Glioblastoma multiforme (GBM) is a deadly brain tumor for which currently there is no cure and the median patient survival after diagnosis is 14.5 months [1]. Testing of new therapies to improve patient outcomes could be greatly facilitated by engineering drug screening platforms that closely mimic the physiological tumor environment [2]. One such model is a hydrogel-encapsulated GBM tumor spheroid that allows one to capture both cell-cell and cell-matrix interactions and their effect on cell drug responsiveness. Multiple groups have built such bioengineered GBM models, using both natural and synthetic hydrogels, and tested drug responses of the encapsulated spheroids [3], [4], [5], [6], [7], [8]. Collectively, previous work suggests that both spheroid formation (aka cell-cell interactions) and matrix encapsulation are important in studying GBM drug responsiveness. For example, it has been shown that dissociated cells grown as both a monolayer culture [3,8] or encapsulated in a hydrogel [5] show differential drug responses compared to spheroid cultures under similar conditions. We have shown differences in drug responses between free-floating and gel-encapsulated spheroids [4], and others have shown that GBM spheroids encapsulated in a hydrogel quickly acquire drug resistance, compared to non-encapsulated spheroids [9]. Further, it has been demonstrated that using multi-cell type spheroid cultures can discern sub-population-specific drug responses [7]. It has also been shown that ex vivo GBM cultures maintain the original tumor growth behavior when embedded in a hydrogel [10] and that encapsulating GBM patient cells in a hydrogel can predict clinical responses unlike cells grown in a monolayer [11].

However, while many studies have focused on the need for developing GBM bioengineered models, less is known about the effect of specific microenvironmental properties on spheroid drug responsiveness. One such critical microenvironmental property is substrate stiffness, as GBM tumors have been shown to respond to matrix stiffness both in vivo and in vitro [12], with some heterogeneity in rigidity sensitivity between various patient-derived GBM cell lines [13]. GBM cells have also shown to respond to interfacial mechanical gradients in hydrogels in vitro [14] and to topography-related biomechanical cues in vivo [15]. The normal brain tissue has a Young's modulus of ∼0.6 – 1.0 kPa, while the GBM tumor typically has a heighted modulus of ∼7 – 26 kPa [16]. However, in certain cases leaky vasculature in GBM tumors can also lead to softening of their environment to a stiffness lower than that of normal brain tissue [17].

Several groups have demonstrated the significant effects of matrix composition and/or stiffness on GBM cell behaviors such as cell morphology, motility, proliferation, and gene expression profiles [12,[18], [19], [20]]. For example, studies have shown that soft hydrogels favor less GBM cell spreading, lower cell motility and proliferation, but higher invasiveness compared to stiff hydrogels [21]. However, few studies have focused on the effect of substrate stiffness on GBM spheroid drug responsiveness. One recent study showed increased resistance to temozolomide (TMZ) with increased hydrogel stiffness (from 0.04 to 26.60 kPa), and noted that dissociated GBM cells formed spheroids only in hydrogels with stiffness >1 kPa [16]. Similarly, a different study showed larger GBM spheroids and higher resistance to TMZ in stiffer hydrogels as well as an increase in expression of drug resistance and invasion related genes in stiffer hydrogels compared to 2D culture [22].

For this work, we were motivated by the ability of GBM cells to sense both bulk and interfacial mechanical properties of their environment [14] and we explored GBM spheroid behaviors in soft, stiff, and dual (soft-stiff) hydrogels, where spheroids were seeded at the stiffness interface. Specifically, we examined spheroid viability, size, invasion, laminin expression, hypoxia, cell proliferation, and response to the chemotherapeutic TMZ as a function of hydrogel stiffness and noted differential cell behaviors, suggesting that matrix stiffness should be considered when designing in vitro GBM models.