(F-240) Glioblastoma Spheroid Growth, Infiltration, Motility, and Chemotherapeutic Responses in Single and Dual-Stiffness Hydrogels

Introduction:: Multicellular spheroids are well-accepted in vitro cancer models that are routinely used for cancer therapy development. Spheroids are typically grown on non-adhesive surfaces in the absence of cell-matrix interactions. Here, we devolved a hydrogel-based glioblastoma spheroid model using polyethylene glycol (PEG) hydrogels, which allows for modulation of the spheroid microenvironment. GBM spheroids encapsulated in the hydrogels were seeded at a stiffness interface to observe the effect of a matrix stiffness mismatch on GBM spheroid growth and motility



Materials and Methods:: Spheroids were formed from U87 cells, C-10 xenoline, or patient-derived cells cultured into Sylgard microwell molds. These cells were seeded at a concentration of 1*10⁶ cells/mL and spun down for 3 min at 3000 rpm. The cells were cultured for 24 h in order to form spheroids and were used to encapsulate in the hydrogels. Hydrogels were made of 4-arm PEG-acrylate (4-arm PEG-Ac), 4-arm PEG-RGDS and PEG-dithiol (PEG-diSH) and an MMP-2 enzymatically degradable peptide crosslinker (VPMS↑MR). Soft hydrogels (Young’s modulus of 1 kPa) and stiff hydrogels (7 kPa) were fabricated to observe the effect of stiffness on spheroid growth. Dual hydrogels consisted of a thin interface of stiffness between 1 and 7 kPa. Cell viability was determined through a live/dead (DiOC/PI) staining, and the size and infiltration of the spheroid were observed from microscopic images. Cells were exposed to the chemotherapeutic drug temozolomide (TMZ; 2 mM) for 48 h. Hypoxia was measured through Hif-1α staining.




Results, Conclusions, and Discussions:: Here we developed an in vitro GBM model featuring a dual stiffness hydrogel in which spheroids were encapsulated within one side of the stiffness interface was successfully created. Figure 1A demonstrates the process for the creation of a dual hydrogel. Figures 1B and 1C exhibit successful encapsulation of spheroids at the stiffness interface. In terms of cell viability, all spheroids exhibited high viability (cell viability > 90%) for up to 7 days in soft, stiff, and dual hydrogels. Live/dead staining for U87 spheroids in dual hydrogels was performed on days 1 and 7. A MatLab code was created for efficient live/dead analysis. The soft hydrogel consisted of a higher cell viability in the core (80.1%) compared to the periphery (48.1%), and the stiff hydrogel consisted of a lower cell viability in the core (47.1%) compared to the periphery (66.2%). This was also seen in the dual hydrogels; however, there was no significant difference in viability between the core and the periphery. In the control dual hydrogels, spheroids were able to show the potential for infiltration past the stiffness interface as well as along the interface with preferential infiltration from soft to stiff, but not stiff to soft.

GBM spheroids infiltrated more in the soft gels than the stiff, and hypoxia occurred in the spheroid core encapsulated in the stiff gels. We suggest that the lower invasion in the stiff gels could be due to the higher crosslinking density compared to the soft gels as well as the higher number of enzymatically degradable sites that cells need to degrade in order to migrate. In terms of motility in the dual hydrogels, the spheroids were able to show potential for infiltration across the stiffness boundary in the controls (stiff-stiff gels). For dual hydrogels of differing stiffnesses, the cells preferentially migrated along the stiffness interface and crossed it only from soft to stiff. Glioblastoma multiforme in vivo are shown to migrate along and prefer areas of higher stiffness, consistent with the data obtained. Overall, replicating the GBM microenvironment is essential to understanding the effect of matrix stiffness on cellular viability, growth, and motility.




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