Programming temporal stiffness cues within extracellular matrix hydrogels for modelling cancer niches

Area of Science:

• Biomaterials Science
• Cancer Biology
• Tissue Engineering

Background:

• Extracellular matrix (ECM) stiffening is a hallmark of diseases like breast cancer.
• Current 3D *in vitro* models often lack dynamic mechanical cues mimicking disease progression.
• There is a need for smart biomaterials that replicate the temporal biomechanical changes of the tumor microenvironment.

Purpose of the Study:

• To develop a preclinical 3D *in vitro* model that replicates the dynamic plasticity of the tumor microenvironment.
• To investigate the effects of progressive extracellular matrix (ECM) stiffening on breast cancer cell behavior and spheroid formation.
• To create a model that allows observation of temporal phenotypic changes in cancer cells within a controlled microenvironment.

Main Methods:

• A composite hydrogel was formulated using adipose-derived decellularized ECM (AdECM) and silk fibroin.
• The hydrogel was crosslinked using visible light and designed to progressively stiffen over 3 weeks via polymer secondary structure interactions.
• MCF-7 breast cancer cells were encapsulated and cultured *in vitro* within the stiffening hydrogel microenvironment.

Main Results:

• Progressive hydrogel stiffening (from initial to 25 kPa) induced growth arrest and phenotypic changes in MCF-7 cells.
• The model fostered the development of mature, organoid-like spheroids with structures mimicking breast epithelium.
• Intratumoral heterogeneity emerged, with central cells showing aggressive phenotypes (increased fibronectin, reduced E-cadherin), reflecting physiological tumors.

Conclusions:

• The developed stiffening hydrogel model successfully replicates temporal biomechanical cues of the tumor microenvironment.
• Dynamic ECM stiffening drives phenotypic heterogeneity and the formation of organoid-like structures in breast cancer spheroids.
• This approach offers a novel strategy for creating advanced 3D *in vitro* disease models with broad applicability.