(31g) Evaluating Biophysical Patterns of GBM-Mediated Matrix Invasion of Microglia Using Microengineered Models

Glioblastoma (GBM) is the most common and aggressive form of primary brain cancer with median survival less than 15 months. Immunosuppression is one of the GBM hallmarks that severely limits the efficacy of current therapeutic strategies, including many immune-modulating treatments. Moreover, the activation, migration, and therapeutic responses are mediated by GBM tumor immune microenvironments (TIME). Microglia, primary brain-resident immune cells that maintain tissue homeostasis of brains, are composed of one-third of total tumor mass in GBM TIME. Nevertheless, while many studies have extensively explored the GBM cancer cell biology and underlying tumorigenesis, microglia have been underappreciated in GBM research. Furthermore, despite several sub-populations of microglia have been recently identified using single-cell technology, the biophysical dynamic of microglia matrix invasion into GBM is still largely unexplored due to the challenges of recapitulating GBM-microglia crosstalk both in vivo and in vitro. Thus, improving our understanding of GBM-microglia crosstalk, such as microglia matrix invasion and ECM remodeling, could potentially develop novel strategies to enhance current GBM therapeutic efficacy. Here, we applied bioengineering approach to fabricate microphysiological models of GBM TIME to evaluate the biophysical patterns of microglia invasion and its ECM remodeling capabilities. In the experimental, the three-parallel channel of microfluidic device was applied to study 3D invasion microglia in GBM TIME. 3 mg/mL type I collagen with or without GBM cancer cells (U87MG) was filled into central channel to mimic the extracellular matrix (ECM) of brain tissue. Human microglia cells (HMC3) were seeded in the lateral channels. Lipopolysaccharides (LPS), IL-4 or U87MG-conditioned medium were supplemented to the lateral channel individually to evaluate microglia invasion in responses to these biochemical signaling in GBM TIME. Matrix invasion of microglia was monitored for 24 hours. In addition, pharmacological inhibitors of matrix metalloproteinase (MMP), ROCK pathway and cellular metabolism were applied to determine the biophysical patterns of microglia matrix invasion. Moreover, the collagen gel contraction assay was used to measure the ECM contractility in responses to GBM TIME. Our finding showed that microglia did not significantly invade into ECM without any biochemical treatments while both LPS and IL-4 treated microglia promoted microglia invasion in terms of invaded length and invasion area. Interestingly, both U87MG-conditioned medium and U87-cocultured conditions significantly increased microglia invasion, suggesting GBM-secreted cytokines alone are potent enough to induce microglia matrix invasion in GBM. In addition, U87 conditioned medium treated microglia promoted ECM contraction, coincidentally with increased matrix invasion. Furthermore, our finding also shows that inhibition of ROCK pathway on HMC3 microglia significantly decreased its matrix invasion while inhibition of both MMP activity and cellular glycolysis metabolism did not alter microglia matrix invasion. The overall results suggest that GBM-mediated microglia invasion is independent of MMP activity (i.e., matrix degradation) and cellular metabolism. On-going work include the profiling the GBM-conditioned medium and evaluating biophysical properties of GBM-treated ECM to account for the GBM-promoted microglia matrix invasion. Collectively, our study provides novel insights of the biophysical dynamics of microglia matrix invasion in the presence of GBM TIME.