Studying Bladder Cancer Spheroids in 3D Collagen-Hyaluronic Hydrogel Exposed to Shear Forces

Cells in tissues are subjected to a variety of mechanical forces generated by their microenvironment, which regulate and influence their properties and behavior. Understanding how cells respond to mechanically altered environments is especially critical in cancer research, as migrating and invading cancer cells can actively modify the viscoelastic properties of surrounding tissues. To fully comprehend metastasis, it is essential to evaluate the dynamic interactions between cells and the mechanics of the extracellular matrix (ECM). Recently, 3D spheroids have been widely used as tumor models because they can mimic the complex physiological properties of living tissues.<br/>In this study, collagen-hyaluronic acid (Col-HA) hydrogels with embedded spheroids were used as a model to study cell migration in a complex 3D environment mimicking the ECM. Spheroids were formed from human non-malignant ureter cells (HCV29), transitional cell carcinoma cells (T24), and bladder carcinoma cells (HT1376). The study aimed to analyze the viscoelastic properties of Col-HA hydrogels and their effect on spheroid deformation and cell migration into the surrounding 3D scaffolds. We used a rheometer to study the elasticity and fluidity of the spheroids, quantified by the storage (<i>G'</i>) and loss (<i>G"</i>) moduli and the transition frequency <i>f_T</i> (<i>G’=G"</i>). The stiffer and softer hydrogels were characterized by G' of 28.8 ± 1.1 Pa and 0.84 ± 0.01 kPa and by G" of 7.6 ± 1.8 Pa and 0.15 ± 0.0 kPa, respectively. They were subjected to shear strain (γ = 1%) and frequency (f = 0.1 ÷ 10 Hz), simulating the strength of physiologically relevant mechanical forces. Incorporating spheroids into Col-HA hydrogel changed the rheological behaviors of the Col-HA 3D system, depending on the spheroid type and Col-HA mechanics. Moreover, our study showed that increased mechanical properties of the hydrogels inhibited spheroid deformation. Cell migration was significantly higher for the highly invasive T24 cells compared to HCV29 and HT1376 cells. These results indicate that Col-HA-based hydrogels are a suitable model for studying the mechanical resistance of spheroids under applied shear forces. We showed that cell escape from the spheroids' surface into the 3D Col-HA matrix depends on both the hydrogel mechanics and cell type. These results may help in understanding the relationship between physicochemical and biological properties at the cell-ECM interface.