Multimodal in vivo imaging of tumor xenografts using novel CMKLR1-targeted peptide tracers

Objectives Targeted multimodal imaging is an emerging approach in personalized cancer medicine. Knowledge about molecular properties such as overexpression of certain receptors is thereby offering new opportunities for targeted imaging and selective treatment of cancers. We utilized novel chemerin-based peptides for receptor-ligand binding mediated targeting of CMKLR1 overexpressing tumors in small animal models. By conjugation with tracer molecules such as near-infrared dye or radiolabeled chelator DOTA we obtained highly specific and affine imaging probes. These novel contrast agents enable targeted multimodal in vivo imaging with near-infrared imaging, positron emission tomography, magnetic resonance imaging and complementary biodistribution studies.

Methods We developed highly specific and affine peptide ligands for the G protein coupled receptor CMKLR1 by substitution of wild type chemerin-9 and structure-activity relationship analysis. The combination of these peptides linked to either near-infrared dye- (ITCC) or the radiolabeled (68Ga) chelator DOTA and an established target positive tumor model in immunodeficient nude mice enabled tumor-specific imaging in vivo. Therefore, we acquired small animal optical and PET/MRI images and assessed biodistribution by ex vivo measurements.

Results Our novel chemerin peptide tracers demonstrated significantly improved properties compared to the wild type peptide concerning biological activity, affinity and metabolic stability. CMKLR1 is known to be over-expressed in different pathologies, including cancer. Beside other tumor entities like esophageal or pancreatic cancer, we could demonstrate CMKLR1 overexpression in breast cancer patient tissue and in the breast cancer cell line Du4475. After establishment of different target-positive cancer models along with target negative tumors, optical near-infrared and PET/MRI imaging along with complementary biodistribution studies revealed a strong CMKLR1 specific uptake of the targeted tracers in positive tumor tissue within twenty-four hours for ITCC-labeled or one hour for 68Ga-labeled peptides. More strikingly, tumor uptake of our tracers could not only be blocked by excess of unlabeled peptide, but also depended significantly on the number of receptors expressed by the tissue. Tumor uptake, organ distribution and corresponding kinetics correlated strongly with the chemical and physical properties of the different tracers. In addition, the tracer conjugates cleared rapidly by predominantly renal excretion and molecular expression analysis of ex vivo tissue confirmed their target selectivity.

Conclusions We found the receptor CMKLR1 to be overexpressed in different tumor entities such as breast cancer. With the cell line Du4475, we had a model endogenously expressing our target to evaluate our optimized chemerin peptides as stable ligands with high affinity. Eventually, we demonstrated the applicability of our novel ITCC- or 68Ga-labeled tracers by visualizing CMKLR1 positive breast cancer tumors in optical and PET/MR imaging and thus developed promising candidates for potential clinical translation.

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