A P P L I C AT I O N N O T E

Development of a Near
Infrared FluorescentLabeled Agent for Assessing
Bombesin Receptor
Expression in Cancer

BombesinRSense 680
AuthorJeffrey D. Peterson, Ph.D.
PerkinElmer, Inc.
Waltham, MA USA

Abstract
Bombesin-like peptides and their G-protein coupled receptors have beenshown to play a role in cancer and are overexpressed in a variety of tumors,
including prostate, breast, lung, CNS, gastric, colon, and renal cell carcinomas.
We developed a novel near infrared (NIR) fluorescent imaging agent,
BombesinRSense™ 680 (BRS 680), comprising a 7-amino acid bombesin peptide
analog, an NIR fluorophore (ex/em 665/691 nm) and a pharmacokinetic modifier
to improve its plasma availability (plasma t1/2 = 30-45 min). This agent can beused to target and quantify upregulation of bombesin receptors (BBR) in vivo
associated with tumor proliferation. Selectivity of agent binding was confirmedby blockade of BRS 680-labeling of human colorectal HT-29 cells with excess
unlabeled native bombesin peptide. BBR-expressing tumors were readily imaged
in nude mice, showing that BRS 680 has the potential to serve as an in vivo realtime early indicator of tumor growth and chemotherapy efficacy.

Materials And Methods

HT-29 Tumor Models

Fluorescent Agents

Six to eight week-old female NU/NU mice were purchasedfrom Charles River Laboratories (Wilmington, MA) andmaintained in a pathogen-free animal facility with water andlow-fluorescence mouse chow (Harlan Tekland, Madison, WI).
Handling of mice and experimental procedures were inaccordance with PerkinElmer IACUC guidelines and approved
veterinarian requirements. HT-29 (ATCC, Manassas, VA) andHT-29-luc2 (PerkinElmer, Inc., Waltham, MA) cell lines weremaintained according to supplier instructions. To induce
tumor growth, mice were injected subcutaneously either inthe dorsal flank or upper chest with 5x106 HT-29 or HT-29luc2 cells, yielding tumor masses within a few weeks (Figure 1).

The BombesinRSense 680 (BRS 680) fluorescent agent is
specific for bombesin receptors (BBRs) expressed in many
types of cancer. This agent was designed to detect andimage BBR expression of tumors implanted in NU/NU mice.
The imaging dose for this agent was 2 nmol/25 g mouse.
Table 1. Basic properties of BombesinRSense 680
fluorescent tumor imaging agent.
BombesinRSense 680
Agent Type

BBR-targeted agent

Molecuar Weight

24,000 gmol-1

Ex/Em

665/691 nm

Blood Half-life

t1/2 30-45 min

Tissue Half-life~ 96-120 hAgent Summary. MW/size, excitation/emission [Ex/Em], andblood/tissue pharmaco-kinetics were determined in multiple
independent studies. Plasma half-life was measured by bloodcollection from mice at different times post-intravenous
injection. Blood samples were measured for fluorescence levels
in a fluorescence microplate reader. Tissue half-lives were
determined by time course FMT 4000 imaging of tumors.
Flow Cytometry and Microscopy
Human colorectal carcinoma HT-29 cells (BBR positive) wereobtained from American Type Culture Collection (ATCC,
Manassas, VA). Cells were routinely cultured in McCoy’s 5A
medium with 10% fetal bovine serum and 1% penicillinstreptomycin in 75 cm2 flasks. Exponentially growing cells
between passages 1-10 were used for all experiments. Cells
were incubated with BRS 680 (1 μM) for 5-15 min. Cells
were analyzed by flow cytometry (Becton Dickinson LSRII) andfluorescence microscopy using appropriate lasers and filters
appropriate for detection of 680 nm wavelength. Cell nuclei
were stained with DAPI (blue).
Frozen tissue sections were obtained 24 h after in vivo agent
intravenous (i.v.) injection (2 nmol per mouse) or labeled exvivo by incubation of 10 micron frozen tumor tissue sections
with 1 µM BRS 680 for 15 min. Some control sections were
stained with a rabbit polyclonal antibody against GRPR (BB2
receptor) for 30 min, followed by a FITC-conjugated secondary
antibody (goat anti rabbit polyclonal secondary antibody).
Sections were imaged by fluorescence microscopy using DAPI
as a nuclear counterstain.

