High throughput absorbance spectra of cancerous cells: a microscopic investigation of spectral artifacts.

FTIR spectroscopy was recently demonstrated to be a useful tool to obtain a unique fingerprint of the effects of several anticancer drugs on cells in culture. While FTIR spectroscopy appears to have a definite potential to sort anticancer drugs on the basis of the metabolic modifications they induced, the present challenge is to evaluate the drug-induced spectral changes in cancer cells on a larger scale. The coupling of FTIR spectroscopy with a high throughput screening extension could become a useful method to generate drug classifications based on their "modes of action". Practically, the robustness of this approach is jeopardized by the variability that can appear from one cell smear to the next. When a few cells are scattered on the support, strong scattering effects are observed and locally dense cell aggregates could result in non-linearity of the signal. A microscopic study using infrared imaging demonstrates that the mean HTS (96-well High Throughput Spectroscopy) spectra recorded on 96 well ZnSe plates are the averages of contributions characterized by a wide absorbance distribution and by Mie scattering effects which significantly vary from point to point. Spectrum quality is at its best at the highest cell concentrations, i.e. from 300 000 to 400 000 cells per well, which present the best S/N and a relatively smaller Mie scattering effect. When the breast cancer cell line MDA-MB-231 was treated with four different polyphenols, spectra showed quite similar variations with respect to control spectra, with more intense variations for the quercetin and EGCG compared to resveratrol and capsaicin. Correction of the spectra with the RMieS algorithm improved their clustering according to the polyphenolic treatment.