Spheroid versus monolayer culture: proteomic insights into divergent pathways in myxofibrosarcoma

Aim: Myxofibrosarcoma (MFS) is characterized by high local recurrence and complex microenvironmental interactions. Although three-dimensional (3D) culture systems better mimic in vivo tumor architecture than conventional two-dimensional (2D) monolayer cultures, the global proteomic consequences of dimensionality in MFS remain incompletely defined. Methods: We performed quantitative mass spectrometry-based proteomic profiling of eight independently established patient-derived MFS cell lines cultured under 2D monolayer and 3D spheroid conditions. Differential protein expression and pathway enrichment analyses were conducted to delineate dimensionality-driven molecular programs. Results: Culture dimensionality emerged as the principal determinant of proteomic variation across all cell lines. Compared with monolayers, 3D spheroids exhibited significant enrichment of hypoxia response, autophagy-related processes, extracellular matrix organization, and PI3K-Akt signaling pathways. In contrast, 2D cultures preferentially upregulated DNA replication, RNA processing, and cell-cycle-associated pathways. These findings indicate that 3D architecture alone is sufficient to induce coordinated stress-adaptive and survival-oriented proteomic reprogramming in MFS cells. Conclusions: This study provides a comprehensive proteomic atlas defining dimensionality-dependent molecular states in MFS. While functional validation was beyond the scope of this work, the pathway rewiring identified here establishes a mechanistic framework for future hypothesis-driven investigations targeting autophagy- and PI3K-Akt-associated vulnerabilities in 3D MFS models.