Current evaluation of histological sections of breast cancer samples remains unsatisfactory. The search for new predictive and prognostic factors is ongoing. Infrared spectroscopy and its potential to probe tissues and cells at the molecular level without requirement for contrast agents could be an attractive tool for clinical and diagnostic analysis of breast cancer. In this study, we report the successful application of FTIR (Fourier transform infrared) imaging for breast tissue component characterization. We show that specific FTIR spectral signatures can be assigned to the major tissue components of breast tumor samples. We demonstrate that a tissue component classifier can be built based on a spectral database of well-annotated tissues and successfully validated on independent breast samples. We also demonstrate that spectral features can reveal subtle differences within a tissue component, capturing for instance lymphocytic and stromal activation. By investigating in parallel lymph nodes, tonsils and wound healing tissues, we prove the uniqueness of the signature of both lymphocytic infiltrate and tumor microenvironment in the breast disease context. Finally, we demonstrate that the biochemical information reflected in the epithelial spectra might be clinically relevant for the grading purpose, suggesting potential to improve breast cancer management in the future.

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Thirteen breast cancer cell lines were grown in traditional two-dimensional (2D) monolayer and three-dimensional (3D) laminin-rich extracellular matrix (lrECM) culture models. Microarray-based transcriptional profiling data were published for these cell lines under both culture conditions. Colonies embedded in Matrigel matrix were fixed in formalin, embedded in paraffin and cut into 4 μm thick sections. The sections were mounted onto infrared-transparent barium fluoride windows and deparaffinized for Fourier transform infrared (FTIR) imaging. Samples consisting of Matrigel-coated 2D-grown cells followed the same processing procedure, simplifying comparison with 3D-cultured cells as well as with routinely prepared formalin-fixed, paraffin-embedded tissue specimens. Gene expression was found to be dominated by the cell line genome. Cluster analysis first groups the same cell line samples, independent of whether cells have been grown in 2D or 3D cultures. FTIR spectroscopy first groups by culture conditions when considering the full spectrum length. The paper reports two important results. First, both gene expression level and FTIR spectroscopy are multivariate techniques that contain sufficient information to identify uniquely both any cell line (among thirteen breast cancer cell lines) and phenotype induced by growing the cells in 2D or 3D lrECM cultures. Second, we established the presence of a strong correlation between gene expression patterns and FTIR spectral data for the thirteen breast cancer cell lines grown in both 2D and 3D lrECM cultures. These results suggest that, although based on completely different principles, the two approaches describe similarly the patterns of variations in cells.

One current challenge in the field of breast cancer infrared imaging is the identification of carcinoma cell subtypes in the tissue. Neither sequencing nor immunochemistry is currently able to provide a cell by cell thorough classification. The latter is needed to build accurate statistical models capable of recognizing the diversity of breast cancer cell lines that may be present in a tissue section. One possible approach for overcoming this problem is to obtain the IR spectral signature of well-characterized tumor cell lines in culture. Cultures in three-dimensional matrices appear to generate an environment that mimics better the in vivo environment. There are, at present, series of breast cancer cell lines that have been thoroughly characterized in two- and three-dimensional (2D and 3D) cultures by full transcriptomics analyses. In this work, we describe the methods used to grow, to process, and to characterize a triple-negative breast cancer cell line, MDA-MB-231, in 3D laminin-rich extracellular matrix (lrECM) culture and compare it with traditional monolayer cultures and tissue sections. While unsupervised analyses did not completely separate spectra of cells grown in 2D from 3D lrECM cultures, a supervised statistical analysis resulted in an almost perfect separation. When IR spectral responses of epithelial tumor cells from clinical triple-negative breast carcinoma samples were added to these data, a principal component analysis indicated that they cluster closer to the spectra of 3D culture cells than to the spectra of cells grown on a flat plastic substrata. This result is encouraging because of correlating well-characterized cell line features with clinical biopsies.

Infrared imaging was applied to investigate a reconstituted basement membrane, known as Matrigel, in three-dimensional cell cultures.

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