Hypoxia effect on proliferative activity of cells in orthotopic xenograft of hepatocellular carcinoma of the liver in the experiment

Purpose of the study. The purpose of this research was to investigate the effect of in vivo hypoxic conditions on the proliferative potential of HepG2 liver cancer cells.

Materials and methods. Human liver cancer cells of the HepG2 line have been cultured. The HepG2 cell suspension was injected subcutaneously into mice in an amount of 5 × 10⁶ to obtain a xenograft. Tumor nodes that had reached the required size were divided into fragments and transplanted into the orthotopic site. Balb/c nude mice with implanted HepG2 liver cancer xenograft were used in this experiment. The mice with tumor implanted in the liver were divided into two groups, intact and hypoxic. Mice from the second group underwent liver blood flow reduction by occlusion of the portal triad for 20 minutes. Tumor nodes were extracted for histological and immunohistochemical staining for proliferation marker Ki-67 on the 4th day after the procedures. The proportion of positively stained cells was calculated, and the results were statistically analyzed using the Statistica 10.0 software.

Results. Orthotopic models of liver cancer in Balb/c Nude mice were obtained. Histological and immunohistochemical studies were carried out. Histological analysis showed that hepatocellular carcinoma is characterized by an average degree of differentiation. In the tissues of these xenografts, by using immunohistochemical analysis for the proliferation marker Ki-67, it was possible to identify statistically significant differences between the two groups, i.e. intact and the one with reduction of blood flow. The proportion of immunopositive cells was 65 [65–70] % and 19 [15–25] %, respectively.

Conclusion. A tendency to decreased proliferative activity of tumor cells after hepatic blood flow reduction, i.e. hypoxia exposure, was demonstrated. Our data indicate that the proliferative activity of tumor cells is directly related to the microenvironment, and to the hypoxic environment in particular. Further study of the effect of hypoxia on the processes of growth and development of malignant tumors may contribute to a deeper understanding of the biological features of tumors and their treatment.

1. Semenza GL. Oxygen sensing, hypoxia-inducible factors, and disease pathophysiology. Annu Rev Pathol. 2014;9:47–71. https://doi.org/10.1146/annurev-pathol-012513-104720

2. Fitzpatrick SF. Immunometabolism and Sepsis: A Role for HIF? Front Mol Biosci. 2019 Sep 6;6:85. https://doi.org/10.3389/fmolb.2019.00085

3. Jing X, Yang F, Shao C, Wei K, Xie M, Shen H, et al. Role of hypoxia in cancer therapy by regulating the tumor microenvironment. Mol Cancer. 2019 Nov 11;18(1):157. https://doi.org/10.1186/s12943-019-1089-9

4. Hsieh CH, Lee CH, Liang JA, Yu CY, Shyu WC. Cycling hypoxia increases U87 glioma cell radioresistance via ROS induced higher and long-term HIF-1 signal transduction activity. Oncol Rep. 2010 Dec;24(6):1629–1636. https://doi.org/10.3892/or_00001027

5. Wu J, Li S, Huang Y, Zeng Z, Mei T, Wang S, et al. MRI features of pituitary adenoma apoplexy and their relationship with hypoxia, proliferation, and pathology. J Clin Ultrasound. 2023;51(6):1078–1086. https://doi.org/10.1002/jcu.23492

6. Dewhirst MW, Ong ET, Braun RD, Smith B, Klitzman B, Evans SM, et al. Quantification of longitudinal tissue pO2 gradients in window chamber tumours: impact on tumour hypoxia. Br J Cancer. 1999 Apr;79(11–12):1717–1722. https://doi.org/10.1038/sj.bjc.6690273

7. Bristow RG, Berlin A, Dal Pra A. An arranged marriage for precision medicine: hypoxia and genomic assays in localized prostate cancer radiotherapy. Br J Radiol. 2014 Mar;87(1035):20130753. https://doi.org/10.1259/bjr.20130753

8. Кит О. И., Дерижанова И. С., Карнаухов Н. С. Вопросы классификации нейроэндокринных опухолей желудка. Вопросы онкологии. 2016;62(5):573-579. EDN: WWOQFF

9. Petrillo M, Patella F, Pesapane F, Suter MB, Ierardi AM, Angileri SA, et al. Hypoxia and tumor angiogenesis in the era of hepatocellular carcinoma transarterial loco-regional treatments. Future Oncol. 2018 Dec;14(28):2957–2967. https://doi.org/10.2217/fon-2017-0739

10. Fei M, Guan J, Xue T, Qin L, Tang C, Cui G, et al. Hypoxia promotes the migration and invasion of human hepatocarcinoma cells through the HIF-1α-IL-8-Akt axis. Cell Mol Biol Lett. 2018;23:46. https://doi.org/10.1186/s11658-018-0100-6

11. Гончарова А. С., Гурова С. В., Кечерюкова Т. М., Максимов А. Ю., Лаптева Т. О., Романова М. В. и др. Моделирование гипоксии печени в эксперименте in vivo. Якутский медицинский журнал. 2023;(2(82)):5–8. https://doi.org/10.25789/YMJ.2023.82.01, EDN: WBXQQT

12. Ron A, Deán-Ben XL, Gottschalk S, Razansky D. Volumetric Optoacoustic Imaging Unveils High-Resolution Patterns of Acute and Cyclic Hypoxia in a Murine Model of Breast Cancer. Cancer Res. 2019 Sep 15;79(18):4767–4775. https://doi.org/10.1158/0008-5472.CAN-18-3769

13. Гурова С. В., Кечерюкова Т. М., Гончарова А. С., Колесников Е. Н., Кожушко М. А., Татимов М. З. Модели рака печени in vivo. Современные проблемы науки и образования. 2023;(4):136. https://doi.org/10.17513/spno.32707, EDN: XFEYBT

14. Кечерюкова Т. М., Гурова С. В., Гончарова А. С., Максимов А. Ю., Галина А. В., Романова М. В. и др. Сравнительная оценка методов создания ортотопической модели рака печени. Современные проблемы науки и образования. 2023;(2):94. https://doi.org/10.17513/spno.32565, EDN: ZFJIHU

15. Sivalingam VN, Latif A, Kitson S, McVey R, Finegan KG, Marshall K, et al. Hypoxia and hyperglycaemia determine why some endometrial tumours fail to respond to metformin. Br J Cancer. 2020 Jan;122(1):62–71. https://doi.org/10.1038/s41416-019-0627-y

16. Li Y, Zhao L, Huo Y, Yang X, Li Y, Xu H, et al. Visualization of hypoxia in cancer cells from effusions in animals and cancer patients. Front Oncol. 2022;12:1019360. https://doi.org/10.3389/fonc.2022.1019360