TMIC-20. Assessing the immune microenvironment in glioma models by correlative high field MRI and light sheet microscopy

Gliomas are malignant brain tumors with an immunosuppressive tumor microenvironment (TME). To induce a proinflammatory shift in the TME, we used the Toll-like receptor 7 agonist CDNP-R848, which effectively treated preclinical Gl261 glioma. MRI, the main clinical tool for monitoring glioma treatment, cannot visualize key immunological components of the TME. Preclinical Gl261 and SB28 gliomas were xenografted in mice, followed by MRI monitoring. The mice received CDNP-R848 intravenously or no treatment as a control. To examine spatial patterns of immune cells in the TME, we propose that light sheet microscopy (LSM) with whole-brain immunostaining of myeloid cells (Iba1) and T cells (CD3) using iDISCO can reveal immune cell influx, distribution, and therapy-induced changes. In the Gl261 tumor model, CDNP-R848 treatment induced glioma regression (ORR: 75%), primarily through macrophage activation during the effector phase (week 3). However, CDNP-R848 did not induce tumor regression in the SB28 tumor model. Cleared mouse brains with Gl261 tumors showed T cell accumulation around peritumoral microvessels and recruitment of distinct myeloid populations, including tumor-associated microglia and infiltrating tumor-associated macrophages. Iba1+ cells prominently accumulated in the corpus callosum, at the tumor border, and peritumorally, with a particularly increased density in the ipsilateral cortex. Our findings suggest white matter tracts as potential routes for immune cell infiltration, particularly for Iba1+ microglia, which may act as therapeutic bystanders. In contrast, myeloid cells located in the tumor core and at the tumor border are primarily blood-derived and represent CDNP-R848-targeted macrophages. Additionally, the data showed a correlation between tumor size and the number of infiltrating immune cells. Untreated tumors showed higher proportions of Iba1+ microglia, while widespread peritumoral myeloid activation appeared therapy-independent. TME complexity appeared to develop prior to therapy. These findings indicate that the recruitment pathways of myeloid and T cells are crucial for understanding potential resistance to glioma immunotherapy.