The presence of an activated stroma surrounding a primary breast tumor is a well-documented phenomenon. Fibroblasts are the cell type responsible for maintaining the stroma, and cancer-associated fibroblasts, characterized by the expression of α-smooth muscle actin (α-SMA) protein, are highly contractile compared with those found in normal tissue. Although the fibroblast to myofibroblast phenotypic transition is drastic and involves changes in cell morphology as well as increased expression of proteins, the best marker for myofibroblasts used in both the laboratory and the clinic is α-SMA. Due to the role of the fibroblast, in particular, and the stroma, in general, in cancer progression, it is important to study this characteristic cellular transformation in vitro. We report a direct comparison between α-SMA expression in primary normal human dermal fibroblasts and molecular spectra obtained through Fourier Transform infrared (FT-IR) spectroscopic imaging. Fibroblasts were stimulated using the growth factor TGFβ1, co-culture with MCF-7 tumorigenic breast epithelial cells, and co-culture with MCF10A normal breast epithelial cells in trans-well co-culture and also three-dimensional cell culture. α-SMA expression was determined using immunofluorescence and the samples were surveyed in a holistic and label-free approach using chemical imaging in two modes: transflection and attenuated total reflectance (ATR) FT-IR. This correlation is also compared with expression of α-SMA and spectra obtained from normal and tumorigenic human breast tissue biopsies
