Effect of hyaluronic acid on the drug resistance of 3D in vitro models of brain tumors



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Abstract

BACKGROUND: One of the key problems in the treatment of brain tumors is the development of drug resistance that appears during treatment, which significantly reduces the efficacy of standard chemotherapy methods. Three-dimensional cell models are a powerful tool in experimental oncology, allowing us to study the mechanisms of drug resistance in tumor cells, including the contribution of signaling pathways activated by extracellular matrix molecules.

AIM: To evaluate the effect of hyaluronic acid on the resistance of 3D in vitro brain tumor models to doxorubicin.

METHODS: 2D monolayer cultures were obtained from continuous brain tumor strains from laboratory animals stored in the unique collection of the A.P. Avtsyn Research Institute of Human Morphology, B.V. Petrovsky Russian Scientific Center of Surgery. 3D spheroids were formed under ultra-low adhesion conditions with or without hyaluronic acid. Doxorubicin cytotoxicity was determined using the CCK-8 assay. Doxorubicin efflux efficiency and CD44 protein production were determined using fluorimetry, and the proportion of CD44+ cells was determined using flow cytometry. The expression of genes associated with drug resistance was assessed using real-time PCR.

RESULTS: The IC50 values of doxorubicin were 180 nM for the 2D model of oligodendroglioma 51/7, 280 nM for glioblastoma 14-60-4, 500 nM for astrocytoma 10-17-2, and 2750 nM for neurinoma NGUK2. Doxorubicin at the IC50 concentration determined for the 2D models did not significantly affect the viability of cells within the spheroids, but a fourfold increase in its concentration resulted in a cytotoxic effect, with its severity depending on the degree of spheroid compaction. Hyaluronic acid dose-dependently increased the resistance of 3D brain tumor models to doxorubicin due to the activation of the CD44 receptor, more efficient drug efflux from cells, provided by changes in the expression of ABC transporter genes (Abcb1, Abcg2, Abcc1), as well as genes associated with DNA repair (Mgmt, Top2a).

CONCLUSION: The obtained data expand the understanding of the role of hyaluronic acid and the CD44 receptor in the formation of drug resistance in brain tumors, which allows for the creation of more relevant in vitro models. The results of the study are of practical importance for the development of strategies to overcome drug resistance, including targeted inhibition of ABC transporters or blockade of the interaction of hyaluronic acid and its receptor CD44.

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About the authors

Irina V. Arutyunyan

Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery, Moscow, Russia; Research Institute of Molecular and Cellular Medicine, Patrice Lumumba Peoples’ Friendship University of Russia, Moscow, Russia

Email: labrosta@yandex.ru
ORCID iD: 0000-0002-4344-8943

MD, Cand. Sci. (Biology), Senior Researcher, Laboratory of Growth and Development

Russian Federation, Moscow, Russia; Moscow, Russia

Georgy A. Lositsky

Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery, Moscow, Russia; Research Institute of Molecular and Cellular Medicine, Patrice Lumumba Peoples’ Friendship University of Russia, Moscow, Russia

Email: glosierror404@gmail.com
Russian Federation, Moscow, Russia; Moscow, Russia

Anna G. Soboleva

Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery, Moscow, Russia; Research Institute of Molecular and Cellular Medicine, Patrice Lumumba Peoples’ Friendship University of Russia, Moscow, Russia

Email: annasobo@mail.ru
Russian Federation, Moscow, Russia; Moscow, Russia

Sofia A. Aleksandrova

Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery, Moscow, Russia; Research Institute of Molecular and Cellular Medicine, Patrice Lumumba Peoples’ Friendship University of Russia, Moscow, Russia

Email: sofia.aleksa@mail.ru
Russian Federation, Moscow, Russia; Moscow, Russia

Dorzhu V. Balchir

Research Institute of Molecular and Cellular Medicine, Patrice Lumumba Peoples’ Friendship University of Russia, Moscow, Russia

Email: dbalchir@mail.ru
Russian Federation, Moscow, Russia

Vera V. Kudelkina

Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery, Moscow, Russia

Email: verakudelkina8047@gmail.com
Russian Federation, Moscow, Russia

Andrey V. Makarov

Research Institute of Molecular and Cellular Medicine, Patrice Lumumba Peoples’ Friendship University of Russia, Moscow, Russia

Email: anvitmak@yandex.ru
Russian Federation, Moscow, Russia

Andrey V. Elchaninov

Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery, Moscow, Russia; Research Institute of Molecular and Cellular Medicine, Patrice Lumumba Peoples’ Friendship University of Russia, Moscow, Russia; National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov, Moscow, Russia

Author for correspondence.
Email: elchandrey@yandex.ru
Russian Federation, Moscow, Russia; Moscow, Russia; Moscow, Russia

