Monitoring innate immune cell dynamics in the glioma microenvironment by magnetic resonance imaging and multiphoton microscopy (MR-MPM)

Abstract

Rationale: Glioblastoma is the most frequent, primary brain tumor that is characterized by a highly immunosuppressive tumor microenvironment (TME). The TME plays a key role for tumor biology and the effectiveness of immunotherapies. Composition of the TME correlates with overall survival and governs therapy response. Non invasive assessment of the TME has been notoriously difficult. Methods: We have designed an in vivo imaging approach to non invasively visualize innate immune cell dynamics in the TME in a mouse glioma model by correlated MRI and multiphoton microscopy (MR-MPM) using a bimodal, fluorescently labeled iron oxide nanoparticle (NP). The introduction of Teflon cranial windows instead of conventional Titanium rings dramatically reduced susceptibility artifacts on MRI and allowed longitudinal MR-MPM imaging for innate immune cell tracking in the same animal. Results: We visualized tumor associated macrophage and microglia (TAM) dynamics in the TME and dissect the single steps of NP uptake by blood-born monocytes that give rise to tumor-associated macrophages. Next to peripheral NP-loading, we identified a second route of direct nanoparticle uptake via the disrupted blood-brain barrier to directly label tissue resident TAMs. Conclusion: Our approach allows innate immune cell tracking by MRI and multiphoton microscopy in the same animal to longitudinally investigate innate immune cell dynamics in the TME.

Keywords: MRI; immunotherapy.; iron oxide nanoparticles; multiphoton microscopy; tumor microenvironment; tumor-associated macrophages.

Figure 1

Comparing MRI properties of Titanium and Teflon cranial windows. Photograph of Titan and Teflon rings used for cranial windows (a). T2 and T2* MRI images of cranial rings made from Titanium, Teflon polymethylmethacrylate (PMMA), polyacetale (POM), polyamide (PA) and polypropylene (PP) (b). Rings were measured in falcon tubes embedded in agarose. Representative MR images of healthy control animals implanted with Titan or Teflon cranial windows for multiphoton microscopy. T1-w and T2-w images (c,d) are shown. Teflon rings reduce metal artifacts compared to conventional titanium rings. Coronal, sagittal and axial plane T2*-w images are shown (e,f). Scale bar is 1mm.

Figure 2

MRI compatible cranial windows for glioma monitoring. T1-w imaging after Gd-contrast administration (upper row), T2-w images (middle row) and T2*-w images (bottom row) at day 4, 16 and 26 after tumor inoculation show the rapid tumor growth (a). Study outline shows the experimental and imaging timepoints (b). T2*-w images before and 48 hours after iron oxide nanoparticle (NP) administration (c). Segmentation and quantification of intratumoral susceptibility signals (ITSS; magenta) before and 48 hours after NP administration (d). n=3 mice. ITSS were quantified separately for the tumor border (grey) and tumor core (black bar). For the subtraction image in (c) the T2* image before NP injection is subtracted from the 48 hours post NP image. This facilitates the detection of the hypointensities mainly present in the tumor border (arrowhead) and the differentiation of preexisting tumor microbleedings from NP uptake. Scale bars are 1mm.

Figure 3

Immune cell dynamics after nanoparticle administration. Multiphoton microscopy images of the same region before, 2 hours after and 48 hours after intravenous CLIO-TAMRA injection in a Cx3cr1-GFP animal (a). Close up with single channels show single Cx3cr1⁺ TAMs before and after NP uptake (arrowheads). Quantification of nanoparticle uptake based on fluorescence intensity in TAMRA channel per Cx3cr1⁺ TAM, normalized to baseline (before NP injection). n=60 cells from 3 mice. Overview image 48 hours after NP injection (recorded as tile scan) (b). Dotted line indicates the tumor border. Uptake of NP in brain resident macrophages/microglia two hours after NP injection (c) Different cytoplasmic appearances of NP in TAMs as dispersed (arrowheads) or clustered NP (arrow) (d). Intravascular migration of a monocyte with phagocytosed NP (e). Monocyte transmigration into the adjacent parenchyma (f). Scale bars are 100µm in a, b and 20µm in c-f and magnified images in a. f.i.: fluorescence intensity. a.u.: arbitrary units.

Figure 4

Immunohistochemistry of TAMs and quantification of NP uptake. Overview image of the tumor bearing hemisphere (a). Close up and magnified images of the tumor core, periphery and adjacent healthy cortex (b). Quantification of Iba-1⁺ cells (c), the NP uptake (d) and proportion of NP-labeled cells (e) in the different regions. n=4 mice. Immunohistochemical staining of the M1 marker CD80 and M2 marker CD206 (f). Quantification of M1-like and M2-like macrophages / microglia in the tumor core vs tumor border. n=6 mice (g). FACS quantification of macrophage polarization as assessed by MHCII and arginase expression. Macrophages were incubated for 24 hours with 100µg of NP and pre-polarized with Il-4/ Il-13 (h). f.i.= fluorescent intensity. Scale bars are 500µm in a, f, 50µm in b and 10µm in close-ups.