Medical Radiology and Radiation Safety. 2025. Vol. 70. № 5
DOI:10.33266/1024-6177-2025-70-5-11-17
E.A. Mysina, D.D. Kolmanovich, N.R. Popova, B.A. Bokl,
N.A. Pivovarov, N.N. Chukavin, I.V. Savintseva, D.A. Vinnik, A.L. Popov
3D Cell Spheroid as a Relevant Experimental Model for Screening Potential Nanoradiosensitizers
Institute of Theoretical and Experimental Biophysics, Pushchino, Russia
Contact person: A.L. Popov, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.
Abstract
Purpose: Cell monolayer (2D culture) has been used to screen biological activity of various biomolecules, nanoconjugates, and other therapeutic agents. However, 2D cell culture cannot fully imitate real physiological structures and states of the human body, in particular, the organization and microenvironment of a solid tumor. This imposes significant limitations on current translational studies of the biological effect of new therapeutic drugs and approaches to tumor radiation therapy. To overcome these limitations, models based on 3D cell spheroids are developed and put into practice. These models allow the most reliable imitation of the structure and state of a solid tumor, including the formation of 3D intercellular matrix, characteristic zonation, and corresponding gene expression.
Purpose of the investigation: To create an experimental model of a 3D spheroid formed on the basis of mouse breast cancer cells EMT6/P line and validate the model under X-ray exposure for screening potential nanoradiosensitizers.
Material and methods: The EMT6/P cell line (mouse carcinoma) was used to form a 3D cell spheroid and evaluate the biological effect of X-ray radiation on it. Cell spheroids were prepared using the "hanging drop" method. An RUT-15 X-ray machine was used to irradiate the spheroids. The radiation doses varied from 0 to 10 Gy. After irradiation, cell viability was analyzed by flow cytometry. Staining was performed with a set of fluorescent dyes Annexin V-FITC/propidium iodide. The migration activity of irradiated spheroid cells was assessed by confluent analysis after transferring the spheroid to adhesive plastic.
Results: A dose-dependent decrease in cell migration activity was shown after X-ray irradiation in the dose range of 1–10 Gy. It has been established that doses of 6–8 Gy are optimal for the analysis of potential radiosensitizers by assessing the migration activity of cells. Using citrate-stabilized cerium oxide (CeO2) nanoparticles as an example, the possibility of using this model for rapid screening of nanomaterials with radiosensitizing action is demonstrated.
Conclusion: A method for forming 3D cell spheroids from EMT6/P cells has been developed and validated. The optimal dose of X-ray irradiation of the resulting cell spheroid has been selected for rapid screening of potential radiosensitizers. The functionality and reproducibility of the developed experimental model have been confirmed.
Keywords: 3D cell spheroid, solid tumor model, X-ray irradiation, radiosensitization
For citation: Mysina EA, Kolmanovich DD, Popova NR, Bokl BA, Pivovarov NA, Chukavin NN, Savintseva IV, Vinnik DA, Popov AL. 3D Cell Spheroid as a Relevant Experimental Model for Screening Potential Nanoradiosensitizers. Medical Radiology and Radiation Safety. 2025;70(5):11–17. (In Russian). DOI:10.33266/1024-6177-2025-70-5-11-17
References
1. Sung H., Ferlay J., Siegel R.L., Laversanne M., Soerjomataram I., Jemal A., Bray F. Global Cancer Statistics 2020: Globocan Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA. A Cancer Journal for Clinicians. 2021. Vol. 71. Global Cancer Statistics 2020;3:209-249.
2. Kapałczyńska M., Kolenda T., Przybyła W., Zajączkowska M., Teresiak A., Filas V., Ibbs M., Bliźniak R., Łuczewski Ł., Lamperska K. 2D and 3D Cell Cultures - a Comparison of Different Types of Cancer Cell Cultures. Archives of Medical Science: AMS. 2018;14;4:910-919.
3. Zhang C., Sui Y., Liu S., Yang M. In Vitro and in Vivo Experimental Models for Cancer Immunotherapy Study. Current Research in Biotechnology. 2024;7:100210.
4. Baker B.M., Chen C.S. Deconstructing the Third Dimension ‒ How 3D Culture Microenvironments Alter Cellular Cues. Journal of Cell Science. 2012;125;13:3015-24. doi: 10.1242/jcs.079509.
5. Gunti S., Hoke A.T.K., Vu K.P., London N.R. Organoid and Spheroid Tumor Models: Techniques and Applications. Cancers. 2021;13;4:874. doi: 10.3390/cancers13040874
6. Rofstad E.K. Growth and Radiosensitivity of Malignant Melanoma Multicellular Spheroids Initiated Directly from Surgical Specimens of Tumours in Man. British Journal of Cancer. 1987; 54;4:569-578.
7. Rofstad E.K., Wahl A., Brustad T. Radiation Response of Human Melanoma Multicellular Spheroids Measured as Single Cell Survival, Growth Delay, and Spheroid Cure: Comparisons with the Parent Tumor Xenograft. International Journal of Radiation Oncology*Biology*Physics. 1986;
12;6:975-982.
8. Wheldon T.E., Livingstone A., Wilson L., O’Donoghue J., Gregor A. The Radiosensitivity of Human Neuroblastoma Cells Estimated from Regrowth Curves of Multicellular Tumour Spheroids. The British Journal of Radiology. 1985;58;691:661-664.
