Abstract
Molecular targeting remains to be a promising approach in cancer medicine. Knowledge about molecular properties such as overexpression of G protein-coupled receptors (GPCRs) is thereby offering a powerful tool for tumor-selective imaging and treatment of cancer cells. We utilized chemerin-based peptides for CMKLR1 receptor targeting in a breast cancer xenograft model. By conjugation with radiolabeled chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of highly specific and affine tracers for hybrid in vivo imaging with positron emission tomography (PET)/ magnetic resonance (MR) and concomitant biodistribution studies.
Methods We developed five highly specific and affine peptide tracers targeting CMKLR1 by linker-based conjugation of chemerin peptide analogs (CG34 and CG36) with radiolabeled (68Ga) chelator DOTA. Our established xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. Therefore, we acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments.
Results The family of five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values being below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be confirmed by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers 68Ga-DOTA-AHX-CG34, 68Ga-DOTA-KCap-CG34 and 68Ga-DOTA-ADX-CG34 with apparent DU4475 tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 4.5% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. More strikingly, the tumor uptake could be blocked by excess of unlabeled peptide (6.4-fold, 7.2-fold and 3.4-fold 1 h post-injection) and further confirmed the CMKLR1 specificity. As our five tracers, each with particular degree of hydrophobicity, showed different results regarding tumor uptake and organ distribution, we identified these three tracers with moderate, balanced properties to be the most potent in receptor-mediated tumor targeting.
Conclusion With the breast cancer cell line DU4475, we established a model endogenously expressing our target CMKLR1 to evaluate our chemerin-based peptide tracers as highly affine and specific targeting agents. Eventually, we demonstrated the applicability of our 68Ga-labeled tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging and thus developed promising theranostics for tumor treatment.
Abbreviations
- % IA/g
- percent injected activity per gram tissue
- 18F-FDG
- 18F-fluoro-deoxyglucose
- ADX
- 10-aminodecanoic acid
- AHX
- 6-aminohexanoic acid
- BERIC
- Berlin Experimental Radionuclide Imaging Center
- BSA
- bovine serum albumin
- CG34
- chemerin analog with amino acid sequence Y-Cha-Hyp-G-Cit-F-a-Tic-S
- CG36
- chemerin analog with amino acid sequence Y-Cha-P-G-M-Y-A-F-f
- Chem9
- chemerin-9
- CMKLR1
- chemokine-like receptor 1
- DMSO
- dimethyl sulfoxide
- DOTA
- 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
- DTPA
- diethylenetriaminepentaacetic acid
- EC50
- half maximal effective concentration
- ECM
- extracellular matrix
- EGFR
- epithelial growth factor receptor
- EGTA
- ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- ESCC
- esophageal squamous cell carcinoma
- G418
- geneticin
- GMP
- good manufacturing practice
- GPCR
- G protein-coupled receptor
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HPLC
- high-performance liquid chromatography
- IC50
- half maximal inhibitory concentration
- KCap
- N-ε-capryloyl-lysine
- MRI
- magnetic resonance imaging
- NET
- neuroendocrine tumor
- NMRI
- Naval Medical Research Institute
- PBS
- phosphate-buffered saline
- PEG
- polyethylene glycol
- PET
- positron emission tomography
- PRRT
- peptide receptor radionuclide therapy
- PSMA
- prostate-specific membrane antigen
- RPMI1640
- Roswell Park Memorial Institute medium 1640
- SD
- standard deviation
- SEM
- standard error of the mean
- SSA
- somatostatin analog
- SSTR
- somatostatin receptor
- T2 FSE 2D
- high-resolution T2-weighted 2D fast spin echo sequence
- TBS
- tris-buffered saline
- TE
- echo time
- TFA
- trifluoroacetic acid
- TR
- repetition time
- TTDS
- 4,7,10-trioxatridecan-succinamic acid
- VOI
- volume of interest