Abstract
INTRODUCTION: Glioma associated macrophages/microglia (GAMs) are the most abundant immune cell type in glioblastoma and play a key role in immunotherapeutic resistance. The tumor microenvironment (TME) is thought to modulate GAM phenotype and function leading to a more pro-tumorigenic immune milieu however the exact nature of this crosstalk is unclear. Existing systems to study human GAMs are suboptimal as primary human GAMs are difficult to culture. There is therefore a need to develop human model systems of sufficient fidelity to probe GAM function. Here we report on the development of a human embryonic stem cell (hES)-based platform to derive microglia without recourse to immortalizing oncogenic transduction. This technology can assess the impact of tumor derived factors on the microglial phenotype and represents a novel, scalable model system to investigate GAM function. METHODS: hES-microglia were generated using a stepwise cytokine-based differentiation protocol. Upon exposure to conditioned media obtained from cultures of a panel of patient-derived glioblastoma cell lines (n=6 biological replicates), hES-GAMs were analyzed using flow cytometry, single cell RNA sequencing (scRNA). Functional assays included phagocytosis of pH rhodo particles, wound scratch migration assays and intracellular proteome analysis. RESULTS: Exposure to tumor media leads to transcriptional changes similar to those seen in primary GAMs with increased chemotactic genes such as CCL2(MCP1), CCL3 and CCL4, with corresponding changes in cytokine profile at the protein level. Other upregulated cytokines of note include VEGF, OSM, CCL4 and IL10RB. Functional impact of co-culture with tumor media included a dramatic increase in GAM motility (>30 fold) and a statistically significant decrease in phagocytic ability (29.1% p < 0.001). CONCLUSIONS: The hES-derived GAM model is a promising novel platform to dissect the impact of the TME on tumor-derived GAMs.
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