Fiber-Dependent Remodeling of Solid Tumor Microenvironment in a 3D Breast Cancer Model

Introduction: :

Introduction: Metastasis is driven by genetic mutations and progressive remodeling of the extracellular matrix (ECM), which alters the biochemical and biophysical architecture of the tumor microenvironment (TME). Among ECM components, collagen I is a key regulator of tumor behavior [1], and it influences cell polarity, migration, invasion, and mechano-signaling by forming dense and aligned networks that define tumor-associated collagen signatures (TACS), often linked to poor progno-sis of breast cancer [2]. Collagen fibers can influence cancer cell migration, polarization, and matrix remodel-ing. While fibrous collagen networks mimic the native stromal architecture, most in vitro platforms fail to sys-tematically recapitulate the influence of fibrous versus non-fibrous collagen architectures on cancer invasion in three-dimensional (3D) contexts. We developed a colla-gen-based screening platform that enables the fabrication of tunable fibrous and non-fibrous microenvironments to investigate the role of ECM fibrillogenesis in breast can-cer remodeling behavior. 3D in vitro models have been used to mimic specific TME aspects and study dynamic ECM evolution. We used real-time screening techniques to record the dynamics of cells and their fibrous ECM.

Materials and

Methods: : Type I collagen was functionalized with methacrylic an-hydride (CMA, Advanced Biomatrix) to allow photo-crosslinking. Fibrous hydrogels were generated by ther-mal self-assembly at 37°C and pH: 7.2, while non-fibrous hydrogels were created using photo-crosslinking of CMA. Turbidity assays were used to confirm fibrillo-genesis kinetics. Microstructural differences in the hy-drogel groups were assessed using scanning electron mi-croscopy (SEM) and reflectance confocal imaging to screen cell-associated fiber density and directionality in real-time. Rheological measurements, such as storage modulus and stress relaxation, were used to quantify their mechanical properties. MDA-MB-231 breast cancer cells were encapsulated in both hydrogel groups at 1 mil-lion cells/ml. Over 14 days, cellular viability (live/dead assay), spreading (phalloidin/DAPI staining), and ECM remodeling were evaluated using confocal imaging and ImageJ-based morphometric analysis. To assess dynamic ECM degradation and crosslinking activity, we per-formed gene expression profiling of key matrix-modifying enzymes, including matrix metalloproteinases (MMPs) and crosslinkers such as transglutaminase 2 (TGM2) and lysyl oxidase (LOX), both of which are in-creasingly recognized as mechanoregulators of metastat-ic potential. Temporal changes in matrix deposition were quantified via immunostaining for type I collagen (COL1A1), a hallmark of tumor desmoplasia and fibrotic progression in breast cancer models.

Results, Conclusions, and Discussions::

Results: Turbidity assays showed a time-dependent in-crease in optical density for fibrous collagen, confirming successful fibrillogenesis, while photo-crosslinked gels remained optically clear (Figure D). SEM imaging re-vealed dense collagen networks in fibrous gels and uni-form pores in non-fibrous models (Figure C). Mechani-cal analysis showed fibrous gels had faster stress relaxa-tion and lower modules than non-fibrous gels. By cell-associated ECM remodeling over time, the fiber density increased, and the turbidity analysis showed changes in the edge location of the hydrogel models. Increased col-lagen fiber density leads to an increase in elasticity and shows stiffening in the fibrous samples (Figure E). To screen the fiber density and alignment, reflectance showed the shape and localization of the ECM masters in the phenotype of the cells, as well as the collagen deposi-tion. At the gene level, the fibrous model showed higher MMP secretion. MDA-MB-231 cells in fibrous matrices exhibited elongated morphology, increased cytoskeletal organization, and active collagen remodeling (Figure F-G). In contrast, cells in non-fibrous matrices remained more rounded and showed limited matrix interaction.

Conclusions: This study presents a robust screening platform to assess breast cancer cell–ECM interactions as a function of collagen microstructure. The findings highlight that collagen fibrillogenesis significantly im-pacts cellular remodeling behavior, with fibrous archi-tecture promoting enhanced tumor-like remodeling. This model offers valuable insight into the biophysical regula-tion of the invasion-metastasis cascade and may inform the design of future ECM-based cancer screening plat-forms and drug response studies

Conclusions: This study presents a robust screening platform to assess breast cancer cell–ECM interactions as a function of collagen microstructure. The findings highlight that collagen fibrillogenesis significantly im-pacts cellular remodeling behavior, with fibrous archi-tecture promoting enhanced tumor-like remodeling. This model offers valuable insight into the biophysical regula-tion of the invasion-metastasis cascade and may inform the design of future ECM-based cancer screening plat-forms and drug response studies.

Acknowledgements and/or References (Optional)::

References: [1] Winkler J, et al. Nat Commun. 2020; [2] Wu PH, et al. ACS Biomater Sci Eng. 2020.