P22 - Extracellular Matrix Regulates Cancer Cell Morphological Transitions in 3D Hydrogel Microtissues.

Introduction: : The extracellular matrix (ECM) provides cues in regulat-ing cancer cell behavior through its mechanical proper-ties, including stiffness and microarchitecture [1]. In solid tumors, ECM remodeling leads to spatial heteroge-neity, impacting how cancer cells proliferate and invade the surrounding tissue. While previous studies in 2D en-vironments highlighted substrate stiffness as a dominant factor in influencing cell morphology, 3D ECMs present a more complex interplay of stiffness and spatial con-finement [2,3]. Our goal is to address the fundamental question of how cancer cells adapt their morphology in response to the physical characteristics of the ECM. Understanding these transitions is essential for modeling cancer invasion and engineering predictive tumor-on-chip models. This work establishes a reliable experi-mental platform for studying cellular markers in triple-negative breast cancer cells, followed by a theoretical modeling of the cell invasion.

Materials and

Methods: : We engineered 3D tumor ECM models using gelatin methacryloyl (GelMA) hydrogels with tunable stiffness (~ 1kPa soft, ~4 kPa stiff) and pore size (50–100 μm soft, 20–50 μm stiff). MDA-MB-231 cells were encapsulated in these hydrogels at 1×10⁶ cells/mL and cultured for 21 days. We optimized the fabrication method to have a stable hydrogel system over 21 days. To show independ-ence of the ECM-associated response from chemical properties we engineered composite GelMA and hyalu-ronic acid methacrylate (HAMA) hydrogels to match the mechanical properties of the homogenous hydrogels. Structural and mechanical stability were validated using swelling, mass residual, and compression testing. To model cell behavior, we developed a theoretical frame-work grounded in the minimum energy principle, calcu-lating total energy contributions (cell and ECM mechan-ical, chemical, and motor-work) across three morpholo-gies: rounded, elongated, and clustered. Cell morphology was analyzed using immunofluorescence (phal-loidin/DAPI), SEM imaging, and image analysis (round-ness, area). Viability and proliferation were assessed using Live/Dead staining and Ki67 immunolabeling. All experiments were conducted in triplicate, and statistical analyses were performed using one-way or two-way ANOVA with GraphPad Prism.

Results, Conclusions, and Discussions::

Results: Our mechanical characterization of the hydrogel ECM showed no significant variations in mechanical properties during tumor cell growth over 21 days for all 4 conditions (Figure B). SEM imaging confirmed a po-rous microstructure in both matrix groups, with quantifi-cation showing larger pore size for the softer hydrogel matrix (Figure B). The analysis of MDA-MB-231 cell phenotype cultured in soft and stiff ECMs showed mor-phological changes over 21 days. Cells in soft ECM tran-sitioned from rounded to elongated shapes, while those in stiff ECM formed compact clusters. Fluorescent imag-ing showed that cell morphology is strongly influenced by the physical properties of the extracellular matrix (Figure D). We used computational models to confirm that ECM stiffness directs cells toward invasive or pro-liferative phenotypes. Using a theoretical energy mini-mization model, we predicted that cells shape the bal-ance of intracellular contractility and ECM deformation energy [4]. In the soft environment, elongated morpholo-gies were favored due to low resistance and higher acto-myosin activity, whereas in stiff environments, cells minimized energy by adopting rounded, clustered forms (Figure E-F). Similar changes were seen in the hydrogels with different compositions but the same mechanical properties, showing stiffness and microstructure, rather than chemical, drive cell behavior. Our computational model closely reflected these trends, confirming that ECM mechanics shape breast cancer cell morphology. These results suggest that matrix stiffness may steer cells toward either an invasive or a proliferative state.

Conclusion: This study provides compelling evidence that ECM physical properties, specifically stiffness and pore size, regulate morphological transitions in an engi-neered 3D cancer model. These morphologies may repre-sent functionally divergent states, with elongation linked to migration and invasion, and clustered (spheroid for-mation) associated with local proliferation and tumor progression. Our approach establishes a quantitative plat-form for predicting and analyzing cell state transitions in heterogeneous tumors, which can inform the develop-ment of predictive tumor-on-chip systems and new ther-apeutic strategies targeting the tumor microenvironment. These results may be extended to other cancers and leveraged to engineer personalized models for drug screen-ing or to manipulate ECM conditions to modulate tumor behavior.

Acknowledgements and/or References (Optional)::

References: [1] Stuelten, et al,. Nat Rev Cancer, 2018. 18(5): p. 296-312. [2] Dogan, E., et al., Acta Biomateria-lia, 2024. 186: p. 156-166 [3] Hong, Y., et al., PNAS, 2025. 122(12): p. e2322762122. [4] Pourmostafa, A., et al, bioRxiv. 2025 May 15.