9. Wei Q., Xu W., Han M., Dong Q., Fu Z., Diao, Liu H., Xu J., Jiang H., Zheng S., Gao J.-Q., Jiang H. Doxorubicin-Mediated Radiosensitivity in Multicellular Spheroids from a Lung Cancer Cell Line is Enhanced by Composite Micelle Encapsulation. International Journal of Nanomedicine. 2012;7:2661-2671.
10. Brüningk S.C., Rivens I., Box C., Oelfke U., Ter Haar G. 3D Tumour Spheroids for the Prediction of the Effects of Radiation and Hyperthermia Treatments. Scientific Reports. 2020;10;1:1653.
11. Bromma K., Beckham W., Chithrani D.B. Utilizing Two-Dimensional Monolayer and Three-Dimensional Spheroids to Enhance Radiotherapeutic Potential by Combining Gold Nanoparticles and Docetaxel. Cancer Nanotechnology. 2023;14;1:80.
12. Higashi Y., Matsumoto K., Saitoh H., Shiro A., Ma Y., Laird M., Chinnathambi S., Birault A., Doan T.L.H., Yasuda R., Tajima T., Kawachi T., Tamanoi F. Iodine Containing Porous Organosilica Nanoparticles Trigger Tumor Spheroids Destruction Upon Monochromatic X-Ray Irradiation: DNA Breaks and K-Edge Energy X-Ray. Scientific Reports. 2021;11;1:14192.
13. Tang J.L.Y., Moonshi S.S., Ta H.T. Nanoceria: an Innovative Strategy for Cancer Treatment. Cellular and Molecular Life Sciences: CMLS. 2023;80;2:46.
14. Ivanova O.S., Shekunova T.O., Ivanov V.K., Shcherbakov A.B., Popov A.L., Davydova G.A., Selezneva I.I., Kopitsa G.P., Tret’yakov Yu.D. One-Stage Synthesis of Ceria Colloid Solutions for Biomedical Use. Doklady Chemistry. 2011;437;2:103-106.
15. Li Y.Q., Guo Y.P., Jay V., Stewart P.A., Wong C.S. Time Course of Radiation-Induced Apoptosis in the Adult Rat Spinal Cord. Radiotherapy and Oncology. 1996;39;1:35-42.
16. Zamyatina E.A., Goryacheva O.A., Popov A.L., Popova N.R. Novel Pyrroloquinoline Quinone-Modified Cerium Oxide Nanoparticles and Their Selective Cytotoxicity Under X-Ray Irradiation. Antioxidants. 2024;13;12:1445.
17. Mysina E., Vinnik D., Pivovarov N., Popova N., Chukavin N., Popov A. Nanoceria Inhibit the Cell Migration from 3D Tumor Spheroid Formed From 4T1 Human Breast Cancer Cells. Biology and Life Sciences, 2025;16;2 (Print).
18. Hirschhaeuser F., Menne H., Dittfeld C., West J., Mueller-Klieser W., Kunz-Schughart L.A. Multicellular Tumor Spheroids: An Underestimated Tool is Catching up Again. Journal of Biotechnology. 2010;148;1:3-15.
19. Wojtkowiak J.W., Verduzco D., Schramm K.J., Gillies R.J. Drug Resistance and Cellular Adaptation to Tumor Acidic PH Microenvironment. Molecular Pharmaceutics. 2011;8;6:2032-2038.
20. West C.M., Sutherland R.M. The Radiation Response of a Human Colon Adenocarcinoma Grown in Monolayer, as Spheroids, and in Nude Mice. Radiation Research. 1987;112;1:105-115.
21. Schwachöfer J.H.M., Hoogenhout J., Kal H.B. The Radiation Response of a Human Lung Adenocarcinoma Grown in Monolayer, as Spheroids, and in Nude Mice. Lung Cancer. 1991;7;4:213-223.
22. Kornienko A.I., Teplonogova M.A., Shevelyova M.P., Popkov M.A., Popov A.L., Ivanov V.E., Popova N.R. Novel Flavin Mononucleotide-Functionalized Cerium Fluoride Nanoparticles for Selective Enhanced X-Ray-Induced Photodynamic Therapy. Journal of Functional Biomaterials. 2024;15;12:373.
23. Kolmanovich D.D., Romanov M.V., Khaustov S.A., Ivanov V.K., Shemyakov A.E., Chukavin N.N., Popov A.L. Proton Beam-Induced Radiosensitizing Effect of Ce0.8Gd0.2O2-x Nanoparticles Against Melanoma Cells in Vitro. Nanosystems: Physics, Chemistry, Mathematics. 2024;15;5:675-682.
PDF (RUS) Full-text article (in Russian)
Conflict of interest. The authors declare no conflict of interest.
Financing. The article was prepared as part of the RSCF grant No 22-73-10231, https://rscf.ru/project/22-73-10231/.
Contribution. E.A. Mysina – work with spheroids (cultivation, irradiation, viability analysis), D.D. Kolmanovich – flow cytometry and data analysis, N.R. Popova – scientific text editing, B.A. Bokle – collection and analysis of literary material, N.A. Pivovarov– collection and analysis of literary material, N.N. Chukavin – scientific text editing, I.V. Savintseva– cell culture, D.A. Vinnik – spheroid irradiation, A.L. Popov – research design development, scientific guidance.
Article received: 20.05.2025. Accepted for publication: 25.06.2025.