Poster S19 - Evaluating Biophysical Patterns of Glioblastoma-Mediated Matrix Invasion of Microglia Using Microengineered Models

Introduction: Glioblastoma (GBM) is the most common and lethal 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, growing evidence suggest that activation, migration, and therapeutic responses are mediated by GBM tumor immune microenvironments (TIME). Microglia, primary brain immune cells that maintain tissue homeostasis of brains, are composed up to one-third of total tumor mass in GBM. Nevertheless, while many studies have extensively explored the fundamental cancer cell biology of GBM, microglia have been underappreciated in brain cancer research. Furthermore, despite several sub-populations of microglia have been recently identified using single-cell technology, the biophysical dynamic of microglia matrix invasion during GBM progression is still largely unexplored due to the challenges of recapitulating GBM-microglia crosstalk both ex vivo and in vitro. Thus, improving our understanding of GBM-microglia crosstalk, such as microglia matrix invasion and ECM remodeling, could potentially inform the future developments of novel strategies to enhance current GBM therapeutic efficacy. Here, we applied integrative bioengineering approach to fabricate microphysiological models of GBM TIME to evaluate the biophysical patterns of microglia invasion and its ECM remodeling.

Materials and

Methods: In the experimental, we used the three-parallel channel of microfluidic devices to study 3D invasion microglia in GBM TIME (Figure A). 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 native brain tissue (Figure B). Human microglia cells (HMC3) were seeded in the lateral channels. Microglia was labeled with cell tracker green and U87MG was labeled with RFP. Lipopolysaccharides (LPS), IL-4 or U87MG-conditioned medium were supplemented to the lateral channels 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.

Results, Conclusions, and Discussions: Our finding showed that microglia did not significantly invade into ECM without any biochemical treatments while both LPS and IL-4 treatments 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 (Figure C). In addition, U87 conditioned medium treatment increased 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.

Acknowledgements (Optional): This work was supported by the NIH (R01CA256481). C.-W.C gratefully acknowledges funding from the IGB Fellow Program at the Institute for Genomic Biology, University of Illinois Urbana-Champaign.