Triple Negative Breast Cancers (TNBCs) account for 20% of breast cancer cases and are characterized by poor prognosis and limited treatment options. The Tumor Microenvironment (TME), particularly the collagen-rich Extracellular Matrix (ECM), plays a critical role in TNBCs progression. Collagen remodeling, including fiber realignment and degradation, significantly influences cancer cell adhesion, migration, and invasion.

Given the importance of collagen in modulating tumor invasion, this study investigates how collagen matrix porosity regulates the invasive behavior of 3D breast cancer cell clusters, spheroids. Spheroids composed purely of breast cancer cells, purely of fibroblast cells, and mixed spheroids containing both cell types in equal proportions, are used to mimic the TME. Spheroid behavior was examined in collagen gels of varying concentrations (1.5 and 4.0 mg/mL for rat tail collagen, and 2.4 and 3.3 mg/mL for bovine collagen). The size and number of pores in the collagen gels were quantified, and changes in spheroid growth and morphology patterns were monitored over time.

Invasion was most pronounced in low-concentration gels with larger pores (1.5 mg/mL rat tail; 2.4– 3.3 mg/mL bovine). Breast cancer-only spheroids were most invasive, fibroblast-only were least invasive, and mixed spheroids showed intermediate behavior more closely resembling that of the fibroblast-only spheroids. In contrast, invasion was greatly restricted in the 4.0 mg/mL rat tail collagen due to reduced porosity and denser networks which acted as physical barriers for all three spheroid types.
These results highlight collagen porosity as a key factor in TNBCs invasion. This has important implications for designing physiologically relevant in vitro tumor models that better capture the complexity of the ECM in cancer progression.