Glioblastoma spheroid growth and chemotherapeutic responses in single and dual-stiffness hydrogels

Glioblastoma (GBM) is the deadliest brain tumor for which there is no cure. Bioengineered GBM models, such as hydrogel-encapsulated spheroids, that capture both cell-cell and cell-matrix interactions could facilitate testing of much needed therapies. Elucidation of specific microenvironment properties on spheroid responsiveness to therapeutics would enhance the usefulness of GBM models as predictive drug screening platforms. Here, GBM spheroids consisting of U87 or patient-derived GBM cells were encapsulated in soft (∼1 kPa), stiff (∼7 kPa), and dual-stiffness polyethylene glycol-based hydrogels, with GBM spheroids seeded at the stiffness interface. Spheroids were cultured for 7 days and examined for viability, size, invasion, laminin expression, hypoxia, proliferation, and response to the chemotherapeutic temozolomide (TMZ). We noted excellent cell viability in all hydrogels, and higher infiltration in soft compared to stiff hydrogels for U87 spheroids. In dual gels spheroids mostly infiltrated away from the stiffness interface with minimal crossing over it and some individual cell migration along the interface. U87 spheroids were equally responsive to TMZ in the soft and stiff hydrogels, but cell viability in the spheroid periphery was higher than the core for stiff hydrogels whereas the opposite was true for soft hydrogels. HIF1A expression was higher in the core of spheroids in the stiff hydrogels, while there was no difference in cell proliferation between spheroids in the stiff vs soft hydrogels. Patient-derived GBM spheroids did not show stiffness-dependent drug responses. U87 cells showed similar laminin expression in soft and stiff hydrogels with higher expression in the spheroid periphery compared to the core. Our results indicate that microenvironment stiffness needs to be considered in bioengineered GBM models including those designed for use in drug screening applications. STATEMENT OF SIGNIFICANCE: Recent work on tumor models engineered for use in drug screening has highlighted the potential of hydrogel-encapsulated spheroids as a simple, yet effective platform that show drug responses similar to native tumors. It has also been shown that substrate stiffness, in vivo and in vitro, affects cancer cell responses to drugs. This is particularly important for glioblastoma (GBM), the deadliest brain cancer, as GBM cells invade by following the stiffer brain structures such as white matter tracks and the perivascular niche. Invading cells have also been associated with higher resistance to chemotherapy. Here we developed GBM spheroid models using soft, stiff and dual-stiffness hydrogels to explore the connection between substrate stiffness, spheroid invasion and drug responsiveness in a controlled environment.