We have studied live non-malignant (MCF10A), mildly malignant (MCF7) and malignant (MDA-MB-231) breast cancer cells and human breast cancer tissue. We demonstrate the first application of Raman imaging and spectroscopy in diagnosing the role of lipid droplets in cell line cultures that closely mimic an in vivo environment of various stages in human breast cancer tissue. We have analyzed the composition of the lipid droplets in non-malignant and malignant human breast epithelial cell lines and discussed the potential of lipid droplets as a prognostic marker in breast cancer. To identify any difference in the lipid droplet-associated biochemistry and to correlate it with different stages of breast cancer, the PCA method was employed. The chemical composition of lipids and proteins, both in the cell line models and in human breast tissue has been analyzed. The paper shows the alterations in lipid metabolism that have been reported in cancer, at both the cellular and tissue levels, and discusses how they contribute to the different aspects of tumourigenesis.

Cytochrome c (Cyt c) is a key protein that is needed to maintain life (respiration) and cell death (apoptosis). The dual-function of Cyt c comes from its capability to act as mitochondrial redox carrier that transfers electrons between the membrane-embedded complexes III and IV and to serve as a cytoplasmic apoptosis-triggering agent, activating the caspase cascade. However, the precise roles of Cyt c in mitochondria, cytoplasm and extracellular matrix under normal and pathological conditions are not completely understood. To date, no pathway of Cyt c release that results in caspase activation has been compellingly demonstrated in any invertebrate. The significance of mitochondrial dysfunctionality has not been studied in ductal carcinoma to the best of our knowledge. We used Raman spectroscopy and imaging to monitor changes in the redox state of the mitochondrial cytochromes in ex vivo surgically resected specimens of human breast tissues, and in vitro human breast cells of normal cells (MCF 10A), slightly malignant cells (MCF7) and highly aggressive cells (MDA-MB-231). We showed that Raman imaging provides insight into the biology of human breast ductal cancer. Here we show that proper concentration of monounsaturated fatty acids, saturated fatty acids, cardiolipin and Cyt c is critical in the correct breast ductal functioning and constitutes an important parameter to assess breast epithelial cells integrity and homeostasis. We look inside human breast ducts by Raman imaging answering fundamental questions about location and distribution of various biochemical components inside the lumen, epithelial cells of the duct and the extracellular matrix around the cancer duct during cancer development in situ. Our results show that human breast cancers demonstrate a redox imbalance compared to normal tissue. The reduced cytochrome c is upregulated in all stages of cancers development. The results of the paper shed light on a largely non-investigated issues regarding cytochromes and mitochondrial function in electron transfer chain. We found in histopathologically controlled breast cancer duct that Cyt c, cardiolipin, and palmitic acid are the main components inside the lumen of cancerous duct in situ. The presented results show direct evidence that Cyt c is released to the lumen from the epithelial cells in cancerous duct. In contrast the lumen in normal duct is empty and free of Cyt c. Our results demonstrate how Cyt c is likely to function in cancer development. We anticipate our results to be a starting point for more sophisticated in vitro and in vivo animal models. For example, the correlation between concentration of Cyt c and cancer grade could be tested in various types of cancer. Furthermore, Cyt c is a target of anti-cancer drug development and a well-defined and quantitative Raman based assay for oxidative phosphorylation and apoptosis will be relevant for such developments.

(1) Background: Novel methods are required for analysing post-translational modifications of protein phosphorylation by visualizing biochemical landscapes of proteins in human normal and cancerous tissues and cells. (2) Methods: A label-free Raman method is presented for detecting spectral changes that arise in proteins due to phosphorylation in the tissue of human breasts, small intestines, and brain tumours, as well as in the normal human astrocytes and primary glioblastoma U-87 MG cell lines. Raman spectroscopy and Raman imaging are effective tools for monitoring and analysing the vibrations of functional groups involved in aberrant phosphorylation in cancer without any phosphorecognition of tag molecules. (3) Results: Our results based on 35 fresh human cancer and normal tissues prove that the aberrant tyrosine phosphorylation monitored by the unique spectral signatures of Raman vibrations is a universal characteristic in the metabolic regulation in different types of cancers. Overexpressed tyrosine phosphorylation in the human breast, small intestine and brain tissues and in the human primary glioblastoma U-87 MG cell line was monitored by using Raman biomarkers. (4) We showed that the bands at 1586 cm⁻¹ and 829 cm⁻¹, corresponding to phosphorylated tyrosine, play a pivotal role as a Raman biomarker of the phosphorylation status in aggressive cancers. We found that the best Raman biomarker of phosphorylation is the 1586/829 ratio showing the statistical significance at p Values of ≤ 0.05. (5) Conclusions: Raman spectroscopy and imaging have the potential to be used as screening functional assays to detect phosphorylated target proteins and will help researchers to understand the role of phosphorylation in cellular processes and cancer progression. The abnormal and excessive high level of tyrosine phosphorylation in cancer samples compared with normal samples was found in the cancerous human tissue of breasts, small intestines and brain tumours, as well as in the mitochondria and lipid droplets of the glioblastoma U-87 MG cell line. Detailed insights are presented into the intracellular oncogenic metabolic pathways mediated by phosphorylated tyrosine.

To monitor redox state changes and biological mechanisms occurring in mitochondrial cytochromes in cancers improving methods are required. We used Raman spectroscopy and Raman imaging to monitor changes in the redox state of the mitochondrial cytochromes in ex vivo human brain and breast tissues at 532 nm, 633 nm, 785 nm. We identified the oncogenic processes that characterize human infiltrating ductal carcinoma (IDC) and human brain tumors: gliomas; astrocytoma and medulloblastoma based on the quantification of cytochrome redox status by exploiting the resonance-enhancement effect of Raman scattering. We visualized localization of cytochromes by Raman imaging in the breast and brain tissues and analyzed cytochrome c vibrations at 750, 1126, 1337 and 1584 cm⁻¹ as a function of malignancy grade. We found that the concentration of reduced cytochrome c becomes abnormally high in human brain tumors and breast cancers and correlates with the grade of cancer. We showed that Raman imaging provides additional insight into the biology of astrocytomas and breast ductal invasive cancer, which can be used for noninvasive grading, differential diagnosis.

We used Raman imaging to monitor changes in the redox state of the mitochondrial cytochromes in ex vivo human brain and breast tissues, surgically resected specimens of human tissues and in vitro human brain cells of normal astrocytes (NHA), astrocytoma (CRL-1718), glioblastoma (U87-MG) and medulloblastoma (Daoy), and human breast cells of normal cells (MCF 10A), slightly malignant cells (MCF7) and highly aggressive cells (MDA-MB-231) by means of Raman microspectroscopy at 532 nm. We visualized localization of cytochromes by Raman imaging in the major organelles in cancer cells. We demonstrated that the “redox state Raman marker” of the ferric low-spin heme in cytochrome c at 1584 cm⁻¹ can serve as a sensitive indicator of cancer aggressiveness. We compared concentration of reduced cytochrome c and the grade of cancer aggressiveness in cancer tissues and single cells and specific organelles in cells: nucleous, mitochondrium, lipid droplets, cytoplasm and membrane. We found that the concentration of reduced cytochrome c becomes abnormally high in human brain tumors and breast cancers in human tissues. Our results reveal the universality of Raman vibrational characteristics of mitochondrial cytochromes in metabolic regulation in cancers that arise from epithelial breast cells and brain glial cells.