The role of Nrf2 in the tumour microenvironment of IDH wild-type glioma

High-grade IDH wild-type gliomas are complex and devastating brain tumours, for which in spite of multiple modes of treatment, including surgery, chemotherapy and radiation therapy, survival times remain very short, on average of ~14 months. Macrophages and microglia are types of immune cells that have been shown to associate with glioma tumours and actively promote glioma growth. However, how they achieve their tumour-promoting activity is not clear. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a protein that is known as the master regulator of the cellular redox homeostasis. Nrf2 is activated by itaconate, a compound produced in mitochondria when macrophages are stimulated by various pathogens, or when macrophages come in contact with tumours cells. The activated Nrf2 then suppresses production of a plethora of factors (known as cytokines and chemokines) that promote inflammation, and thus Nrf2 activation plays a crucial role in the resolution of inflammation and in preventing chronic inflammation, which could otherwise lead to cell death and tissue damage. Here, we propose that association with glioma cells increases the levels of itaconate in macrophages and microglia, which in turn activates Nrf2, creating an anti-inflammatory environment that favours tumour growth.

We aim to answer the following research questions:
1. What are the molecular mechanisms that, upon association with glioma cells activate Nrf2 in brain macrophages and microglia? How does Nrf2 affect the interactions between glioma cells and brain macrophages and microglia?
2. Does association with glioma cells activate Nrf2 in macrophages and microglia in a zebrafish model? How? At which stages during tumour development does this occur?

To achieve these aims, we will use macrophage and microglia cells with different levels of Nrf2 activity. We will expose these immune cells to media from glioma cells, or grow them together with glioma cells, and quantitatively determine the expression of pro- and anti-inflammatory factors. We will then assess the effect of macrophage and microglia cells with different levels of Nrf2 on glioma cell growth, migration and invasion. Using zebrafish glioma models, combined with live imaging, we will analyse glioma development and determine the effect of Nrf2 (activation or suppression) on the interactions between macrophages and tumour cells, and on the growth of the tumour cells. This knowledge will open possibilities to suppress the pro-tumoural activity of macrophages and microglia, and design effective anti-cancer treatments.

If as hypothesized, Nrf2 activation in macrophage and microglia cells is responsible for promoting glioma development, the availability of compounds that inhibit the activity of Nrf2, provides an exciting opportunity to test their effect on glioma development in preclinical models and ultimately, in clinical trials.

High-grade, IDH wild-type gliomas are devastating brain tumours with survival times of ~14 months. Macrophages and microglia promote glioma growth, however how they achieve their tumour-promoting activity is unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2) is activated in macrophages upon upregulation of inducible immunoresponsive gene 1 (IRG1) and inhibits production of pro-inflammatory cytokines. Recent data suggest increased levels of IRG1 and the Nrf2-target gene NQO1 in microglia from human gliomas. This project will test the hypothesis that association with tumour cells upregulates IRG1 in tumour-associated macrophages/microglia, which in turn activates Nrf2, creating immunosuppressive microenvironment.

To test this hypothesis, we will combine analysis of human glioma tumours with cell culture and in vivo zebrafish glioma models. We will perform multiplex immunofluorescence of human IDH wild-type gliomas and quantify the levels of IRG1, Nrf2 and target genes in macrophages/microglia. Nrf2 will be manipulated genetically or pharmacologically in primary macrophages/microglial cells grown in glioma-conditioned medium. We will quantify the expression of IRG1, pro- and anti-inflammatory genes and investigate the potential involvement of the IRG1 product, itaconate. A co-culture system will be used to assess glioma cell proliferation, migration and invasion. Using zebrafish glioma models, combined with live imaging, we will analyse glioma initiation and determine the effect of Nrf2 (activation or suppression) on the interactions between macrophages/microglia and pre-neoplastic cells, and on proliferation of oncogenic cells. Detailed understanding of the role of Nrf2 will open possibilities to inhibit the pro-tumoural activity of macrophages/microglia and design effective anticancer treatments. The availability of compounds that inhibit the transcriptional activity of Nrf2 provides unique opportunity to test their effect on glioma development.