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.

Breast cancer is one of the most dangerous forms of cancer worldwide. Continuous research is therefore essential in order to find realistic treatment options. 3D tumor spheroids have been proven to imitate the structural and physiological TME of in vivo tumors relatively more accurate than the more widely used 2D cell cultures.
During this study, the aim was to find the optimal conditions for single spheroid formation, employing breast cancer cells and utilizing GrowDex. Different concentrations and cell densities were used, as
well as differing methods (GrowDex addition prior or (days) after cell cluster formation). The liquid overlay technique was used to grow the spheroids, which were observed for a maximum of 14-16 days. Finally, an ATP viability assay was performed to determine whether the spheroids contained a proliferation zone, which corresponds to active cells.
Single spheroids were obtained for all cell densities. The relatively high GrowDex concentrations responded well to a cell density of 2000 cells, while the relatively low GrowDex concentrations responded well to cell densities of 5000 and 10000 cells. Through the ATP viability assay the samples illustrated
the presence of a proliferation zone, and thus active cells.