Introduction: : Tumor spheroids grow under mechanical constraints imposed by their surrounding extracellular matrix (ECM), which they actively deform over time. Understanding how this mechanical feedback varies across tumor types is crucial for decoding the role of physical forces in cancer progression. Here, we present a quantitative framework to assess the mechanical deformation generated by multicellular tumor spheroids (MCTS) embedded in bioinert agarose hydrogels. By comparing two breast cancer lines (MCF10A DCIS and SUM149PT) with a colon cancer line (HCT116), we investigated how different tumor types interact with their matrix during growth. We employed 1 µm-diameter polystyrene microbeads as passive markers of matrix deformation and tracked their displacements over a 48-hour period using time-lapse microscopy and image processing. Our findings reveal that deformation magnitude and spatial decay differ significantly by cell type and hydrogel stiffness, suggesting a correlation between tumor aggressiveness and force generation. This platform provides a tractable system to model tumor–ECM interactions in vitro and serves as a foundation for future studies exploring the mechanical regulation of cancer cell behavior.
Materials and
Methods: : Spheroid growth and induced matrix deformation were highly dependent on both cell line and gel stiffness. As shown in Figure 2, MCF10A DCIS spheroids expanded the most in free culture, decreased in 0.4% agarose, and increased again in 1% agarose—indicating a non-monotonic response to stiffness. HCT116 grew more in stiffer gels, while SUM149PT exhibited reduced growth. Displacement tracking (Figure 3) revealed that all MCTS generated increasing deformation over time, with highest displacements near the tumor boundary. MCF10A DCIS consistently showed the strongest deformation field, followed by SUM149PT and HCT116. Additionally, as illustrated in Figure 4, increased agarose stiffness amplified the bead displacement magnitudes, suggesting stronger mechanical resistance augments force propagation.
Results, Conclusions, and Discussions:: Spheroid growth and induced matrix deformation were highly dependent on both cell line and gel stiffness. As shown in Figure 2, MCF10A DCIS spheroids expanded the most in free culture, decreased in 0.4% agarose, and increased again in 1% agarose—indicating a non-monotonic response to stiffness. HCT116 grew more in stiffer gels, while SUM149PT exhibited reduced growth. Displacement tracking (Figure 3) revealed that all MCTS generated increasing deformation over time, with highest displacements near the tumor boundary. MCF10A DCIS consistently showed the strongest deformation field, followed by SUM149PT and HCT116. Additionally, as illustrated in Figure 4, increased agarose stiffness amplified the bead displacement magnitudes, suggesting stronger mechanical resistance augments force propagation This study provides a quantitative framework for evaluating tumor-induced mechanical deformation field using embedded microbeads and time-lapse microscopy. Our findings confirm that both tumor-intrinsic properties and matrix stiffness regulate how force is transmitted into the extracellular environment. MCF10A DCIS spheroids, representative of a more aggressive breast cancer phenotype, generated the highest displacement fields. Changes in hydrogel stiffness altered not just spheroid growth but also the degree of mechanical deformation. These results suggest that tumor–matrix interactions are context-dependent and nonlinearly modulated by mechanical resistance. The platform is generalizable for comparing biomechanical behavior across cell types and conditions and can be used to explore how tumors respond to confined growth environments.
The differential growth and deformation behaviors observed suggest that mechanical confinement is not merely a passive boundary condition but an active regulator of tumor biomechanics. The enhanced deformation in stiffer gels—despite variable growth outcomes—points to a possible mechanical feedback mechanism where matrix resistance increases the force output from spheroids. This may reflect underlying changes in cytoskeletal tension or contractility. The stronger deformation fields generated by breast cancer spheroids relative to colon cancer ones may also be tied to differences in actomyosin activity or invasiveness. Importantly, the mismatch between growth and displacement trends underscores the need to consider force transmission independently of size. These insights lay the groundwork for mechanotransduction studies in cancer progression models.
