Monika Kopec

Development of a new diagnostic Raman method for monitoring epigenetic modifications in the cancer cells of human breast tissue

Cancer diagnosis requires better screening of early stages of pathology and monitoring patient responses to treatment. Current technologies in the clinical sector are expensive, sophisticated and time consuming. This paper develops a novel Raman based alternative for currently existing epigenetics research approaches. The proposed Raman approach can ‘upgrade’ cancer epigenetic tests and answer many questions by monitoring the biochemistry of cancer cells. We will show that Raman spectroscopy and Raman imaging can detect the relative amounts of acetylated and methylated lysine by monitoring the vibrations of the acetyl and methyl chemical functional groups. In comparison to existing tests and assays used to monitor molecular processes of acetylation and methylation of all proteins, Raman-based methods have the potential to be a powerful alternative for conventional methods of cell biology, because they allow non-invasive detection of cellular acetylation and methylation processes that are not limited to only those events that are sensitive to a specific antibody. Vibrational Raman signatures of acetylation and methylation processes in epithelial cells of human breast tissue (ductal and lobular carcinoma) have been used to identify and discriminate structures in normal and cancerous tissues. Our results demonstrate that the stretching vibration of the acetyl group observed at around 2938–2942 cm−1 and the methyl group at 2970 cm−1 by Raman spectroscopy is useful in monitoring these epigenetic molecular processes in cancer cells. The Raman vibrational marker is markedly blue-shifted from 2905 cm−1 (non-acetylated C–H vibrations) for normal cells to 2942 cm−1 (vibrations of the acetylated functional group) in cancer cells. The sensitivity and specificity obtained directly from PLSDA and cross-validation for a chosen model gives a sensitivity and specificity of 86.1% and 91.3% for calibration and 85.3% and 91.3% for cross-validation, respectively. The results presented in the paper provide strong evidence that the global acetylation level of histone and non-histone proteins increases in human breast cancer cells.

Angiogenesis - a crucial step in breast cancer growth, progression and dissemination by Raman imaging

Combined micro-Raman imaging and AFM imaging are efficient methods for analyzing human tissue due to their high spatial and spectral resolution as well as sensitivity to subtle chemical, structural and topographical changes. The aim of this study was to determine biochemical composition and mechanical topography around blood vessels in the tumor mass of human breast tissue. Significant alterations of the chemical composition and structural architecture around the blood vessel were found compared to the normal breast tissue. A pronounced increase of collagen-fibroblast-glycocalyx network, as well as enhanced lactic acid, and glycogen activity in patients affected by breast cancer were reported.

Surface-Enhanced Raman Spectroscopy Analysis of Human Breast Cancer via Silver Nanoparticles: An Examination of Fabrication Methods

Breast cancer in a traditional way is diagnosed using mammography, computer tomography, ultrasounds, biopsy, and finally, histopathological analysis. Histopathological analysis is a gold standard in breast cancer diagnostics; however, it is time consuming and prone to the human interpretations. That is why new methods based on optical properties of analyzed human tissue samples are needed to be introduced to the clinical practice objective, costless and fast diagnostic protocols. Nowadays, Raman spectroscopy-based methods are gaining more and more importance. Raman spectroscopy and imaging allow to characterize human tissue samples using an electromagnetic radiation from a safe range, and simultaneously, a minimal sample preparation is required. During measurements, a natural differentiation in tissues components’ scattering cross sections is used to build 2D and 3D maps of the chemical component distribution. The paper presents the application of SERS (surface-enhanced Raman spectroscopy) measurements for analysis of human breast cancer (adenocarcinoma). The advantages of SERS application in cancer diagnostics are also discussed. Moreover, the detailed chemical composition of human breast cancer tissue based on Raman bands of DNA/RNA, amino acids, lipids, and proteins which are significantly enhanced is presented. Three different methods of NP preparation are presented, and the effectiveness of Raman signal enhancement of Ag nanoparticles synthetized by these methods is compared. The enhancement effect of NPs synthetized by reduction of silver nitrate with sodium borohydride (method no. 1) and silver nitrate-hydroxylamine hydrochloride reduction (method no. 2) was stronger when compared with the polyol method (method no. 3). Presented SERS results confirmed that the clearly resolved and high-intensity Raman spectra of cancer human breast tissue can be recorded using integration times of the order of fractional seconds and one milliwatt of the excitation laser power.