In Vivo Type 2 Cannabinoid Receptor-Targeted Tumor Optical Imaging Using a Near Infrared Fluorescent Probe

Abstract

The type 2 cannabinoid receptor (CB2R) plays a vital role in carcinogenesis and progression and is emerging as a therapeutic target for cancers. However, the exact role of CB2R in cancer progression and therapy remains unclear. This has driven the increasing efforts to study CB2R and cancers using molecular imaging tools. In addition, many types of cancers overexpress CB2R, and the expression levels of CB2R appear to be associated with tumor aggressiveness. Such upregulation of the receptor in cancer cells provides opportunities for CB2R-targeted imaging with high contrast and for therapy with low side effects. In the present study, we report the first in vivo tumor-targeted optical imaging using a novel CB2R-targeted near-infrared probe. In vitro cell fluorescent imaging and a competitive binding assay indicated specific binding of NIR760-mbc94 to CB2R in CB2-mid delayed brain tumor (DBT) cells. NIR760-mbc94 also preferentially labeled CB2-mid DBT tumors in vivo, with a 3.7-fold tumor-to-normal contrast enhancement at 72 h postinjection, whereas the fluorescence signal from the tumors of the mice treated with NIR760 free dye was nearly at the background level at the same time point. SR144528, a CB2R competitor, significantly inhibited tumor uptake of NIR760-mbc94, indicating that NIR760-mbc94 binds to CB2R specifically. In summary, NIR760-mbc94 specifically binds to CB2R in vitro and in vivo and appears to be a promising molecular tool that may have great potential for use in diagnostic imaging of CB2R-positive cancers and therapeutic monitoring as well as in elucidating the role of CB2R in cancer progression and therapy.

Targeted zwitterionic near infrared fluorescent probe for improved imaging of type 2 cannabinoid receptors

“…28 Previous studies indicate that heptamethine cyanine dyes with an enol ether linkage have significant stability issues, which can be overcome by replacing the enol ether with a C─C linkage. 26,30 In addition, the carboxylate group on ZW760 allows for universal conjugation to various targeting moieties.…”

“…26 Briefly, CB 2 -mid DBT cells were seeded onto 96 well optical bottom plates with 5000 cells per well and incubated for 24 h. Hank's balanced salt solution with 1 mM Mg 2þ , 0.1% BSA, 0.1% NaN 3 was used as the binding buffer. Cells were incubated for 30 min with an increasing concentration of ZW760-mbc94 (2.0, 5.0, 10.0, 40.0, 100.0, and 160.0 nM) at 37°C.…”

“…We recently reported a CB 2 R-targeted near-infrared (NIR) fluorescent probe, NIR760-mbc94, which was successfully used to image the receptor in vitro and in vivo. 26 Although the specific binding of NIR760-mbc94 to CB 2 R has been demonstrated, the probe also showed nonspecific binding. Specifically, SR144528, a CB 2 R ligand, partially inhibited the uptake of NIR760-mbc94 in vitro (40%) and ex vivo (35%).…”

“…Specifically, SR144528, a CB 2 R ligand, partially inhibited the uptake of NIR760-mbc94 in vitro (40%) and ex vivo (35%). 26 Recently, Dr. Frangioni's group found that replacing charged NIR fluorescent dyes with a zwitterionic (net charge ¼ 0) NIR dye significantly reduced the nonspecific binding of integrin α v β 3 -targeted imaging agents. 27 Inspired by this report, in this study, we introduced zwitterionic properties into our CB 2 R-targeted agents.…”

Fluorescence-guided Tumor Visualization Using the Tumor Paint BLZ-100

“…This was demonstrated by the use of a fluorescently labeled affibody, which had more clear differentiation of the brain-tumor interface than the significantly larger full antibody to the same receptor. Most recently, a novel type 2 cannabinoid receptor (CB 2 R)-targeted probe that is highly expressed in invasive cancer has been tested as another fluorescent probe [35], with many more in development. Even if BLZ-100 fails to work in clinical settings, our SIRIS system is ideally suited to be utilized with all other NIR targeted fluorescent probes, and can easily be optimized for any specific wavelength as needed.…”