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The study of metabolic properties of mouse hepatocellular carcinoma cell culture using fluorescence lifetime microscopy

Ksenia Y. Kandurova,1 Alexander A. Palalov,1 Elena V. Potapova,1 Andrey V. Dunaev, 1 Gerhard Holst,2 Andrey Y. Abramov,1,3 Evgeny A. Zherebtsov,1,4 1 Orel State University, Orel, 302026, Russia 2 PCO AG, Science and Research, Kelheim, 93309, Germany 3 UCL Queen Square Institute of Neurology, London, WC1N 3BG, UK 4 University of Oulu, Oulu, 90014, Finland


Currently, the diagnosis of such malignancy as liver cancer relies on the mandatory procedure of percutaneous puncture biopsy. Despite its "gold standard" status in verifying the presence and nature of tumour pathology, the probability of significant level of false-negative results remains a significant issue. Liver cancer is characterized by its rapid progression, so the issues of timely diagnosis and further treatment do not lose their relevance. Improvement of medical diagnostic technology and the introduction of new modern methods can help identify pathological changes at the initial stages and increase the effectiveness of traditional diagnostic procedures.
Biophotonics methods can be used to obtain data on the metabolic state of the liver and tumor tissues. In particular, fluorescence methods demonstrate high sensitivity to changes in the metabolic state of tissues. For this reason, evaluation of spectral and temporal parameters of fluorescence can serve as a diagnostic criterion, including in oncology. Another advantage of these methods is the possibility to be integrated into standard biopsy tools.
An important step in developing this approach is a fundamental study of the metabolic state of malignant cells to understand their biochemical processes better and correctly interpret the data obtained at the tissue and organ levels. The work aimed to study bioenergetics parameters using hepatocellular carcinoma cell culture experimentally.
Cell culture of murine hepatocellular carcinoma H33 was used as the object of study. The cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% of fetal bovine serum, penicillin, and streptomycin, at 37°C, 100% relative humidity, under an atmosphere containing 5% CO2 and 95% air. Cells were placed in 35-mm dishes and measured after 3-4 days. The measured cells included the control group (N=9), the one treated with iodoacetic acid (20 μM, N=4) 2 h prior to measurements, and the one treated with dehydroepiandrosterone (DHEA, 10 μM, N=4) 15 min prior to measurements. Iodoacetic acid was used to inhibit glycolysis, while DHEA was used as an inhibitor of the pentose phosphate pathway. The measurements included the recording of basal fluorescence signal, fluorescence signal after addition of oxidative phosphorylation uncoupler FCCP (0.2 mM) and complex I inhibitor rotenone (0.02 mM). The measurements lasted for 15 min.
In order to evaluate mitochondrial NADH autofluorescence intensity and lifetime parameters, the method of fluorescence lifetime microscopy (FLIM) in the frequency domain was used. The experimental setup included frequency-domain FLIM camera pco.flim (PCO AG, Germany) operated at the modulation frequency of 40 MHz and image resolution of 1008 x 1008 pixels. A 375 nm laser (PCO AG, Germany) was used to excite the autofluorescence in cells. The camera was connected to inverted biological microscope IX73 (Olympus Corporation, Japan) with a 40x objective. The received data were processed using OriginPro 2015 (OriginLab Corporation) software. The analyzed parameters included NADH pool, NADH redox index, and phase and modulation shifts characterizing the first and second components of fluorescence lifetime decay. The statistical significance of the differences was assessed using one-way ANOVA test (p<0.05).
The results demonstrated decrease of mean NADH pool both in iodoacetic and DHEA group (41±27 a.u., 37±7 a.u., 30±18 a.u., respectively), while NADH redox index increased by more in the iodoacetic acid group (53±27 %, 76±16 %, 70±17 %). Lower values of the redox index indicate more significant respiratory activity of cells. Therefore it can be concluded that the application of inhibitors worsened the condition of the cells to different degrees. The observed changes may indicate that liver cancer cells use the process of glycolysis for glucose oxidation and energy production in the form of ATP to a greater extent than the pentose phosphate pathway. Inhibition of glycolysis is more critical for the viability of this cell culture. Comparison of lifetime values at the same stages of measurements in different groups of cells showed that adding iodoacetic acid resulted in a statistically significant decrease in the average values of both components throughout the experiment.
In contrast, DHEA addition led to the opposite effect. All three groups show similar dynamics of changes. However, a significant scatter of the obtained values was observed in the control group, which made it difficult to assess in more detail possible changes in the ratio of bound and unbound NADH.
Thus, the obtained data on the application of the new type of frequency domain CMOS camera showed the feasibility of imaging the endogenous fluorescence and fluorescence lifetime parameters to be used for evaluating metabolic processes in cells. The preliminary study allowed us to observe the criticality of glycolysis for the functioning of malignant cells. The study was supported by the Russian Science Foundation under the project № 21-15-00325.

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Ksenia Kandurova
Orel State University


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