2

In Vivo Imaging and Analysis
Tumor-bearing mice were anesthetized using inhaledisoflurane, injected IV with BRS 680, and positioned for
imaging in either the IVIS® Spectrum or the FMT 4000™
in vivo imaging system (both systems from PerkinElmer,
Waltham, MA). Fluorescence images were captured at avariety of time points post-agent injection as described in
Figure legends.
Imaging Data Analysis
The collected FMT fluorescence images were reconstructedby FMT system software (TrueQuant™ v4.0, PerkinElmer, Inc.
Waltham, MA) for the quantification of three-dimensional
fluorescence signal within the tumors. Three-dimensional
regions of interest (ROI) were drawn encompassing therelevant flank or chest regions. IVIS Spectrum data wereanalyzed by Living Image® software (PerkinElmer, Inc.,
Waltham, MA) and analyzed for 2D epifluorescence.
HT-29 orHT-29-luc2Tumor Cells

A few
weeks

24 h

NU/NU mice
IV Imaging Agent
Injection
Figure 1. Tumor xenograft model.

Tumor MiceBombesin receptors (BBRs), which play a role in a variety ofphysiological systems in mammals, have been the subjectof investigation for the past two decades because of theoverexpression of the three characterized receptor types(BB1R, BB2R, and BB3R) in several prevalent solid tumors,
including prostate, breast, small cell lung, and gastrointestinal
cancers. The bombesin family of peptide ligands for these
BBRs, first described in the 1970s in amphibian skin, include
the mammalian analogs, Neuromedin B (NMB), gastrinreleasing peptide (GRP), and phyllolitorins. Receptors
are increased in cancer as a marker of proliferation, andbombesin peptides serve as autocrine growth factors,
signaling either calcium mobilization or proliferation in tumor
cells (findings particularly attributed to GRP signaling throughBB2R). Several radio-labeled analogs of these peptides havebeen used for PET or SPECT imaging in preclinical cancer
research as well as in imaging of patients. Cytotoxic analogs
of bombesin, as well as bombesin receptor antagonists, arebeing explored as novel therapeutic approaches.
PerkinElmer developed a NIR fluorescent agent for detectionof upregulation of BBRs in vivo, comprising a 7-aminoacid bombesin peptide analog, an NIR fluorophore (ex/em
665/691 nm) and a pharmacokinetic modifier. The NIRlabeled bombesin peptide (BombesinRSense 680 [BRS 680])
effectively labeled HT-29 human colon adenocarcinoma cells,
and the specificity of this binding was confirmed by control
experiments using competitive blockade with unlabeled
excess bombesin. Fluorescence microscopy confirmed the
expected BRS 680 localization and uptake of fluorescence.
In vivo and ex vivo imaging (FMT 4000 and IVIS Spectrum),showed significant accumulation within tumors, peaking at
24 h following intravenous injection of 2 nmol of the agent,decreasing thereafter with a tissue half-life of ~5 days.
Other tissues known to express BBRs (e.g. pancreas, skin)also showed preferential accumulation of BRS 680, and theshort half-life in blood was in agreement with high signal
in the kidneys, suggesting rapid clearance via the kidneys
and bladder. In summary, BRS 680 selectively targets BBR
expression, allowing both imaging in vitro and the noninvasive real-time NIR imaging and quantification in vivo.
Microscopy and Flow Cytometry
Figure 2 shows that BBR-positive HT-29 cells can beeffectively labeled with BRS 680 with only a short 5 minuteincubation. The fluorescent signal of labeled cells wasreadily quantified by flow cytometry as ~40-fold increase influorescence (Figure 2A). In contrast, a peptide-scrambled
version of BRS 680 (Scrambled 680 Control) showed only
background cell labeling. BRS 680 binding specificity
was confirmed by prior blockade with native unlabeled
bombesin; the ability of BRS 680 to label HT-29 cells wasinhibited ~90%. Results were confirmed by fluorescencemicroscopy, and Figure 2B illustrates the strong labeling oftumor cells as assessed by fluorescence microscopy.

A.
3000
Mean Fluorescence (RFUs)

Introduction and Results

2500
2000
1500
1000
500
0

Cells only
BRS 680
BRS 680			with 100X
			Bombesin

B.

BombesinRSense 680

Scrambled
680 Control

100X Bombesin Competition

Figure 2. HT-29 cells were incubated with 1 µM BRS 680 or negative control
agent (scrambled peptide,) for 5 min at RT , with or withour prior incubation
with 100 µM of native, unlabeled Bombesin (15 min at 37 ˚C) to compete forbinding. Cells were rinsed and imaged by both flow cytometry (A.) and byfluorescence microscopy (B.) Red signal is BRS 680, and blue signal
represents DAPI nuclear counterstain.

