The use of high-frequency ultrasound imaging and biofluorescence for in vivoevaluation of gene therapy vectors

The use of non-invasive and accurate methods to determine tumour volume, as well as biodistribution and transduction imaging of novel therapeutics, is essential in experimental models in vivo. In particular, for gene therapy studies, knowledge of maintenance, expression and efficacy of the vector is a fundamental part of the testing process [1]. However, this is rarely achieved during the in vivo study of a novel gene therapy strategy, as often only longitudinal calliper measurements of xenograft growth or final histology after treatment are carried out. The spread or loss of a vector is rarely detected during the course of the experiment and for cancer treatment, not all therapies will result in a reduction in tumour volume. Therefore it is important to be able to examine the impact of a gene therapy vector during the in vivo testing phase using different assessment criteria, whilst being mindful of adhering to the principles of reduction, refinement and replacement in animal experiments.

Ultrasound is a non-invasive method that has been utilised recently for tumour growth studies in vivo and is used in the clinic for staging colorectal cancer among others [2, 3]. High-frequency ultrasound (HF-US) machines are available for small animal imaging. They are relatively easy to use and give high resolution greyscale images of mouse anatomy [4]. They also give functional information on the vascular structure of xenografts through the use of contrast agents and are relatively inexpensive and portable compared to MRI machines [5]. Mechanical callipers, however, are still utilised extensively for therapeutic agent testing, especially in gene therapy applications on xenografts [6]. These are very cheap, non-invasive and allow multiple repeated measurements with no anaesthetic required. However, mechanical callipers assume that the growth of xenografts is always ellipsoid and can only measure growth above the skin surface of the animal. In addition, calliper measurements are also affected by skin thickness, subcutaneous fat layer thickness and compressibility of the tumour [7]. From our experience of xenograft growth in gene therapy and other therapeutic studies, we know that this ellipsoid growth pattern is rarely observed, especially as the tumour volume becomes large (above approximately 300mm³).

A gene encoding a fluorescent or luminescent protein is often incorporated into gene therapy vectors in order to enumerate transduction efficiencies in vitro[8, 9]. Moreover, these markers are also very useful for in vivo studies. Optical imaging chambers can be used to image the biodistribution of a vector when administered and can give an indication of the transduction efficiency in the target cells [10]. Optical imaging systems also allow the maintenance of a vector to be determined throughout the course of treatment, as well as examining the genetic stability of the vector over time. The first paper to prove that optical imaging could be used to measure tumour growth used bioluminescence of tumour cells in rat brain and was compared to MRI scans for tumour volume [11]. Imaging of stably-transfected cell lines containing red or green fluorescent protein (RFP or GFP) has been used to measure tumour and metastatic growth [12, 13]. Recent work has also shown that fluorescent intensity correlates better with tumour volume than fluorescent area [14].

In the study described herein, we aimed to determine whether the use of HF-US measurements were more accurate than mechanical callipers in assessing xenograft volumes of tumour cells which were infected before injection with an experimental gene therapy vector. The use of HF-US to provide anatomical information on tumour growth and BFI to monitor expression of a gene therapy vector in longitudinal studies, were also analysed. The vector we used was a Herpesvirus saimiri (HVS) amplicon which contains the minimal elements for episomal maintenance without infectious capabilities [9, 15]. This gamma-2 Herpesvirus amplicon can incorporate large amounts of heterologous DNA using a HVS-BAC (bacterial artificial chromosome) system and infects a broad range of human cells. The amplicon was previously stably transfected into the SW480 colorectal cancer cell line and contains a constitutively active GFP gene [16]. The presence of the GFP gene enabled monitoring of its persistence during xenograft growth in this study.

Multimodal imaging in gene therapy applications is a useful tool to shed light on the behaviour of vectors during in vivo testing. In this study, the use of HF-US imaging identified anatomical differences during growth between the parental cell line and the vector-transfected cell line in a xenograft model, even from day 8 after implantation. It has been shown that HF-US can more accurately measure tumour volume compared to the traditional mechanical callipers, as demonstrated in this paper and by others [2, 18]. The use of different ellipsoid volume formulae to generate the tumour volumes from calliper measurements made small differences in accuracy where the highest correlation to mass was found using π/6 × (L × W)^3/2 rather than the more commonly used 0.5 × L × W² as described previously (although based on only one paper [17]). Correlation to determining volume by water displacement would be the gold standard and would be a useful addition to this study. HF-US volume generation and mechanical calliper measurements by multiple operators would also be valuable for determining variability as these measurements are subject to bias from operators. Jensen and colleagues compared volumes determined by microCT, ¹⁸F-FDG-microPET and external callipers, to an ex vivo reference volume calculated by weight and density [19]. They demonstrated that micro-CT was more accurate and reproducible between observers than either external callipers or ¹⁸F-FDG-microPET. They also showed that ¹⁸F-FDG-microPET was not so useful for determining tumour size, although there was some correlation (R² = 0.75). This was similar to our findings with biofluorescence imaging. As with our study, this functional tumour imaging modality is useful for metabolic imaging and should give an indication of the effect of a gene therapy vector on tumour viability. In the current study, HF-US accurately showed the slower tumour growth of the vector-transfected cell line compared to the parental cell line, as predicted from in vitro cell growth curves [16]. However, lobe formation was unexpected. We are currently investigating whether this is due to the GFP gene or other components of the vector backbone. We also demonstrated the utility of the different greyscale textures in monitoring different patterns of growth. The discrimination of areas of necrosis and high vascularity (using contrast agents) was also possible. This should allow real-time monitoring of agents that currently have little apparent effect on tumour volume but may have useful effects of anti-angiogenesis or inducing cell senescence. HF-US would be of particular use for very small xenografts, orthotopic models to in transgenic mice such as the Apc ^Min/+ mouse, where callipers cannot access the tumour. Indeed, gene therapy vectors are also used in non-cancer applications such as diabetes or organ regeneration, where callipers may not be used to measure disease progress or regression. In these cases, HF-US would be invaluable in monitoring progress longitudinally without sacrifice of mice.

In addition to HF-US images, the use of biofluorescence allowed monitoring of tumour growth patterns and correlated well with final tumour volumes (although it must be noted this was based on small numbers with a wide variation). This technique is a simple and very quick method of visualising the tumour and much less expensive than ¹⁸F-FDG-microPET, for example. Bio-fluorescence is also applicable to patients. It is currently being trialled in surgery on human tumours to define tumour margins for resection [20]. The monitoring of these two cell lines grown as xenografts showed that the presence and expression of the vector was maintained within the tumour over the duration of the experiment. This information is of great value for gene therapy applications as silencing of the vector can occur, which may not be evident from growth curves or even from immunohistochemistry on ex vivo tumour sections for vector proteins. Linkage of the therapeutic gene of interest to a fluorescent marker gene via an IRES (internal ribosomal entry site) sequence or as a fusion protein would yield valuable information on the efficacy of expression during the time course of an in vivo experiment. It may also be used to reduce costs by eliminating animals in which the introduction of a vector by injection has not been successful.