In vivo mr imaging and quantification of primary and metastatic cancers using biomodal lentiviral vector encoding human ferritin and green fluorescent protein

Abstract

A combination of reporter genes for magnetic resonance imaging (MRI) and optical imaging can provide an additional level of noninvasive and quantitative information about biological processes occurring in deep tissues. Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest. I developed a bimodal lentiviral vector to monitor deep tissue events using MRI to detect myc tagged human ferritin heavy chain (myc-hFTH) expression and fluorescence imaging to detect green fluorescent protein (GFP) expression. No cellular toxicity due to overexpression of myc-hFTH and GFP was observed in MTT and trypan blue exclusion assays. Iron accumulation was observed in myc-hFTH cells and tumors by Prussian blue staining and iron binding assays. First of all, for primary tumor imaging, the transgene construct was stably transfected into MCF-7 and F-98 cells. After transplantation of the cells expressing myc-hFTH and GFP into mice or rats, serial MRI and fluorescence imaging were performed with a human wrist coil on a 1.5T MR scanner and optical imaging analyzer for 4 weeks. The myc-hFTH cells and tumors had significantly lower signal intensities in T2 weighted MRI than mock-transfected controls (P ≤ 0.05). This is direct evidence that myc-hFTH expression can be visualized noninvasively with a 1.5T clinical MR scanner. Second, for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice, a B16F10 murine melanoma cell line expressing hFTH and GFP was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4T MRI in vitro and in vivo, yielding significant changes in T2* relative to control cells. In BALB/c nude mice, the presence of hFTH and GFP expressing metastatic melanoma cells in deep seated axillary LNs was demonstrated as areas of low T2* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T2* MRI data, correlated well with tumor burden, which was determined by histology (r = -0.8773, p = 0.0001). This study shows that MRI and fluorescence imaging of transplanted cells or metastatic cancer cells at molecular and cellular levels can be performed simultaneously using my bimodal lentiviral vector system. In addition, this techniques can be used to monitor tumor growth, regression during cell and gene-based therapy in deep tissues.