Comparative proteomics reveals distinct architecture-associated states in spheroid and bioprinted cervical cancer models

Abstract

Three-dimensional cancer models are widely used to improve the biological relevance of in vitro studies, yet scaffold-free spheroids and hydrogel-supported bioprinted constructs are often grouped under the same “3D” category. Here, we compared HeLa and SiHa cervical cancer cells cultured as adherent monolayers, scaffold-free spheroids, and 3D bioprinted alginate/gelatin constructs. Morphology, resazurin-based relative metabolic activity, and DIA-PASEF label-free quantitative proteomics were integrated with differential protein abundance analysis, pathway enrichment, direct 3D-to-3D comparison, and selected protein-level assessment. The global proteomic dataset comprised 8,837 protein groups and showed architecture-dependent organization in both cell lines. Spheroid and bioprinted models differed morphologically and metabolically and diverged markedly from adherent cultures at the proteome level. Spheroids were preferentially associated with glycolytic remodeling, metabolic adaptation, and hypoxia-associated signatures, compatible with compact multicellular organization and diffusion-limited architecture. In contrast, bioprinted models showed greater representation of ECM/cell-matrix-associated proteins and selected extracellular-remodeling proteins. Direct 3D-to-3D comparison confirmed that spheroid and bioprinted architectures generated distinct proteomic states. These findings support proteome-informed selection of 3D models according to the biological question under investigation.

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