References

  1. Kim S, Son Y, Oh J, et al. Global burden of brain and central nervous system cancer in 185 countries, and projections up to 2050: a population-based systematic analysis of GLOBOCAN 2022. J Neurooncol. 2025:175(2):673–685. doi: 10.1007/s11060-025-05164-0
  2. Kaprin AD, Starinsky VV, Shakhzadova AO, editors. Malignant Neoplasms in Russia in 2023 (Incidence and Mortality). Moscow: P.A. Herzen Moscow Research Institute of Oncology — Branch of the National Medical Research Radiological Centre of the Ministry of Health of the Russian Federation; 2024. 276 p. (In Russ.)
  3. Debinski W, editor. Gliomas. Brisbane: Exon Publications; 2021. doi: 10.36255/exonpublications.gliomas.2021
  4. Holbeck SL, Collins JM, Doroshow JH. Analysis of Food and Drug Administration-approved anticancer agents in the NCI60 panel of human tumor cell lines. Mol Cancer Ther. 2010 May;9(5):1451–60. doi: 10.1158/1535-7163.MCT-10-0106
  5. Thomas G, Rahman R. Evolution of Preclinical Models for Glioblastoma Modelling and Drug Screening. Curr Oncol Rep. 2025;27(5):601–624. doi: 10.1007/s11912-025-01672-4
  6. Peters A, Sherman LS. Diverse Roles for Hyaluronan and Hyaluronan Receptors in the Developing and Adult Nervous System. Int J Mol Sci. 2020;21(17):5988. doi: 10.3390/ijms21175988
  7. Inoue A, Ohnishi T, Nishikawa M, et al. A Narrative Review on CD44's Role in Glioblastoma Invasion, Proliferation, and Tumor Recurrence. Cancers (Basel). 2023;15(19):4898. doi: 10.3390/cancers15194898
  8. Ravindranath AK, Kaur S, Wernyj RP, et al. CD44 promotes multi-drug resistance by protecting P-glycoprotein from FBXO21-mediated ubiquitination. Oncotarget. 2015;6(28):26308–21. doi: 10.18632/oncotarget.4763
  9. Mahboubi E, Mondanizadeh M, Almasi-Hashiani A, Ghasemi E. Exploring the effects of doxorubicin on survival rates in glioma patients: a comprehensive systematic review. Eur J Med Res. 2025;30(1):425. doi: 10.1186/s40001-025-02674-5
  10. Sarkaria JN, Hu LS, Parney IF, et al. Is the blood-brain barrier really disrupted in all glioblastomas? A critical assessment of existing clinical data. Neuro Oncol. 2018;20(2):184–191. doi: 10.1093/neuonc/nox175
  11. Alekseeva AI, Khalansky A, Miroshnichenko E, et al. The effect of therapy regimen on antitumor efficacy of the nanosomal doxorubicin against rat glioblastoma 101.8. Bulletin of Experimental Biology and Medicine. 2024;176(5):697–702. doi: 10.47056/1814-3490-2023-4-276-282
  12. Arrieta VA, Gould A, Kim KS, et al. Ultrasound-mediated delivery of doxorubicin to the brain results in immune modulation and improved responses to PD-1 blockade in gliomas. Nat Commun. 2024;15(1):4698. doi: 10.1038/s41467-024-48326-w
  13. Liao WH, Hsiao MY, Kung Y, Huang AP, Chen WS. Investigation of the Therapeutic Effect of Doxorubicin Combined With Focused Shockwave on Glioblastoma. Front Oncol. 2021;11:711088. doi: 10.3389/fonc.2021.711088
  14. Potez M, Rome C, Lemasson B, et al. Microbeam Radiation Therapy Opens a Several Days' Vessel Permeability Window for Small Molecules in Brain Tumor Vessels. Int J Radiat Oncol Biol Phys. 2024;119(5):1506–1516. doi: 10.1016/j.ijrobp.2024.02.007
  15. Shah S, Chandra A, Kaur A, et al. Fluorescence properties of doxorubicin in PBS buffer and PVA films. J Photochem Photobiol B. 2017;170:65–69. doi: 10.1016/j.jphotobiol.2017.03.024
  16. Sharma S, Mathur K, Mittal A, et al. Study of Surrogate Immunohistochemical Markers IDH1, ATRX, BRAF V600E, and p53 Mutation in Astrocytic and Oligodendroglial Tumors. Indian journal of neurosurgery 2022. Indian Journal of Neurosurgery. 2023;12(02):137–146. doi: 10.1055/s-0042-1743265
  17. Uceda-Castro R, van Asperen JV, Vennin C, et al. GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence. Sci Rep. 2022;12(1):424. doi: 10.1038/s41598-021-04127-5
  18. Pekmezci M, Reuss DE, Hirbe AC, et al. Morphologic and immunohistochemical features of malignant peripheral nerve sheath tumors and cellular schwannomas. Mod Pathol. 2015;28(2):187–200. doi: 10.1038/modpathol.2014.109