In Vivo Imaging: Cross-validation with Bioluminescence
Figure 3 highlights the in vivo ability of BRS 680 to detect
BBR expression in subcutaneously implanted colorectal
tumor xenografts in a manner similar to the detection oftumor luciferase expression. Three different mice, bearing
three different levels of tumor burden, showed clear
bioluminescence signal defining the tumor localization(Figure 3A) with signal to background ratios exceeding
100. The same mice, imaged for 2D epifluorescence by
IVIS Spectrum 24 h after injection with BRS 680, showed
the same tumor masses by virtue of their expression of
BBRs (Figure 3B). These NIR fluorescence imaging results
confirmed the appropriate localization of signal, with goodtumor definition and ~3.5 fold target to background ratios.
In addition, BRS 680 detected the normal, expected BBR
expression in the pancreas. The optimal imaging time in thisstudy was 24 h post-injection, with a tissue residence halflife of approximately 96 h (not shown).

3

A.

B.

2D Bioluminescence

2D Epifluorescence

Figure 3. NU/NU mice were implanted with human colorectal HT-29 luc tumors, injected IV with 2 nmol BRS 680, and imaged by IVIS Spectrum CT 24 hlater; (A.) bioluminescence and (B.) epifluorescence. The same three mice per group were imaged to show tumor specific localization of BRS 680. Red and blue
arrows indicate tumor masses and pancreatic tissue, respectively. Upper torso signal may be attributed to either lymph nodes or salivary glands. Tumor signal asassessed by BRS 680 correlates strongly with bioluminescence intensity (r2 = 0.91).

In Vivo Imaging: Timecourse Optimization
To establish a full range of imaging datasets for defining
optimal imaging time and the kinetics of agent clearance,
HT-29-bearing mice were injected with 2 nmol of BRS 680
and imaged 15 and 30 minutes, 1 and 4 h, and 1, 2, 5, and12 days post injection. As shown in Figure 4, most of thefluorescent signal is in circulation prior to 4 h, yielding poor
tumor definition by epifluorescence imaging (Figure 4A).
Tomographic imaging by FMT detected and quantified thisearly heart signal, providing a kinetic imaging profile in goodA.

agreement with the established plasma pharmacokinetics(Figures 4A and B). The kinetic changes of liver fluorescencealso closely agreed with heart imaging data, suggesting thatthe apparent liver signal was due to signal within the bloodvessels of the liver rather than due to agent extravasation
into liver tissue. The optimal imaging time was 24 h postinjection, with a tissue residence half-life of approximately
96-120 h. These data generated from a single tumor-bearing
mouse were in close agreement with averages of all 4 mice
within the study group (data not shown)B.

Imaging HT-29 Tumors (FMT 4000)

Tomography Quantification

Epifluorescence (2D)

600

0.00
[Count/Energy]

Fluorescence Tomography (3D)
1000
756
513
269

Heart Fluorescence (pmol)

0.06
0.03

Heart
Liver Region
Left Tumor
Right Tumor

700

0.09

70
60

500

50

400

40

300

30

200

20

100

10

0

0

24

48
72
96
120
Time Post-Injection (h)Tumor Fluorescence (pmol)

0.12

0
144

25
[nm]

Figure 4. NU/NU mice were implanted subcutaneously with human colorectal HT-29 tumors (2 tumors per mouse). When tumors reached the desired volume,
mice were injected IV with 2 nmol BRS 680 and imaged tomographically (FMT 4000) at times ranging from 15 minutes to 12 days. (A.) Shown is 1 representative
mouse imaged at all timepoints by both epifluorescence (upper panels) and fluorescence tomography (lower panels). (B.) Tomographic imaging datasets wereused to quantify kinetic fluorescence changes in the upper liver region and the heart as well as targeted accumulation in the tumors.
4

In Vivo Multiplex Imaging

Blood PharmacokineticsA.

To better understand the in vivo performance of BRS 680,
normal mice were injected with the agent and blood wascollected at a variety of times spanning 24 h. Changes
in the fluorescent signal in the blood over time revealeda circulation half-life of 30-45 minutes (Figure 6A inset).
This relatively short half-life is supported by the observed
increased fluorescence in the kidneys of HT-29-bearing
NU/NU mice at 24 h post-injection (Figure 6A). Most tissues
showed low fluorescent signal at 24 h, except for pancreasand skin, supporting studies showing expression of BBRs
in pancreatic acinar tissue and in skin mast cells. Naturally,
HT-29 tumors also showed increased BRS 680 signal inagreement with data in Figures 3, 4, and 5. Epifluorescence
images of excised organs and tissues (Figure 6B) shows
clearly the brighter signal in these tissues collected from a
representative mouse.

0.3

Mean Intensity (Counts/Energy)

0.25

Tumor tissues were also collected from mice 24 h after
receiving an IV injection of BRS 680. Serial 10 micron
sections were prepared to determine whether the pattern ofsignal generated by the agent in vivo correlated with ex vivo
staining of serial sections with anti-BBR antibody. Figure 7Bshows excellent co-localization of BRS 680 and ex vivo antiBBR staining, supporting the contention that in vivo BRS 680
localizes selectively to BBR-expressing cells.

7000

t1/2 = 30 h

6000
5000
4000
3000
2000
1000
0
0.01

0.15

0.1

1
10
Time (h)

100

0.1

0.05

0

Detection of BBR Expression in Frozen Tumor Tissue Sections
To further confirm the specificity of BRS 680 binding andlocalization within tumors, we performed studies using
frozen HT-29 tumor xenograft sections. Figure 7A shows
tumor sections which were incubated ex vivo with BRS
680 in the presence or absence of 100X excess of native
unlabeled bombesin as a receptor blocker. BRS 680 signal
in unblocked tissue showed a heterogeneous distributionof signal within the tissue, however excess native bombesin
blocked this tissue labeling completely.

0.2

8000

Mean Fluorescence
Intensity

In Vivo Biodistribution and Pharmacokinetics

Figure 5. NU/NU mice implanted subcutaneously with human colorectal
HT-29 tumors were injected intravenously with 2 nmol BombesinRSense 680
and 2 nmol of AngioSense 750 EX. Epifluorescence imaging by FMT 4000 at
24 h post IV injection shows overlapping intratumoral distribution of these
two imaging agents.

brain
clavaria
sal gland
heart
lung
spleenliver
kidneylymph nodes
tumor
musclebladderintestines
pancreasfatuterusskin
blood

The BRS 680 imaging agent can be paired with otherimaging agents to better provide a more complete view of
the complex biology of a tumor. For example, in Figure 5,
BRS 680 (which provides a measure of increased proliferative
status of the tumor) was co-imaged with AngioSense 750
EX (which shows areas of increased vascular leak). As
expected for this pair of agents, there is a good degreeof overlap in localization with some other regions ofdifferential localization. Tumor metabolism and proliferation
are expected to be higher in well perfused tumor regions.
However, vascular leak is also generally high in regions
around the tumor where you see less BRS 680 accumulation.

B.
Tumor

Pancreas

Liver

0.00

0.15
Epifluorescence (counts/energy)

Kidney0.30

Figure 6. Twelve HT-29-bearing NU/NU mice were injected IV with 2 nmol
BRS 680. Blood was collected at multiple times, and tissues were removed at
24 h, to assess blood and tissue biodistribution, respectively. (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.
5Conclusions
Cancer is a term used for diseases in which cells
spontaneously proliferate out of control and invade othertissues via the blood and lymph systems. Bombesin-like
peptides and bombesin receptors (BBRs) have been thesubject of investigation for the past two decades becauseof the association between these molecules and theproliferative status of tumor cells. Overexpression of thethree characterized receptor types (BB1R, BB2R, and BB3R) is
prevalent in several solid tumors, including prostate, breast,
small cell lung, and gastrointestinal cancers.
The ability to detect expression of BBRs non-invasively inanimal models of cancer has provided an important tool forbiomedical research. Adapting bombesin as a fluorescenceimaging tool, however, offers further advantages in ex vivo
microscopy as well as in the replacement of hazardous
radioactive labels. For this reason, we developed a NIR
fluorescent imaging agent specific for tumor BBRs by
conjugating a NIR fluorescent dye to a bombesin peptide
analog. This imaging agent was profiled extensively in vitro
and in vivo for performance and selectivity, offering a useful
tool for examining the proliferative status of tumors. The
further pairing of BombesinRSense with PerkinElmer imaging
systems, and other novel red and near infrared (NIR) imaging
agents, allows the detection and quantification of multiple
important biological processes in the context of tumor
BBR expression.

A.
BRS 680

B.

BRS 680 + native Bombesin

BRS 680

Anti-BB2R Ab

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Figure 7. (A.) To assess binding specificity of BRS 680 tumors were removed
from HT-29-tumor bearing mice, and frozen sections (10 micron thickness)were incubated with 1 µM BRS 680 for 15 min. at RT, without or withprevious incubation (for 15 min. at 37 ˚C) with 100 µM native bombesin,
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