CEST-MRI studies of cells loaded with lanthanide shift reagents

Abstract

Purpose: Magnetic resonance imaging has been used extensively to track in vivo implanted cells that have been previously labeled with relaxation enhancers. However, this approach is not suitable to track multiple cell populations, as it may lead to confounding results in case the contrast agent is released from the labeled cells. This paper demonstrates how the use of CEST agents can overcome these issues. After encapsulating paramagnetic lanthanide shift reagents, we may shift the absorption frequency of the intracellular water resonance (δ^In ), thus generating frequency-encoding CEST responsive cells that can be visualized in the MR image by applying the proper RF irradiation.

Methods: Eu-HPDO3A, Dy-HPDO3A, and Tm-HPDO3A were used as shift reagents for labeling murine breast cancer cells and murine macrophages by hypotonic swelling and pinocytosis. The CEST-MR images were acquired at 7 T, and the saturation transfer effect was measured. Samples at different dilution of cells were analyzed to quantify the detection threshold. In vitro experiments of cell proliferation were carried out. Finally, murine breast cancer cells were injected subcutaneously in mice, and MR images were acquired to assess the proliferation index in vivo.

Results: It was found that entrapment of the paramagnetic complexes into endosomes (i.e., using the pinocytosis route) leads to an enhanced shift of the intracellular water resonance. δ^In appears to be proportional to the effective magnetic moment (μ^eff ) and to the concentration of the loaded lanthanide complex. Moreover, a higher shift is present when the complexes are entrapped in the endosomes. The cell proliferation index was assessed both in vitro and in vivo by evaluating the reduction of δ^In value in the days after the cell labeling.

Conclusion: Cells can be visualized by CEST MRI after loading with paramagnetic shift reagent, by exploiting the large ensemble of the properly shifted intracellular water molecules. A better performance is obtained when the complexes are entrapped inside the endosomes. The observed (δ^In ) value is strongly correlated to the chemical nature of the probe, and to its concentration and cellular localization. Two applications of this method are reported in this paper: (1) for in vivo cell visualization and (2) for the monitoring of the cellular proliferation process, as this method is accompanied by a change in δ^In that may be exploited as a longitudinal reporter of the proliferation rate.

Keywords: CEST; Cell-CEST; MRI; cell labeling; lanthanide; shift reagents.