Research

A targeted imaging agent for detecting cell death

The agent is based on the 14 kDa C2A domain of the protein synaptotagmin, which binds to the phosphatidylserine (PS) exposed by dying cells. Initially we labelled the molecule with tags that could be detected using magnetic resonance imaging (MRI) and have subsequently labelled it with radioactive metals for radionuclide imaging. We made a site-directed mutant (C2Am) in which a serine residue distant from the active site was replaced with a cysteine residue (S78C) (Alam et al., Bioconjug Chem 2010; 21: 884). The mutant has a similar affinity for PS as the wildtype protein - using sulphydryl-selective reagents, we have produced homogeneous preparations that have been labelled at this single site with a DOTA chelate. A patent application has been filed on this mutant. This year, using the 111In-loaded chelate and following installation of a single photon computed tomography (SPECT) instrument in the CRI, we have demonstrated that we can detect cell death in a drug treated tumour using SPECT (Figure 1). Targeted radionuclide-labelled imaging agents based on Annexin V, which like C2Am binds to PS, have been trialled in the clinic for the detection of cell death in tumours post treatment. However, there were problems with their biodistribution. Initial studies suggest that C2Am has better specificity for detecting cell death than Annexin V, in that it shows lower binding to viable cells (Alam et al., Bioconjug Chem 2010; 21: 884).

Figure 1
Imaging cell death in EL-4 murine lymphoma tumours using 111In-labelled C2Am and SPECT. Representative images from untreated (A and C) and drug-treated (B and D) animals at 4 and 24 hours after injection of 111In-C2Am-malDOTA. The locations of the tumour (T), kidneys (K) and bladder (B) are indicated. The arrows indicate the presence of radioactivity in the tumour of the treated mouse.

Imaging metabolism with hyperpolarised 13C-labelled cell substrates

MRI gives excellent images of soft tissues, such as tumours. The technique works by mapping, in 3D, the distribution and MR properties of tissue water protons, which are very abundant (60 - 70 M in tissues). However, we have known since the 1970s that we can also use MR to detect metabolites in vivo. The problem is that these molecules are present at 10,000x lower concentration than the protons in tissue water. This makes them hard to detect and almost impossible to image, except at very low resolution. We have been collaborating with GE Healthcare in the development of a technique, termed 'hyperpolarisation', that increases sensitivity in the MRI experiment by more than 10,000x. With this technique we inject a hyperpolarised 13C-labelled molecule and now have sufficient sensitivity to image its distribution in the body and the distribution of the metabolites produced from it. We have shown that we can detect early treatment response in lymphoma tumours by monitoring decreased tumour utilisation of one cell metabolite, pyruvate, and then detect subsequent cell death by watching the increased metabolism of another molecule, fumarate (Gallagher et al., Proc Natl Acad Sci USA 2009; 106: 19801). This year we consolidated this work by demonstrating that the technique will work with other tumour types such as breast (Witney et al., Br J Cancer 2010; 103: 1400) and glioma (Day et al., Magn Reson Med 2010; 18 Nov EPub), and with other types of drugs, notably combretastatin A4 phosphate, which is an anti-vascular drug (Bohndiek et al., Mol Cancer Ther 2010; 9: 3278). In the latter case we showed that measurements of hyperpolarised pyruvate and fumarate metabolism could provide a more sustained and sensitive indicator of response to a vascular disrupting agent than dynamic contrast agent enhanced (DCE)- or diffusion weighted (DW)-MRI respectively, which are used currently in the clinic to detect the action of anti-vascular and anti-angiogenic drugs.

Imaging tumour cell glycosylation

Aberrant glycosylation is a hallmark of cancer. We are currently developing a novel molecular imaging platform for the non-invasive assessment of tumour glycosylation, in which sugar analogues are incorporated metabolically by tumour cells in vivo and subsequently detected by a highly selective chemical reaction with a reporter probe that has been labelled with an imaging agent. This year we demonstrated that this technique can be used to image tumour glycans in vivo, using both fluorescence and radionuclide (SPECT) imaging. This methodology could provide new insights into tumour cell proliferation, response to therapy, and perhaps more importantly, metastasis. The technique also has the potential for subsequent translation into a clinical setting, using nuclear imaging techniques.

Genetically engineered models of disease

We are developing a platform technology that enables rapid bioluminescent imaging of autochthonous Cre/loxP-dependent tumour models in vivo. We have designed lentiviral and transposon-based vectors to couple tumour induction with the establishment of expression of the luciferase reporter transgene. By simply varying the route of vector administration, we will be able to selectively induce tumour formation in various organ types. Bioluminescent imaging will allow individual animals to be identified very early during tumour development and then selected for analysis with lower throughput but clinically applicable imaging modalities, such as CT, MRI and PET.

Future directions

With the arrival of microPET and SPECT scanners in the CRI we will continue the development of radiolabelled C2Am with a view to translating the most promising derivative to the clinic. We will apply the glycan imaging approach to animal models of early dysplasia to determine whether the technique can give prognostic information on tumour progression. We will extend an image analysis method that we have developed previously in animal models of disease to the clinic, in particular to determine whether it can detect early treatment response in glioma. We will introduce new hyperpolarized 13C-labelled cell substrates for imaging metabolism and evaluate these for detecting treatment response in new animal models of disease.