  19. Kudelkina VV, Kosyreva AM, Gulyaev MV, et al. Models of a unique scientific installation “Collection of Experimental Tumors of the Nervous System and Neural Tumor Cell Lines” and their application possibilities. In: Morphology in the 21st Century: Theory, Methodology, and Practice. Collection of Papers from the International Scientific and Practical Conference. Moscow: Moscow State Academy of Veterinary Medicine and Biotechnology — K.I. Skryabin Moscow Veterinary Academy; 2024:105–109. (In Russ.) EDN: JPKENQ
  20. Kamińska K, Cudnoch-Jędrzejewska A. A Review on the Neurotoxic Effects of Doxorubicin. Neurotox Res. 2023;41(5):383–397. doi: 10.1007/s12640-023-00652-5
  21. Harahap Y, Ardiningsih P, Corintias Winarti A, et al. Analysis of the Doxorubicin and Doxorubicinol in the Plasma of Breast Cancer Patients for Monitoring the Toxicity of Doxorubicin. Drug Des Devel Ther. 2020;14:3469–3475. doi: 10.2147/DDDT.S251144
  22. Kullenberg F, Degerstedt O, Calitz C, et al. In Vitro Cell Toxicity and Intracellular Uptake of Doxorubicin Exposed as a Solution or Liposomes: Implications for Treatment of Hepatocellular Carcinoma. Cells. 2021;10(7):1717. doi: 10.3390/cells10071717
  23. Pasupuleti V, Vora L, Prasad R, et al. Glioblastoma preclinical models: Strengths and weaknesses. Biochim Biophys Acta Rev Cancer. 2024;1879(1):189059. doi: 10.1016/j.bbcan.2023.189059
  24. Deen DF, Hoshino T, Williams ME, et al. Development of a 9L rat brain tumor cell multicellular spheroid system and its response to 1,3-bis(2-chloroethyl)-1-nitrosourea and radiation. J Natl Cancer Inst. 1980;64(6):1373–82. doi: 10.1093/jnci/64.6.1373
  25. Lanskikh D, Kuziakova O, Baklanov I, et al. Cell-Based Glioma Models for Anticancer Drug Screening: From Conventional Adherent Cell Cultures to Tumor-Specific Three-Dimensional Constructs. Cells. 2024;13(24):2085. doi: 10.3390/cells13242085
  26. Ncube KN, Jurgens T, Steenkamp V, et al. Comparative Evaluation of the Cytotoxicity of Doxorubicin in BT-20 Triple-Negative Breast Carcinoma Monolayer and Spheroid Cultures. Biomedicines. 2023;11(5):1484. doi: 10.3390/biomedicines11051484
  27. Doublier S, Belisario DC, Polimeni M, et al. HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast. BMC Cancer. 2012;12:4. doi: 10.1186/1471-2407-12-4
  28. Świerczewska M, Nowacka M, Stasiak P, et al. Doxorubicin and topotecan resistance in ovarian cancer: Gene expression and microenvironment analysis in 2D and 3D models. Biomed Pharmacother. 2025;183:117804. doi: 10.1016/j.biopha.2024.117804
  29. Nowacka M, Sterzynska K, Andrzejewska M, et al. Drug resistance evaluation in novel 3D in vitro model. Biomed Pharmacother. 2021;138:111536. doi: 10.1016/j.biopha.2021.111536
  30. Daum AK, Schlicker L, Schneider MA, et al. Cancer-associated fibroblasts promote drug resistance in ALK-driven lung adenocarcinoma cells by upregulating lipid biosynthesis. Cancer Metab. 2025;13(1):28. doi: 10.1186/s40170-025-00400-7
  31. Lo Cicero A, Campora S, Lo Buglio G, et al. Enhancing therapeutic efficacy through degradation of endogenous extracellular matrix in primary breast tumor spheroids. FEBS J. 2025;292(13):3494–3507. doi: 10.1111/febs.70069
  32. Liu Y, Li L, Wang L, et al. 'Two-faces' of hyaluronan, a dynamic barometer of disease progression in tumor microenvironment. Discov Oncol. 2023;14(1):11. doi: 10.1007/s12672-023-00618-1
  33. Suryandari DA, Yunaini L, Muharam R, et al. Identification and Validation of Chemoresistance-Associated Genes in A2780 Ovarian Cancer Cells Using Integrated Transcriptomic and qPCR Analysis. Journal of Experimental Biology and Agricultural Sciences. 2025;13(3):425–434. doi: 10.18006/2025.13(3).425.434

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СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
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