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In this paper, we present Raman imaging as a non-invasive approach for studying changes in mitochondrial metabolism caused by cardiolipin–cytochrome c interactions. We investigated the effect of mitochondrial dysregulation on cardiolipin (CL) and cytochrome c (Cyt c) interactions for a brain cancer cell line (U-87 MG). Mitochondrial metabolism was monitored by checking the intensities of the Raman bands at 750 cm−1, 1126 cm−1, 1310 cm−1, 1337 cm−1, 1444 cm−1 and 1584 cm−1. The presented results indicate that under pathological conditions, the content and redox status of Cyt c in mitochondria can be used as a Raman marker to characterize changes in cellular metabolism. This work provides evidence that cardiolipin–cytochrome c interactions are crucial for mitochondrial energy homeostasis by controlling the redox status of Cyt c in the electron transport chain, switching from disabling Cyt c reduction and enabling peroxidase activity. This paper provides experimental support for the hypothesis of how cardiolipin–cytochrome c interactions regulate electron transfer in the respiratory chain, apoptosis and mROS production in mitochondria. [ABSTRACT FROM AUTHOR]

Endothelial cells (EC) in vivo buffer and regulate the transfer of plasma fatty acid (FA) to the underlying tissues. We hypothesize that inflammation could alter the functionality of the EC, i.e., their capacity and uptake of different FA. The aim of this work is to verify the functionality of inflamed cells by analyzing their ability to uptake and accumulate exogenous saturated FA. Control and inflammatory human microvascular endothelial cells stimulated in vitro with two deuterium-labeled saturated FA (D-FA), i.e., palmitic (D31-PA) and myristic (D27-MA) acids. Cells were measured both by spontaneous and stimulated Raman imaging to extract detailed information about uptaken FA, whereas coherent anti-Stokes Raman scattering and fluorescence imaging showed the global content of FA in cells. Additionally, we employed atomic force microscopy to obtain a morphological image of the cells. The results indicate that the uptake of D-FA in inflamed cells is dependent on their concentration and type. Cells accumulated D-FA when treated with a low concentration, and the effect was more pronounced for D27-MA, in normal cells, but even more so, in inflamed cells. In the case of D31-PA, a slightly increased uptake was observed for inflamed cells when administered at higher concentration. The results provide a better understanding of the EC inflammation and indicate the impact of the pathological state of the EC on their capacity to buffer fat. All the microscopic methods used showed complementarity in the analysis of FA uptake by EC, but each method recognized this process from a different perspective. [ABSTRACT FROM AUTHOR]

Modern diagnostics of acute myeloid leukemia (AML) lack rapid, label-free methods that provide information about the metabolic state of cells. Raman spectroscopy could fill this gap and enrich the diagnostic process with new data. Raman spectra serve as "molecular fingerprints" characterizing the biochemical composition of complex cellular structures. The study aimed to identify the spectroscopic Raman signatures of molecular changes induced by IDH1/2 oncogenes in AML models and to analyze the effect of a single mutation on the molecular structure of leukemia cells in vitro. Somatic mutations in the IDH1/2 gene are among the most common in adult AML patients. The effect on energy metabolism and the cell cycle make IDH mutants an ideal candidate for analysis with Raman spectroscopy. The HEL and HL-60 leukemic cell lines were retrovirally transduced with the IDH1//R132H, IDH2/R140Q, and IDH2/R172K constructs in pMIG vector. Cells were imaged using WITec Alpha 300 confocal Raman system. Recorded Raman images were subjected to clustering analysis using the k-means (KMCA) method, and then principal components analysis (PCA) and partial least squares discrimination (PLS-DA) were done on averaged spectra of cells and selected subcellular regions. PCA analysis showed that cells with mutated IDH1/2 genes showed differences in the content of proteins and nucleic acids compared to control cells. The most significant differences were observed in the Raman spectra of cells with the IDH1 R132H mutation. Using the PLS-DA method, a mathematical model was built to distinguish mutant transgenic cells from wild-type IDH1 and IDH2 cells. The obtained results confirm the effectiveness of Raman spectroscopy in identifying changes between mutant and wild-type forms of IDH1/2. Potential applications of Raman spectroscopy could be monitoring the progress of targeted therapy or action of specific inhibitors. To improve and optimize the method, more data ought to be collected for machine learning. This work was supported by „Label-free and rapid optical imaging, detection and sorting of leukemia cells" project carried out within the Team-Net program of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. [ABSTRACT FROM AUTHOR]

Simple Summary: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy originating from abnormal lymphoid progenitor cells. Since ALL is genetically highly heterogenous, more sensitive and rapid methods for identifying the molecular subtype of ALL are still being searched, and Raman spectroscopy (RS) has a chance of becoming a valuable tool for this purpose. Herein, the RS was applied to analyze normal B cells and three subtypes of B-ALL, characterized by the presence of the product of gene fusion, i.e., BCR-ABL1, TEL-AML1, and TCF3-PBX1. The classification and discrimination of normal and neoplastic cells were carried out with the chemometric approach. Normal B cells were characterized mostly by bands assigned to nucleic acids and proteins, whereas three subtypes of ALL appeared to contain a higher lipid content. Spectral differences between particular ALL subtypes were modest. The results lead to the conclusion that RS has the potential as a diagnostic tool in clinical practice. Acute lymphoblastic leukemia (ALL) is the most common type of malignant neoplasms in the pediatric population. B-cell precursor ALLs (BCP-ALLs) are derived from the progenitors of B lymphocytes. Traditionally, risk factors stratifying therapy in ALL patients included age at diagnosis, initial leukocytosis, and the response to chemotherapy. Currently, treatment intensity is modified according to the presence of specific gene alterations in the leukemic genome. Raman imaging is a promising diagnostic tool, which enables the molecular characterization of cells and differentiation of subtypes of leukemia in clinical samples. This study aimed to characterize and distinguish cells isolated from the bone marrow of patients suffering from three subtypes of BCP-ALL, defined by gene rearrangements, i.e., BCR-ABL1 (Philadelphia-positive, t(9;22)), TEL-AML1 (t(12;21)) and TCF3-PBX1 (t(1;19)), using single-cell Raman imaging combined with multivariate statistical analysis. Spectra collected from clinical samples were compared with single-cell spectra of B-cells collected from healthy donors, constituting the control group. We demonstrated that Raman spectra of normal B cells strongly differ from spectra of their malignant counterparts, especially in the intensity of bands, which can be assigned to nucleic acids. We also showed that the identification of leukemia subtypes could be automated with the use of chemometric methods. Results prove the clinical suitability of Raman imaging for the identification of spectroscopic markers characterizing leukemia cells. [ABSTRACT FROM AUTHOR]