Fast-field-cycling NMR at very low magnetic fields: water molecular dynamic biomarkers of glioma cell invasion and migration

Abstract

Our objective was to study NMR relaxometry of glioma invasion/migration at very low field (<2 mT) by fast-field-cycling NMR (FFC-NMR) and to decipher the pathophysiological processes of glioma that are responsible for relaxation changes in order to open a new diagnostic method that can be extended to imaging. The phenotypes of two new glioma mouse models, Glio6 and Glio96, were characterized by T_2w -MRI, HE histology, Ki-67 immunohistochemistry (IHC) and CXCR4 RT-qPCR, and were compared with the U87 model. R₁ dispersions of glioma tissues were acquired at low field (0.1 mT-0.8 T) ex vivo and were fitted with Lorentzian and power-law models to extract FFC biomarkers related to the molecular dynamics of water. In order to decipher relaxation changes, three main invasion/migration pathophysiological processes were studied: hypoxia, H₂ O₂ function and the water-channel aquaporin-4 (AQP4). Glio6 and Glio96 were characterized with invasion/migration phenotype and U87 with high cell proliferation as a solid glioma. At very low field, invasion/migration versus proliferation was characterized by a decrease in the relaxation-rate constant (ΔR₁ ≈ -32% at 0.1 mT) and correlation time (≈-40%). These decreases corroborated the AQP4-IHC overexpression (Glio6/Glio96: +92%/+46%), suggesting rapid transcytolemmal water exchange, which was confirmed by the intracellular water-lifetime τ_IN decrease (Δτ_IN ≈ -30%). In functional experiments, AQP4 expression, τ_IN and the relaxation-rate constant at very low field were all found to be sensitive to hypoxia and to H₂ O₂ stimuli. At very low field the role of water exchanges in relaxation modulation was confirmed, and for the first time it was linked to the glioma invasion/migration and to its main pathophysiological processes: hypoxia, H₂ O₂ redox signaling and AQP4 expression. The method appears appropriate to evaluate the effect of drugs that can target these pathophysiological mechanisms. Finally, FFC-NMR operating at low field is demonstrated to be sensitive to invasion glioma phenotype and can be straightforwardly extended to FFC-MRI as a new cancer invasion imaging method in the clinic.

Keywords: FFC-NMR; H2O2 redox-signaling pathway; aquaporin-4; glioma invasion/migration; hypoxia; relaxometry; transcytolemmal water exchange; very low magnetic field.