Cell tracking by magnetic resonance imaging using ferritin in atherosclerosis and cancer mouse models

Abstract

Molecular imaging is a type of innovative medical imaging that provides detailed images of the in vivo occurrences at a molecular or cellular level through real-time imaging. Among the various molecular imaging modalities, magnetic resonance imaging(MRI) can use in detection of cell movement, proliferation, and division through cell labeling, and diagnose and evaluate the treatment effectiveness of tumors or inflammations depending the labeling cell type. MRI provides high tissue contrast and superior resolution but it has relatively low sensitivity, and therefore, contrast agents are used to overcome this challenge. However, problems such as dilution of contrast agents within the body, instability of cell labeling, and cytotoxicity suggest that there is a need for a more safe and efficient cell labeling technique. The reporter-gene based technique is being proposed as a method to overcome these problems. Ferritin is an intracellular, iron-binding protein that accumulates extracellular iron entering the cell and exhibits magnetic properties, allowing T2 contrast effects under MRI. This makes it a good candidate as an MR contrast agent for cell tracking. This study aims to understand the effectiveness of ferritin as a contrast agent and also a potential reporter gene, by tracking macrophages and tumor cells using ferritin in arteriosclerosis and tumor mouse models. In a comparison experiment with the currently used T2 contrast agent Feridex, ferritin showed 50% lower relaxivity (Feridex vs ferritin = 0.00298 ml/µg Fe∙1/sec vs 0.00159 ml/µg Fe∙1/sec), which was sufficient to act as the MR contrast agent for cellular imaging. Macrophage cell line Raw264.7, labeled with ferritin, showed T1 and T2 contrast effects in cell pellet imaging. In vivo imaging of ApoE knockout (ApoE-/-) mice 24 hr after intravenous injection of the cells revealed negative contrast effects in the aortic arch walls. Therefore, ferritin can be used to diagnose inflammations by tracking ferritin-labeled macrophages. Since the contrast effects of ferritin were confirmed, the use of ferritin as a reporter gene and its use as an adenovirus-based expression system were assessed in this experiment. First, a Mock (control group) and an adenoviral human ferritin heavy chain ([Ad-FTH] experimental group) were injected into the subcutaneous tumor models made using the colorectal cancer cell line HCT116. T1 and T2 -weighted images were obtained every two days after injection. Day 2 post-injection images showed negative contrast areas within the tumor, and by day 4 post-injection, the enhanced contrast areas were clearly observed. The results of the Prussian blue staining performed using the same tissue samples demonstrated overlapping of the negative contrast-enhanced region and iron absorption region, thereby confirming that the contrast effects were due to ferritin gene expression. Considering the fact that the iron concentration increases within the tumor, a glioma cell line U87MG was infected with Ad-FTH and ferritin expression was induced. Then, cell-pellet MRI was performed using cells cultured with 1mM FeCl3 and 3mM FeCl3; cells cultured under higher iron concentration were confirmed to show stronger negative contrast enhancing effects. In order to validate the negative contrast effects of ferritin through T2*-weighted images, brain tumor models were established by injecting U87MG cells into mouse brain and Ad-FTH was injected into the tumor; T1, T2 and T2*- weighted images were simultaneously obtained from days 1 through 6. Day 2 post-injection, T2 and T2*- images showed negative contrast effects at the tumor margins, and maintained until day 6. In conclusion, through cellular level investigation and in vivo experiments using animal models, this study strongly suggests the effectiveness of ferritin as an endogenous MR contrast agent and a potential reporter gene, which is capable of maintaining cell labeling stability and cellular safety.