ИСТИНА

The success of brain tumor removal depends on the detection of tumor borders. Investigation of the intracellular processes in cancer cells and the development of label-free techniques for the distinguishing of the brain tumors from the surrounding normal tissue can help to increase the accuracy in the detection of borders of the infiltrated peritumoral regions and of the dense tumor. We applied Raman microspectroscopy with laser excitations at 532 and 633 nm, confocal fluorescent microscopy with Alexa Fluor-4 intracellular astrocyte staining, and patch-clamp in the whole-cell configuration to compare cells in the normal cortex, peritumoral regions and in the dense tumor in patients with II-IV grades of gliomas. The studied preparations were 200-250 μm brain slices prepared from the patient brain tissue removed during the surgery. All studies were carried out with the permission of the Ethical Committee of the Privolzhskiy Research Medical University on the basis of informed consent of patients about the transfer of the material for the research in accordance with the necessary ethical conventions. We demonstrated that Raman spectroscopy allows: (i) to discriminate between healthy cells in cortex and tumor cells (region of dense cancer, II and IV types of glioma) and (ii) to distinguish peritumoral regions from the normal cortex in patients with IV grade glioma. Using specified Raman peaks, we demonstrated that under IV grade gliomas, the relative amount of proteins is significantly higher in the dense tumor regions compared to the healthy cortex. Besides, under IV grade glioma, we observed a difference in the redox state of mitochondrial cytochromes in the peritumoral regions compared to the cortex and dense tumor regions. Raman images of peritumoral regions in IV grade glioma provide a clear visualization of cancer cells infiltrated these brain parts possessing a higher amount of proteins and altered redox state of mitochondria. On the contrary, the peritumoral regions in II grade glioma do not demonstrate a change in the mitochondria redox status. We found that the input resistance of tumor cells in IV grade gliomas is increased compared to cortex astrocytes that can be due to the decrease in the cell size, smaller amount of processes and gap-junctions. We also observed changes in the spontaneous Ca2+ dynamics in tumor cells in high-grade gliomas: the area of Ca2+ events was smaller and the amplitude of Ca2+ concentration changes was higher in tumor cells than in cortex astrocytes. We suppose that observed changes in electrophysiological properties, amount of proteins and Ca2+ dynamics of IV grade glioma cancer cells can be due to the high rate of their growth, smaller cell size, decreased amount of intracellular contacts and modulation of mitochondria function.