Optoacoustic Imaging of Glucagon-like Peptide-1 Receptor with a Near-Infrared Exendin-4 Analog

Abstract

Limitations in current imaging tools have long challenged the imaging of small pancreatic islets in animal models. Here, we report the first development and in vivo validation testing of a broad spectrum and high absorbance near infrared optoacoustic contrast agent, E4x12-Cy7. Our near infrared tracer (E4x12-Cy7) is based on the amino acid sequence of exendin-4 and targets the glucagon-like peptide-1 receptor (GLP-1R). Cell assays confirmed that E4x12-Cy7 has a high binding affinity (IC50 = 4.6 ± 0.8 nM). Using the multispectral optoacoustic tomography (MSOT), we imaged E4x12-Cy7 and optoacoustically visualized ß-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for ß-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.

Pathways of Glucagon Secretion and Trafficking in the Pancreatic Alpha Cell: Novel Pathways, Proteins, and Targets for Hyperglucagonemia

“…GLP-1 appears to mildly reduce action potentials in the alpha cell membrane at 1 mM glucose in isolated mouse alpha cells, and this effect is blocked by the GLP-1R antagonist exendin (9-39) ( 110 ), therefore suggesting the presence of GLP-1R, perhaps at a very low density, on a small proportion of alpha cells. The development of near infra-red and fluorescent analogues of GLP-1R ligands has enabled both in vivo ( 111 , 112 ) and high-resolution tissue imaging ( 113 , 114 ) of GLP-1R with high specificity, sensitivity, and reproducibility. Coupled with super-resolution microscopy, the fluorescent analog LUXendin645 revealed that 5% of alpha cells in murine islets expressed GLP-1R ( 114 ).…”

“…Created with BioRender.com. (111,112) and high-resolution tissue imaging (113,114) of GLP-1R with high specificity, sensitivity, and reproducibility. Coupled with super-resolution microscopy, the fluorescent analog LUXendin645 revealed that 5% of alpha cells in murine islets expressed GLP-1R (114).…”

Reagents and models for detecting endogenous GLP1R and GIPR

“…GLP1R/GIPR antisera, fluorescent agonists/antagonists and mouse models validated according to known cell localizations, enzyme self-labels, pharmacology, Glp1r/Gipr expression in enriched fractions, Glp1r − / − tissue, Glp1r − / − cells or GLP1R/GIPR-transfected cells. Reagent Name Source Reported cross-reactivity Validation GLP1R antibody [39] Mab 3F52 Iowa DSHB Human, primate GL1PR_BHK cells (and wild-type cells) GLP1R antibody [50] Mab 7F38 Iowa DSHB Human, Mouse, Rabbit, Rat Glp1r −/− mice GLP1R antagonistic antibody [ 27 , 48 ] Glp1R0017, GLP1R-APC University of Cambridge, Duke University Mouse Glp1r −/− mice, pharmacology, antibody co-localization GLP1R antibody [53] ab218532 Abcam Mouse, Rat Glp1r −/− mice GLP1R antibody * [18] MAB2814 RnD Systems Human SNAP_hGLP1R-U2OS (and wild-type cells) GLP1R agonist [ 57 , 58 , 60 , 61 ] E4 K12 -Fl, E4 ×12 -VT750, EP12-TR, EP12-BTMR, EP12-BTMR-X, EP40-BF, EP40-TR Harvard University Mouse, human GLP1R_HEK293 cells, MIN6, insulin reporter/staining, pharmacology GLP1R agonist [59] E4 x12 -Cy7 Memorial Sloan Kettering Cancer Center Mouse, human GLP1R_HEK293, insulin reporter, pharmacology GLP1R agonist [97] Ex4-Cy3, Ex4-Cy5 Novo Nordisk Mouse Glp1r −/− mice GLP1R agonist [ 65 , …”

“…The probe, termed E4 K12 -Fl, was able to label GLP1R_HEK293 cells, MIN6 beta cells and pancreatic islets in vivo, overlapping with an insulin reporter or insulin protein [57] . The same authors used click chemistry on a K12-substituted Ex4 to install an azide-functionalized VT750 or Cy7 fluorophores, showing utility for estimating in vivo beta cell mass in NOD type 1 diabetes and insulinoma xenograft models [ 58 , 59 ]. Demonstrating the importance of modification site, an Exendin4 (Ex4)-like neopeptide was substituted (or added) at positions 12, 27 and 40, before reaction with various NHS-fluorophores spanning green through near-infrared wavelengths [ 60 , 61 ].…”

Indocyanine Green-Conjugated Superparamagnetic Iron Oxide Nanoworm for Multimodality Breast Cancer Imaging

“…A contrast agent such as ICG can absorb the energy from the incident laser in PAI. Part of the absorbed energy is converted to heat, causing a thermoelastic expansion, which generates an acoustic wave for detection. − An advantage of ICG is that it is a water-soluble near-infrared (NIR) dye approved by the Food and Drug Administration (FDA) as a clinical imaging agent with a good safety profile. − …”