3D Cell Spheroid as a Relevant Experimental Model for Screening Potential Nanoradiosensitizers

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.