• Overview
• Products and catalog numbers
• Using BombesinRSense 680 in vivo/ex vivo
• Using BombesinRSense 680 in vitro
• Application notes and posters
• Citations

Overview

Bombesin receptors (BBRs) play a role in a variety of physiological systems in mammals. Three characterized receptor types (BB1R, BB2R, and BB3R) are overexpressed in several prevalent solid tumors, including prostate, breast, small cell lung, and gastrointestinal cancers. The ability to detect expression of BBRs non-invasively in animal models of cancer has provided an important tool for biomedical research. There is a bombesin family of peptides that serve as ligands for these BBRs. When the BBRs are overexpressed, the bombesin peptides serve as autocrine growth factors, signaling either calcium mobilization or proliferation in tumor. Several radio-labeled analogs of these peptides have been used for PET or SPECT imaging in preclinical cancer research as well as in imaging of patients. Cytotoxic analogs of bombesin and bombesin receptor antagonists have been explored as novel therapeutics.

BombesinRSense™ 680 is a fluorescently-labeled peptide designed to bind specifically to bombesin receptors involved in cancer cell stimulation of proliferation. It is comprised of a 7-amino acid bombesin peptide analog, a near infra-red (NIR) fluorophore and a pharmacokinetic modifier to improve its plasma availability. This agent can be used to specifically target and quantify the upregulation of bombesin receptors (BBR) in vivo associated with tumor proliferation. Selectivity of agent binding was confirmed by blocking BombesinRSense 680-labeled human colorectal HT-29 cells with excess unlabeled native bombesin peptide.

BombesinRSense 680 has the potential to serve as an in vivo real-time early indicator of tumor growth and chemotherapy efficacy.

Products and catalog numbers

Using BombesinRSense 680 in vivo/ex vivo

The generally recommended procedure for in vivo imaging with BombesinRSense 680 is administration via intravenous injection and imaging 24-48 hours post injection.

Repeat injection and imaging may be performed every 7 days for longitudinal studies. It is recommended that a pre-injection baseline image be taken prior to re-injection and imaging if repeat injections are to be performed less than 1 week apart. 

BombesinRSense 680 also enables imaging of HT-29 tumors implanted subcutaneously in nude mice.

Instructions on setting up an in vivo mouse experiment with BombesinRSense 680 and imaging on an IVIS or FMT system.

In Vivo Imaging

Figure 1: In Vivo Imaging: Cross-validation with Bioluminescence. NU/NU mice were implanted with human colorectal HT-29 luc tumors, injected intravenously with 2 nmol of BombesinRSense 680, and imaged by IVIS Spectrum CT 24 hours later. (A) Bioluminescence and (B) Epifluorescence. The same three mice per group were imaged to show tumor specific localization of BombesinRSense 680. Arrows indicate tumor masses (red) and pancreatic tissue (blue). Upper torso signal may be attributed to either lymph nodes or salivary glands. Tumor signal as assessed by BombesinRSense 680 correlates strongly with bioluminescence intensity (r2 = 0.91).

Figure 2: In Vivo Imaging: Time-course Optimization. NU/NU mice were implanted subcutaneously with human colorectal HT-29 tumors (2 tumors per mouse). When the tumors reached the desired volume, the mice were injected intravenously with 2 nmol of BombesinRSense 680 and imaged tomographically (FMT 4000) at times ranging from 15 minutes to 12 days. A) Shown is one representative mouse imaged at all time-points by both epifluorescence (upper panels) and fluorescence tomography (lower panels). B) Tomographic imaging datasets were used to quantify kinetic fluorescence changes in the upper liver region and the heart as well as targeted accumulation in the tumors.

Figure 3: In Vivo Multiplex Imaging. NU/NU mice implanted subcutaneously with human colorectal HT-29 tumors were injected intravenously with 2 nmol of BombesinRSense 680 and 2 nmol of AngioSense 750 EX. Epifluorescence imaging by FMT 4000 at 24 hours post intravenous injection shows overlapping intratumoral distribution of these two imaging agents.

Figure 4: In Vivo Biodistribution and Pharmacokinetics. Twelve HT-29-bearing NU/NU mice were injected intravenously with 2 nmol of BombesinRSense 680. Blood was collected at multiple times and tissues were removed at 24 hours. A) Quantified tissue biodistribution measured by FMT epifluorescence. Inset shows plasma pharmacokinetics measured by microplate assay. B) Epifluorescence tissue images using tissues from a representative mouse.

Ex Vivo Imaging

Figure 5a: Detection of BBR Expression in Frozen Tumor Tissue Sections. To assess binding specificity of BombesinRSense 680 (BRS 680),tumors were removed from HT-29-tumor bearing mice, and frozen sections (10 micron thickness) were incubated with 1 μM BombesinRSense 680 for 15 minutes at room temperature, with or without previous incubation (for 15 minutes at 37 ˚C) with 100 μM native bombesin. The tissue was rinsed and imaged by fluorescence microscopy with BombesinRSense 680 signal shown in red and DAPI (shown in blue) as a nuclear counterstain.

Figure 5b: Detection of BBR Expression in Frozen Tumor Tissue Sections. To confirm proper tumor localization of BombesinRSense 680 in vivo, HT-29 tumor bearing mice were injected with 2 nmoles of BombesinRSense 680. Tumors were excised 24 hours later and 10 micron sections were compared to serial sections stained with a rabbit polyclonal antibody against GRPR (BB2 receptor). Sections were imaged by fluorescence microscopy with BombesinRSense 680 signal shown in red and DAPI (shown in blue) as a nuclear counterstain.

Frozen Tissue Protocol

We have validated BombesinRSense 680 for use with frozen tissue samples. Here is a brief protocol with a recommended concentration of agent to use:

1. Freeze tumor/tissue (without agent) and section 5-10 µm by cryostat.  For specificity, pre-incubate sample with native bombesin 15 min prior incubation with agent.
2. Incubate with 1 µM BombesinRSense 680 at 37ºC for 15 min.
3. Wash 2x with PBS. 
4. Mount with anti-fade reagent.
5. Fluorescence microscopy filter: Cy5.5*

Using BombesinRSense 680 in vitro

Flow cytometry and in vitro microscopy

We have validated BombesinRSense 680 for use with fluorescence microscopes and flow cytometers. Here is a brief protocol with a recommended concentration of agent to use:

1. Resuspend cells in 1x PBS. For competition study (with excess native bombesin), treat with 50 μg/ml Mitomycin C for 3 hours prior incubation with agent. 
2. Incubate cells with 1 µM BombesinRSense 680 for 5 min at 37°C 
3. Wash 1-2x with PBS. 
4. Flow cytometry filter settings: 712/21
   Fluorescence micrsocopy filter: Cy 5.5

Figure 6: HT-29 cells were incubated with either 1 μM BombesinRSense 680 or a negative control agent (scrambled peptide) for 5 min at room temperature, with or without prior incubation with 100 μM of native unlabeled bombesin (15 min at 37 ˚C) to compete for binding. Cells were rinsed and imaged by both flow cytometry (A) and by fluorescence microscopy (B). Red signal is BombesinRSense 680 and blue signal represents DAPI nuclear counterstain.

Application notes and posters

• Application Note: Development of a Near Infrared Fluorescent Labeled Agent for Assessing Bombesin Receptor Expression in Cancer

Citations

Please visit our Citations Library for references using BombesinRSense 680 on the IVIS or on the FMT.