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Characterising hepatocellular carcinoma inoculated in mice by fluorescence lifetime measurements

Ksenia Kandurova (Research and Development Center of Biomedical Photonics, Orel State University, Russia);
Valery Shupletsov (Research and Development Center of Biomedical Photonics, Orel State University, Russia);
Elena Potapova (Research and Development Center of Biomedical Photonics, Orel State University, Russia);
Evgeny Zherebtsov (Research and Development Center of Biomedical Photonics, Orel State University, Russia; Optoelectronics and Measurement Techniques Unit, University of Oulu, Finland).


Currently, cancer remains the second leading cause of death in the world. One of the widely prevalent groups of malignant tumours in abdominal organs is liver cancer. The most significant features of the cancer type are the difficulties of early determination and rapid growth, which leads to a high level of mortality. In order to determine treatment tactics, in most cases, it is necessary to analyse the type and histological structure of the tumour. Histological analysis is usually performed after obtaining tissue samples by puncture biopsy. That procedure has been widely used for years. However, the method still suffers from the recognisable probability of getting a non-diagnostic sample. The time factor is also crucial as the following study can take 5-10 days.
The introduction of biophotonics methods into standard procedures of minimally invasive surgery seems to be promising for the minimisation of the non-diagnostic sample taking. In particular, fluorescence methods are applied to assess the metabolic state of biological tissues as several endogenous fluorophores have decent fluorescence spectra and are actively involved in cell metabolism. One of them is nicotinamide adenine dinucleotide (NADH) coenzyme of mitochondria. The fluorescence intensity and lifetime decay function of NADH bear direct information on the metabolic state of the cells. These parameters indicate NADH concentration and its biochemical interaction with surrounding substances, which changes due to cancer formation.
The information about the metabolic state at the cellular level obtained in the real-time can be a valuable addition to assist a surgeon during a routine biopsy. Therefore, the aim of preliminary experiments was to integrate fluorescence intensity and lifetime measurements into the optical needle probe and test its capabilities in an animal model of liver cancer.
In order to assess both fluorescence intensity and lifetime data, the measurements were carried out using the time-correlated single-photon counting technique. The system used (Becker & Hickl, Germany) included two HPM-100-40 photomultiplier detectors and BDL-SMN ps laser. The fluorescence excitation wavelength was 365 nm, the emission range was 445 ± 25 nm. The laser source and detectors were connected to the specially designed fibre needle probe. Its distal end has 1 mm diameter, and it is designed to be used through 17.5 G standard needle with 20° bevel. To compare the fluorescence lifetime parameters of healthy and malignant tissues, the tumours were modelled in laboratory mice. The cells of H33 mouse hepatocellular carcinoma (50000 cells/μl, 100 μl per mouse,) were inoculated into the middle lobe of the liver of eight BDF mice. The experiments were carried in vivo three months later during laparotomy. The needle probe was introduced into several areas of intact liver and tumour tissues. After that, the samples of the organs studied were extracted for further histological study. Its results were used to confirm the state of tissues for processing the data. The studies were approved by the ethics committee of Orel State University (minutes of the meeting No 12, September 6, 2018).
The results showed high individual variability and insignificant increase of average fluorescence intensity in carcinoma tissues compared to the healthy ones. Two fluorescence lifetime decay components τ1 and τ2 demonstrated a statistically significant difference between the tissue type with an average decrease by 35 and 22% respectively in tumour tissue. The values of τ1 were identified to be the liver 848±26 and in tumour 552±15 ps. The values of τ1 reached 3259±60 ps in liver and 2532±26 ps in the tumour. The amplitude contribution of fast component α1 increased from 63.0 ± 0.7 to 75.6 ± 0.6 %, leading to decrease of slow component contribution α2 from 36.9 ± 0.7 to 24.4 ± 0.6 %.
The obtained data showed that atypical metabolic activity of malignant cells has led to changes in the ratio of bound and unbound NADH, which was possible to observe with time-correlated single-photon counting. The wide range of fluorescence intensities and lifetime decay components also can indicate that the growth of every malignant tumour has its individual features. The further studying of fluorescence parameters of different malignant tumours and the development of diagnostic criteria seems promising for analysis of different stages and types of tumours for minimally invasive diagnostics procedures
The study was supported by the Russian Science Foundation under the project №18-15-00201.


Ksenia Kandurova
Orel State University


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