RESUMO
BACKGROUND: Image-based diagnosis of tumours can be advanced and improved by targeted strategies addressing malignant molecular structures. A promising molecular target is the cholecystokinin-2-receptor (CCK2R) which can be targeted by high-affinity peptides called minigastrins. Here we present how the imaging properties of minigastrins tagged with near-infrared fluorescence (NIRF) dyes can be modulated by the introduction of different spacer sequences. We identify interactions of different probe variants with regard to target affinity, specificity and pharmacokinetic properties to optimize early detection of CCK2R-expressing tumours under clinical conditions. METHODS: Two minigastrin probes with the same near-infrared hemicyanine fluorescence dye (DY-754) for signalling and the same CCK2R-binding peptide A-Y-G-W-M/Nle-N-F-amide but different spacers were designed as follows: 'QE' with three alternating d-glutamines and d-glutamic acids and 'bivQ' with two minigastrins, each preceded by three d-glutamines. They were tested for affinity and specificity in vitro on CCK2R-expressing and CCK2R-non-expressing cells. In vivo imaging was conducted with subcutaneous tumour-bearing nude mice after i.v. probe injection (54 to 108 nmol/kg) and under competitive conditions with non-fluorescent minigastrin (n = 5/group). We also assessed probe biodistribution as well as NIRF distribution in tumour sections. RESULTS: Both probes showed high affinity and specificity to CCK2R-expressing cells in vitro. In vivo tumour-to-background contrasts (tumour/background ratios (TBRs) of around 6) enabled identification of CCK2R-expressing tumours by both probes with low accumulation in CCK2R-negative tumours (TBR of around 2). Specificity of the in vivo accumulation, revealed by competition, was higher for QE. Besides renal retention, probe uptake into organs was very low. CONCLUSION: The properties of optical minigastrin probes can be specifically modified by the introduction of spacer sequences. A spacer of six hydrophilic amino acids increases affinity. A mix of d-glutamic and d-glutamine acids increased target-to-background contrast. Multimerization could not increase affinity but supposedly lowered stability. The probe QE is a promising candidate for clinical evaluation in terms of diagnosis of CCK2R-expressing tumours.
RESUMO
Optical imaging-based diagnostics identify malignancies based on molecular changes instead of morphological criteria in a non-invasive, irradiation free process. The aim of this study was to improve imaging efficiency by the development of a new Cholecystokinin-2-receptor targeted fluorescent peptide that matches the clinical needs regarding biodistribution and pharmacokinetics while displaying superior target specificity. Furthermore we performed multifactorial imaging of Cholecystokinin-2-receptor and tumor metabolism, since simultaneous targeting of various tumor biomarkers could intensely increase tumor identification and characterization. Affinity and specificity of the fluorescent Cholecystokinin-2-receptor targeted minigastrin (dQ-MG-754) were tested in vitro. We conducted in vivo imaging of the dQ-MG-754 probe alone and in a multifactorial approach with a GLUT-1 targeted probe (IR800 2-DG) on subcutaneous xenograft bearing athymic nude mice up to 24 h after intravenous injection (n = 5/group), followed by ex vivo biodistribution analysis and histological examination. We found specific, high affinity binding (Kd = 1.77 nM ± 0.6 nM) of dQ-MG-754 to Cholecystokinin-2-receptor expressing cells and xenografts as well as favorable pharmacokinetics for fluorescence-guided endoscopy. We successfully performed multifactorial imaging for the simultaneous detection of the Cholecystokinin-2-receptor and GLUT-1 targeted probe. Prominent differences in uptake patterns of the two contrast agents could be detected. The results were validated by histological examinations. The multifactorial imaging approach presented in this study could facilitate cancer detection in diagnostic imaging and intraoperative and endoscopic applications. Especially the dQ-MG-754 probe bears great potential for translation to clinical endoscopy imaging, because it combines specific high affinity binding with renal elimination and a favorable biodistribution.
Assuntos
Diagnóstico por Imagem/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptor de Colecistocinina B/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Linhagem Celular Tumoral , Feminino , Glicólise , Humanos , Camundongos , Camundongos Nus , Sondas Moleculares/química , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Peritonitis is an inflammatory process characterized by massive monocytes-macrophages infiltration. Since early diagnosis is important for a successful therapeutic outcome, the feasibility for a selective labeling and imaging of macrophages for highly sensitive optical imaging was assessed. After in vitro incubation of mouse macrophages J774A.1 with the far-red/near-infrared fluorochrome DY-676, distinct fluorescence intensities (1026+/-142 a.u.) were detected as compared to controls (552+/-54 a.u.) using a whole-body small animal near-infrared fluorescence (NIRF) imaging system. Macrophage labeling was confirmed by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting, (FACS). The fluorochrome was also found to be predominantly distributed within compartments in the cytoplasm. Additionally, peritonitis was induced in mice by intraperitoneal injection of zymosanA. After intravenous injection of fluorochrome (55 nmol/kg) and using whole-body fluorescence imaging, higher fluorescence intensities (869+/-151 a.u.) were detected in the peritoneal area of diseased mice as compared to controls (188+/-41 a.u.). Furthermore, cells isolated from peritoneal lavage revealed the presence of labeled monocytes-macrophages. The results indicate that in vivo diagnosis of peritonitis by near-infrared optical imaging of labeled monocytes-macrophages is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic method in the long term.