Radiotherapy induces layer-specific molecular and ultrastructural alterations in triple-negative breast cancer spheroids.

Reliable in vitro models to study radiotherapy response in triple negative breast cancer (TNBC) remain elusive. Traditional 2D cultures fail to reproduce the complex architecture and heterogeneity of solid tumors, which are essential factors influencing radiotherapy outcome. In contrast, 3D tumor spheroids better mimic key features of the tumor microenvironment, as cell-cell interactions and gradients of oxygen and nutrients. These features contribute to the formation of distinct zones within the spheroid - proliferative, quiescent, and necrotic, closely resembling the in vivo tumor structure and affecting radiotherapy efficacy. However, further investigation is needed to understand how radiotherapy affects cancer cells ultrastructure across the distinct layers of the tumor, as well as associated molecular pathways. Herein, we present a 3D TNBC spheroid platform that enables spatial characterization of tumor cell responses to clinically relevant radiotherapy across distinct spheroid regions. Radiotherapy decreased spheroids viability, while increasing cell size and the number of organelles, such as mitochondria, lipid droplets, rough endoplasmic reticulum and autophagic vesicles in surviving cells. Spheroids periphery, with higher oxygen availability and proliferation, exhibited the most pronounced organelle alterations after irradiation. Proteomic profiling revealed upregulated pathways linked to cell cycle progression and organelle fission, supporting G2/M cell cycle arrest. Enrichment of mitotic nuclear division, oxidative phosphorylation, ATP synthesis and lysosomal activity pathways suggest activation of repair and survival mechanisms following radiation-induced stress. Overall, this TNBC 3D model represents a promising platform for radiobiology research and for high-throughput screening of combinatory treatments to overcome radioresistance.