Normalisation to Blood Activity Is Required for the Accurate Quantification of Na/I Symporter Ectopic Expression by SPECT/CT in Individual Subjects

Peggy Richard-Fiardo,Audrey Lamit,Béatrice Cambien,Thierry Pourcher,Robert Marsault,Jacques Darcourt,Julien Guglielmi,Sabine Lindenthal,Philippe R Franken,Fanny Graslin,Georges Vassaux,Juri G Gelovani

doi:10.1371/journal.pone.0034086

Abstract

The utilisation of the Na/I symporter (NIS) and associated radiotracers as a reporter system for imaging gene expression is now reaching the clinical setting in cancer gene therapy applications. However, a formal assessment of the methodology in terms of normalisation of the data still remains to be performed, particularly in the context of the assessment of activities in individual subjects in longitudinal studies. In this context, we administered to mice a recombinant, replication-incompetent adenovirus encoding rat NIS, or a human colorectal carcinoma cell line (HT29) encoding mouse NIS. We used 99mTc pertechnetate as a radiotracer for SPECT/CT imaging to determine the pattern of ectopic NIS expression in longitudinal kinetic studies. Some animals of the cohort were culled and NIS expression was measured by quantitative RT-PCR and immunohistochemistry. The radioactive content of some liver biopsies was also measured ex vivo. Our results show that in longitudinal studies involving datasets taken from individual mice, the presentation of non-normalised data (activity expressed as %ID/g or %ID/cc) leads to ‘noisy’, and sometimes incoherent, results. This variability is due to the fact that the blood pertechnetate concentration can vary up to three-fold from day to day. Normalisation of these data with blood activities corrects for these inconsistencies. We advocate that, blood pertechnetate activity should be determined and used to normalise the activity measured in the organ/region of interest that expresses NIS ectopically. Considering that NIS imaging has already reached the clinical setting in the context of cancer gene therapy, this normalisation may be essential in order to obtain accurate and predictive information in future longitudinal clinical studies in biotherapy.

Highlights

The sodium iodide symporter (NIS) is an integral membrane glycoprotein that mediates the uptake and concentration of iodide into cells
The status of Na/I symporter (NIS) as a relevant reporter gene for SPECT imaging in cancer gene therapy has been validated recently in phase 1 clinical trials using conditionally replicating adenovirus [24,25], vector design and dose are critical to the successful visualisation of transgene expression [26]
As expected from previous studies [33,34], significant activity was noted in the liver when the animals were injected with 16109 PFU virus, as a result of ectopic NIS expression in this organ

Summary

Introduction

The sodium iodide symporter (NIS) is an integral membrane glycoprotein that mediates the uptake and concentration of iodide into cells. In gene and cell therapies, ectopic NIS expression, associated with relevant radioisotopes such as 123I2, 124I2 or 99mTcO42, has been used extensively in rodent models to monitor the efficacy of gene transfer using various imaging modalities (PET and SPECT) [1,4] With this technology, it is possible to evaluate the patterns of gene expression allowed by different gene delivery vectors [5,6,7,8,9,10,11,12] or to assess the specificities of particular promoters [13,14,15,16]. The status of NIS as a relevant reporter gene for SPECT imaging in cancer gene therapy has been validated recently in phase 1 clinical trials using conditionally replicating adenovirus [24,25], vector design and dose are critical to the successful visualisation of transgene expression [26]

Methods

Results

Conclusion