Recent advances in non-invasive imaging, including the introduction of genetically encoded reporters into cells and the use of different imaging techniques, had a critical role in understanding normal physiology, disease progression and response to therapy2–4
. Imaging modalities are becoming increasingly important in the field of cancer as it can allow a fast quantitative measurement of tumor volume and treatment response, thereby accelerating the development of experimental therapeutic strategies1
. Since every imaging modality has its own characteristics and limitations (depth, resolution, cost, throughput), a multimodal reporter provides the option to select the best modality for a particular application, thereby giving insight into the anatomy, physiology and functions of living organisms, facilitating the validation of the study in experimental models and its translation into the clinic11
In this study, we present a very small multimodal reporter based on a membrane-bound form of the sensitive Gaussia luciferase reporter fused to a biotin acceptor peptide. Upon expression, biotin ligase tags the BAP peptide with a single biotin moiety at a specific lysine residue followed by processing through the secretory pathway and binding on the cell surface. The biotin now serves as a “molecular beacon” to attract any imaging agent coupled to streptavidin. Further, the presence of Gluc on the cell surface can be imaged with bioluminescence imaging upon injection of its substrate, coelenterazine. We showed that brain tumors expressing this reporter were tracked with high sensitivity with most commonly used molecular imaging modalities including bioluminescence, fluorescence, radionuclide (SPECT) and magnetic resonance.
Fusion proteins consisting of two or more reporters, generated through recombinant DNA expression constructs, and which expression can be imaged with different imaging modalities in both individual cells and living organisms have been described. For instance, fusions between a fluorescent protein and a PET reporter protein37,38
, a triple fusion protein between luciferase, RFP and thymidine kinase (TK)12
or a hybrid between the sodium iodide symporter (NIS) and eGFP linked with an IRES element13
were developed and applied for multimodal imaging of tumors. Although such fusions or linked proteins have proven to be applicable for experimental purposes, a considerable amount of activity was lost (80% for RFP, and 50% for luciferase) for each reporter due to steric hindrance and/or lower level of expression due to reduced translation of coding sequences following the IRES element39
. Thus, the use of these multi-fusions reduces detection sensitivity. Further, the size of these and similar fusion proteins is considerably large (Rluc-mRFP-TK; 2.4 kb; 90 kDa; NIS-IRES-eGFP; 3.7 kb; 140 kDa) making the expression cassettes too large to be carried in some viral vectors such as AAV vectors which are widely used in clinical trials for gene transfer.
The interaction of biotin with streptavidin is one of the strongest non-covalent binding known being orders of magnitude higher than most antibodies40
. Naturally, biotin in its physiologically active form is covalently attached at the active site of a class of important metabolic enzymes, biotin carboxylase and decarboxylases. These enzymes are found either in the cytoplasm (acetyl coA carboxylase) or mitochondria (pyruvate carboxylase, propionyl-CoA caboxylase and 3-methylcrotonyl-CoA carboxylase)41
and therefore do not interfere with imaging of the mbGluc-biotin reporter which is expressed on the cell surface. The attachment of biotin to the BAP sequence, through biotin ligase, is a highly selective process and has already been used in different fields ranging from protein/vectors purification to cell tracking19,20,23
. Further, the streptavidin-biotin system has been used in clinical trials for targeted tumor therapy as well as for imaging using magnetic nanoparticles and radionuclides42,43,14,17
. The mbGluc-biotin reporter described here has several advantages compared to commonly used multimodal reporters: (1) the reporter has a small size (~40 kDa) which makes it a valuable reporter for several delivery systems; (2) This reporter can be imaged with four different imaging modalities including BLI, FMT, MR and SPECT and can be extended for PET and IVM imaging; (3) the very strong binding of biotin to streptavidin makes the reporter very sensitive even in detecting tumors in deep tissues such as the brain. The mbGluc-biotin reporter provides a strong platform for multimodal imaging of tumors and can be extended to many different fields including stem cells tracking for gene and cell therapy.