(C-104) Granular hydrogel scaffolds for assessing the effect of cellular iron content on glioblastoma cell migration

Glioblastoma is a lethal brain cancer that infiltrates healthy brain tissues, making complete surgical removal almost impossible and posing significant therapeutic challenges. The effect of cellular iron on glioblastoma cell migration remains inadequately understood. Granular hydrogel scaffolds (GHS) made up of chemically assembled microparticles (microgels) provide a unique platform to investigate cell migration in response to different stimuli. GHS provides interconnected cell-scale void spaces, ameliorating oxygen and nutrient transport and facilitating nutrient gradient generation to promote cell migration within the scaffold. This study sought to develop GHS using gelatin methacryloyl (GelMA) photocrosslinkable microgels, a protein-based biomaterial that partially resembles the extracellular matrix (ECM). This platform was employed to investigate the impact of cellular iron content on cancer cell migration.


Materials and Methods:

3D GHS were fabricated using GelMA microgels with optimized rheological and mechanical properties designed to closely resemble the tumor microenvironment. To assess cell migration, CellTracker™ Green 5-chloromethylfluorescein diacetate (CMFDA) dye was used to label the cells, which were then seeded topically onto the 3D GelMA GHS. Prior to seeding, the scaffolds were immersed in DPBS supplemented with 1% v/v antibiotic for 3 hours in a 24-well non-treated cell culture plate. Afterward, the DPBS was removed, and 20 μL of stained cell suspension (5×10⁶ cells per mL of FAC-treated or untreated culture media) was applied to each scaffold. The scaffolds were then incubated for 30 minutes to allow the cells to attach to the microgels. Subsequently, complete culture media was added, and the scaffolds were further incubated at 37 °C, under a 5% v/v CO₂ atmosphere for 72 hours. Finally, the samples were cross-sectioned and imaged using a Leica DMi8 THUNDERED™ microscope (Germany) with an excitation wavelength of 470 nm (blue) and an emission wavelength of 550 nm (green). Image analysis was performed using the ImageJ software (FIJI, version 1.53c, NIH, MD, USA).


Results, Conclusions, and Discussions:

The effect of iron on cell migration in three dimensions was assessed using fluorescently labeled T98G and LN229 human glioblastoma cell lines, seeded topically on GHS and subjected to varying concentrations of ferric ammonium citrate (FAC). Results showed that iron treatment significantly reduced the average migration length of both cell lines compared with untreated groups. In conclusion, GHS is a suitable platform for understanding the role of cellular iron in glioblastoma pathogenesis and developing new therapeutic approaches.
 

Acknowledgements (Optional): We would like to acknowledge the funding received from the Meghan Rose Bradly Foundation.

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