The rise of 3D spheroids in radiobiology for assessing tumour radioresistance

Authors

  • Alexandra Charalampopoulou Radiobiology Unit, Research and Development Department, CNAO National Center for Oncological Hadrontherapy, Pavia, Italy; Hadron Academy PhD Course, School for Advanced Studies (IUSS), Pavia, Italy https://orcid.org/0000-0002-8690-9420
  • Fabrizio De Luca Department of Biology and Biotechnology L. Spallanzani, University of Pavia, Pavia, Italy https://orcid.org/0000-0002-6462-2373
  • Giuseppe Magro Medical Physics Unit, Clinical Department, CNAO National Center for Oncological Hadrontherapy, Pavia, Italy https://orcid.org/0000-0001-7140-8642
  • Niloufar Matoor Department of Biology and Biotechnology L. Spallanzani, University of Pavia, Pavia, Italy
  • Amelia Barcellini Radiation Oncology Unit, Clinical Department, CNAO National Center for Oncological Hadrontherapy, Radiation Oncology Unit, Clinical Department, Pavia, Italy; Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy https://orcid.org/0000-0002-1595-104X
  • Giorgio Butella Research and Development Department, CNAO National Center for Oncological Hadrontherapy, Pavia, Italy https://orcid.org/0009-0008-4506-9497
  • Giovanni Battista Ivaldi Radiation Oncology Department, Istituti Clinici Scientific Maugeri IRCCS, Pavia, Italy https://orcid.org/0000-0002-7280-5698
  • Sara Lillo Radiation Oncology Unit, Clinical Department, CNAO National Center for Oncological Hadrontherapy, Radiation Oncology Unit, Clinical Department, Pavia, Italy; Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy https://orcid.org/0000-0001-7300-1168
  • Lorenzo Manti Department of Mathematics and Physics, University of Campania “L. Vanvitelli”, Caserta, Italy; National Institute for Nuclear Physics (INFN), Naples Section, Naples, Italy https://orcid.org/0000-0003-0168-5040
  • Alessio Mereghetti Research and Development Department, CNAO National Center for Oncological Hadrontherapy, Pavia, Italy https://orcid.org/0000-0002-0586-257X
  • Paola Tabarelli De Fatis Medical Physics Unit, Clinical Scientific Institutes Maugeri IRCCS, Pavia, Italy https://orcid.org/0000-0002-5721-9295
  • Maria Grazia Bottone Department of Biology and Biotechnology L. Spallanzani, University of Pavia, Pavia, Italy https://orcid.org/0000-0003-4570-8785
  • Angelica Facoetti Radiobiology Unit, Research and Development Department, CNAO National Center for Oncological Hadrontherapy, Pavia, Italy https://orcid.org/0000-0001-8215-9258

DOI:

https://doi.org/10.2340/1651-226X.2026.45079

Abstract

Background and purpose: Particle therapy (PT), including proton (PRT) and carbon ion radiotherapy (CIRT), offers physical and biological advantages over photon radiotherapy (XRT), particularly for radioresistant tumours such as glioblastoma and osteosarcoma. However, systematic preclinical comparisons using physiologically relevant models remain limited.
Material and methods: T98G (glioblastoma), Saos-2 and U2-OS (osteosarcoma) cells were cultured as two-dimensional (2D) monolayers and three-dimensional (3D) spheroids and irradiated with XRT, PRT or CIRT at 2, 4 or 6 Gy. Clonogenic survival, metabolic activity (PrestoBlue), invasion and spheroid growth kinetics were quantified. Relative biological effectiveness (RBE) was derived from survival data, and spheroid sections were analysed histologically (H&E).
Results: CIRT induced the strongest cytotoxic and anti-invasive effects across all models. In 2D cultures, the surviving fraction at 2 Gy decreased from 0.62 to 0.69 after XRT to 0.16–0.28 following CIRT (RBE = 2.1–2.4 vs. 1.1 for PRT; p < 0.0001). 3D spheroids exhibited overall higher radioresistance, yet CIRT markedly reduced growth and invasion, lowering normalised indices to 0.52 ± 0.12 (T98G) and 0.58 ± 0.09 (Saos-2) at 6 Gy, while photons often promoted invasion (> 1.2; p < 0.001). RBE values in 3D reached 3.6–4.0. H&E staining confirmed dose-dependent architectural disruption, with carbon ions inducing extensive necrosis and cellular degeneration.
Interpretation: This study introduces a robust 3D preclinical platform for radiobiological assessment of particle therapy. CIRT consistently overcame intrinsic and microenvironment-mediated resistance, outperforming photons and protons in suppressing viability, invasion and spheroid integrity, thus reinforcing the translational relevance of 3D models and the therapeutic promise of carbon ion therapy for resistant malignancies.

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2026-02-03

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Charalampopoulou, A., De Luca, F., Magro, G., Matoor, N., Barcellini, A., Butella , G., … Facoetti , A. (2026). The rise of 3D spheroids in radiobiology for assessing tumour radioresistance. Acta Oncologica, 65, 46–58. https://doi.org/10.2340/1651-226X.2026.45079

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Vol. 65 (2026): Acta Oncologica

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Copyright (c) 2026 Alexandra Charalampopoulou, Fabrizio De Luca, Giuseppe Magro, Niloufar Matoor, Amelia Barcellini, Giorgio Butella , Giovanni Battista Ivaldi , Sara Lillo, Lorenzo Manti, Alessio Mereghetti , Paola Tabarelli De Fatis , Maria Grazia Bottone , Angelica Facoetti

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