics were used to analyze changes in cellular spectra as a function of cell cycle phase. Our results demonstrate that all data measured on cells can be classified in one out of two groups: i) one group contains spectra recorded on untreated cells, cells blocked in G0G1 phase and cells treated with 0.2 µM of etoposide during 24, 48 and 72 hours, ii) the second group includes spectra recorded on cells blocked in S and G2-M cell division cycle, and cells treated with 2 µM and 20 µM of etoposide. We are not able to discriminate between cells treated with 2 and 20 µM of etoposide, G2-M and S phase. In fact, cells accumulated in S and G2-M in response to those etoposide concentrations. FACS analysis revealed that treatment with 0.2, 2, and 20 µM of etoposide caused cells to accumulate mostly in G0G1, G2-M and S phases, respectively. These results are in agreement with the spectroscopic analysis where cells accumulated in G0G1 phase and cells treated with 0.2 µM of etoposide were pooled in the same class. Qualitative changes in cellular components associated with the different phases of cell cycle and with drug effect were also investigated. Our results show that Raman spectra are very sensitive to cell cycle phase due to drug treatment and are associated to molecular changes in protein, DNA and/or RNA content. These results may prove to be useful in the future to evaluate the effect and efficacy of chemotherapeutic treatment.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA