Comparative radiobiological characterization of multiple ion irradiation in 2D and 3D in vitro models: Assessing radioresistance profiles and metastatic processes.

La radioterapia (RT) rappresenta un pilastro del trattamento oncologico, ma la radioresistenza tumorale e l’eterogeneità del microambiente ne limitano l’efficacia, in particolare nei tumori solidi. Sebbene l’adroterapia offra vantaggi fisici rispetto alla RT convenzionale con fotoni, l’efficacia biologica delle diverse qualità di radiazione e la loro interazione con l’architettura tumorale non sono completamente chiarite. I modelli in vitro 2D non ricapitolano adeguatamente la complessità strutturale dei tumori solidi, mentre i modelli 3D forniscono un contesto biologicamente e traslazionalmente più rilevante. Questa tesi ha avuto l’obiettivo di valutare sistematicamente l’impatto della qualità della radiazione su sopravvivenza, crescita tumorale e processi metastatici in tumori umani intrinsecamente radioresistenti. A tal fine, irraggiamenti con fotoni, protoni, ioni elio e ioni carbonio sono stati integrati in colture 2D e in modelli di sferoidi tumorali 3D, analizzando undici linee cellulari rappresentative di diversi istotipi solidi. Nelle colture 2D sono state valutate sopravvivenza clonogenica, vitalità metabolica, migrazione e invasione, mentre negli sferoidi 3D sono state analizzate dinamiche di crescita, vitalità e comportamento invasivo. L’efficacia biologica relativa (RBE) è stata quantificata dalla sopravvivenza clonogenica in 2D e, per la prima volta, derivata anche dalla crescita degli sferoidi 3D. In tutti i modelli sperimentali, l’aumento del trasferimento lineare di energia (LET) ha potenziato l’efficacia biologica. Gli ioni carbonio hanno mostrato la maggiore efficacia, con curve di sopravvivenza più ripide, valori di RBE fino a 3.0 e una marcata soppressione di crescita e invasione, mentre protoni e ioni elio hanno evidenziato effetti più moderati. Negli sferoidi 3D, la radioresistenza è risultata amplificata rispetto alle colture 2D, in particolare per fotoni e protoni, mentre l’irraggiamento ad alto LET ha attenuato radioresistenza, ipossia ed eterogeneità tumorale. Complessivamente, questi risultati dimostrano che la qualità della radiazione, più della sola dose, rappresenta il determinante principale del destino delle cellule tumorali e supportano l’integrazione di strategie di radioterapia con particelle biologicamente informate, basate su LET e approcci multi-ione, per il trattamento dei tumori solidi resistenti.

Radiotherapy (RT) remains a cornerstone of cancer treatment, yet tumour radioresistance and microenvironmental heterogeneity continue to limit therapeutic efficacy, particularly in solid tumours. While particle therapy offers clear physical advantages over conventional photon irradiation, the biological effectiveness of different radiation qualities, and their interaction with tumour architecture, have not been fully elucidated. Conventional in vitro 2D models inadequately capture key features of solid tumours, including cell-cell and cell-matrix interactions, three-dimensional organisation and spatial complexity, thereby limiting their ability to predict biologically relevant radiation responses. In contrast, 3D culture models offer clear advantages by better recapitulating tumour architecture and contextual interactions, providing a more biologically and translationally relevant framework for radiobiological research. This doctoral thesis aimed to systematically evaluate the impact of radiation quality on tumour cell survival, tumour growth and metastatic processes across a broad spectrum of intrinsically radioresistant human cancers, integrating photon, proton, helium ion and carbon ion irradiation in both 2D monolayer cultures and 3D tumour spheroid models. Eleven human cancer cell lines representing glioblastoma, osteosarcoma, pancreatic, ovarian, breast and vulvar carcinomas were analysed. In 2D cultures, clonogenic survival, metabolism-based viability, migration and invasion were assessed following irradiation with photons, protons, helium ions and carbon ions. Relative biological effectiveness (RBE) was quantified from clonogenic survival in 2D cultures and, for the first time, also derived from growth dynamics in 3D tumour spheroids. In 3D spheroids, growth, metabolism-based viability and invasive behaviour were evaluated. Comparative analyses were performed to delineate linear energy transfer (LET)-dependent effects and tumour-specific response patterns. Across all models, increasing LET enhanced biological effectiveness. Carbon ions produced the steepest clonogenic survival curves and the lowest surviving fractions at 2 Gy with RBE values of 1.1-3.0, while protons showed moderate effects with RBE of 1.0-1.2 and helium ions displayed intermediate effectiveness. High-LET irradiation increased α and α/β ratios, reflecting reduced repair capacity and fractionation dependence. In 3D spheroids, radioresistance was amplified relative to 2D cultures, particularly for photons and protons, whereas carbon ions strongly suppressed spheroid growth and invasion. In contrast to photons, which often promoted migratory and invasive phenotypes, carbon ions consistently inhibited tumour cell migration and invasion. This thesis demonstrates that radiation quality, more than dose alone, is the dominant determinant of tumour cell fate, governing survival, growth and invasive behaviour. High-LET carbon ions attenuate intrinsic radioresistance, hypoxia and tumour heterogeneity, producing uniform and sustained biological effects across diverse tumour types. Importantly, this work provides the first experimental evidence that clinically relevant RBE values derived from 3D spheroid growth exceed those obtained from conventional 2D clonogenic assays, highlighting the limitations of simplified models in predicting therapeutic gain. Collectively, these findings support the integration of biologically informed approaches, including LET-based optimisation and multi-ion strategies, to improve the precision and efficacy of particle radiotherapy in resistant and heterogeneous solid tumours.