Using 3D Spheroid Coculture Assays to Model Glioma-Macrophage Interactions​

Eleftheria Mahlis

Co-Presenters: Katilyn Sookoo, Lamont Lee, Jazmin Mendoza-Ortega

College: Hennings College of Science Mathematics and Technology

Major: BS.SCI/TEC/MOLBIO

Faculty Research Mentor: Coniglio, Salvatore  

Abstract:

Glioblastoma Multiforme (GBM) is the most aggressive form of adult brain tumor with a median survival time of twelve months. GBM is highly resistant to conventional therapy which includes surgical resection of the tumor, radiation treatment and chemotherapy. GBM cells are highly motile and invasive resulting in infiltrative tumors with poorly defined borders. GBM tumors are heavily infiltrated with microglia cells which are known to stimulate glioma cell invasion. Our laboratory has previously demonstrated that macrophages strongly stimulates glioma invasion both in-vitro and in orthotopic animal models. This interaction was found to be dependent on CSF-1R which is expressed on all tumor infiltrating macrophages/microglia. Blockade of the CSF-1R using compounds such as pexidartinib (PLX3397) can inhibit microglia/macrophage-stimulated glioma invasion in-vitro and in vivo. Here we generate glioma cell spheroids using the “hanging drop” method on a 96 well U bottom plate. After 24 hours, spheroids are embedded in matrigel containing GFP-expressing macrophages. Time lapse imaging is used to measure glioma cell- macrophage interactions including trafficking and stimulation of invasive protrusions. A variety of chemokines are upregulated in the GBM tumor microenvironment and facilitate “cross-talk” between macrophages and glioma cells eliciting a chemotactic response. Using novel CCR-1 specific antagonists, we show that macrophage-stimulated glioblastoma invasion in-vitro is dependent on CCR-1. Furthermore, using quantitative-PCR, immunofluorescence and ELISA assays we observe that treatment of microglia with glioblastoma conditioned media strongly induces the expression of CCR-1 and several ligands for CCR-1 including CCL-3 and CCL-5. Finally, we investigated potential effectors of CCR-1 signaling which mediates glioblastoma invasion. Among these, the matricellular protein osteopontin (OPN) emerges as a promising candidate. OPN is highly upregulated in glioma conditioned media stimulated macrophage cell lines and human monocyte derived macrophages. Furthermore, the upregulation of OPN is dependent on CCR-1 as shown using two different pharmacological inhibitors of CCR-1.