Subcellular localization of early biochemical transformations in cancer-activated fibroblasts using infrared spectroscopic imaging

Abstract

The tumor microenvironment, or stroma, is chemically and morphologically modified during carcinoma progression. The predominant cell type in the stroma, the fibroblast, maintains collagen properties in normal tissue and often transformed during tumor progression. Biochemical changes within fibroblasts upon initial cancer activation, however, are relatively poorly defined. Here, we hypothesized that Fourier transform infrared (FT-IR) spectroscopic imaging could potentially be employed to examine these early transformations. Further, we employ attenuated total reflectance (ATR) microscopy to characterize subcellular spectra and their changes upon transformation. We characterized fibroblast transitions upon stimulation with both a molecular agent and a carcinoma-mimicking cellular co-culture system. Changes were predominantly observed in the 1080 cm−1 and 1224 cm−1 peak absorbance, commonly associated with nucleic acids, as well as in the band at 2930 cm−1 associated with the C-H stretching of proteins in the cytoplasmic compartment. In conclusion, biochemical changes in cancer-associated fibroblasts that express a-SMA are dominated by the cytoplasm, rather than the nucleus. This ensures that spectral changes are not associated with proliferation or cell cycle processes of the cells and the cells are undergoing a true phenotypic change denoted by protein modifications in the cell body.

FTIR Imaging of Tissues: Techniques and Methods of Analysis

“…Compared to transmission or transflection modes, AtR FtIR spectra do not show artefacts due to mie scattering and the sample preparation is generally straight forward. Potential applications of AtR FtIR imaging have been reported for both cells and tissues [45][46][47][48]. the other advantage of this technique is that normalisation for the sample path length is usually not required because the depth of penetration is constant throughout the medium.…”

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

“…There are a number of modifications that can be made to the imaging system including imaging in ATR mode 10,24,26 and using nanoscale thermal approaches 52,53 to allow for high resolution IR imaging. The main limitation with high resolution IR imaging is that the tissues must be carefully prepared and thin enough for IR to pass through (typically 4 μm thickness).…”

“…In addition, FT-IR provides a novel route to obtaining additional biochemical information that may not be readily accessible using conventional staining techniques. , with many very exciting studies showing its applications [19][20][21][22][23][24][25] . In addition, it was recently demonstrated that the increased spatial resolution associated with ATR imaging can allow for the visualization and classification of endothelial and myoepithelial cells in breast tissue which form a key component of breast cancer diagnosis 26 .…”

Study of plasma-induced peripheral blood mononuclear cells survival using Fourier transform infrared microspectroscopy

“…34 Spectroscopic techniques are increasingly being used to study cell metabolism in different systems. [35][36][37] Of the different groups of WBCs present in the peripheral blood, the monocytes are important as they have a propensity to differentiate into antigen-presenting cells (APCs), like macrophages and dendritic cells (DCs), that elicit adaptive immune responses. These cells require the presence of specific amounts and types of cytokines for their differentiation, being affected by the relative abundance of cytokines which decide their functionality.…”