Radiolabeled RGD Tracer Kinetics Annotates Differential αvβ3 Integrin Expression Linked to Cell Intrinsic and Vessel Expression

Mol Imaging Biol (2013)

DOI: 10.1007/s11307-013-0710-3

* World Molecular Imaging Society, 2013

RESEARCH ARTICLE

Radiolabeled RGD Tracer Kinetics AnnotatesDifferential αvβ3 Integrin Expression Linkedto Cell Intrinsic and Vessel Expression

Israt S. Alam, Timothy H. Witney, Giampaolo Tomasi, Laurence Carroll,

Frazer J. Twyman, Quang-Dé Nguyen, Eric O. Aboagye

Comprehensive Cancer Imaging Centre, Faculty of Medicine, Imperial College London, London W12 0NN, UK

Abstract

Purpose: The purpose of this paper is to study the association between RGD binding kinetics

and αvβ3 integrin receptor density in the complex tumor milieu.

Procedures: We assessed αvβ3 in vitro and by 68Ga-DOTA-[c(RGDfK)]2 positron emission

tomography (PET) in tumors with varying αvβ3.

Results: Intrinsic αvβ3 expression decreased in the order of M21999MDA-MB-2319M21L in

cells. Tumor volume of distribution by PET, VT, was significantly higher in M21 compared to

isogenic M21L tumors (0.40±0.01 versus 0.25±0.02; pG0.01) despite similar microvessel

density (MVD) likely due to higher αvβ3. VT for MDA-MB-231 (0.40±0.04) was comparable to

M21 despite lower αvβ3 but in keeping with the higher MVD, suggesting superior tracer

distribution.

Conclusions: This study demonstrates that radioligand binding kinetics of PET data can be used

to discriminate tumors with different αvβ3 integrin expression—a key component of the

angiogenesis phenotype—in vivo.

Key words: Integrin, RGD peptide, Positron emission tomography, Kinetic modeling,

Vasculature

Introduction

The αvβ3 integrin is a heterodimeric protein that is

preferentially expressed on proliferating vascular endo-

thelial cells, as well as in a variety of cancers such as breast [1,2], ovarian [3], and melanoma [4, 5]. The αvβ3 integrin

receptor preferentially binds to ECM components such as

fibronectin, fibrinogen and laminin via recognition of the

arginine–glycine–aspartic acid (RGD) sequence [6]. Given

the role of αvβ3 integrin in angiogenesis, invasion, and

metastasis, an array of cyclic RGD-based peptides have been

developed to date to probe integrin expression by nuclear

imaging [7–11]. Radiolabeled RGD peptides have been

exploited as diagnostic radiopharmaceuticals for the assess-

ment of angiogenic activity of solid tumors, as well as for

monitoring response of tumors to integrin-targeted and

antiangiogenic therapies [10, 12, 13].

Some aspects of integrin receptor research in the context

of diagnostic imaging remain relatively underexplored. In

particular, it is unknown whether radiolabeled RGD peptides

largely provide a blood volume-independent readout of αvβ3integrin receptor on cells within the tumor or primarily on

neovasculature, making their role as imaging agents for the

angiogenenic phenotype unclear. Furthermore, despite the

widely reported use of the RGD peptides in molecular

Electronic supplementary material The online version of this article(doi:10.1007/s11307-013-0710-3) contains supplementary material, whichis available to authorized users.

Correspondence to: Eric Aboagye; e-mail: [email protected]

http://dx.doi.org/10.1007/s11307-013-0710-3

imaging, there is paucity of studies directly assessing the

relationship between RGD binding kinetics and αvβ3 integrin

receptor density in the complex tumor milieu comprising

both receptor expressing cancer cells and neovasculature. A

number of recent [14, 15] and earlier reports [16] describe

the use of kinetic variables—volume of distribution (VT) and

binding potential (BPND)—to allow quantitative compari-

sons of the behavior of RGD tracers in somatic tumors of

patients and animal models. In an attempt to evaluate

whether RGD radioligand binding provides a blood vol-

ume-independent readout of αvβ3 integrin receptor, we

utilized kinetic modeling of PET data from Gallium-68,

DOTA-cyclo [RGDfK]2 (68Ga-DOTA-[c(RGDfK)]2) to de-

tect varying physiological expression of αvβ3 integrin in

different tumor models, to enable assessment of the

contribution of tumor cell and vascular compartments in

vivo, with the latter being more relevant to the angiogenic

phenotype.

Materials and Methods

Cell Lines

Isogenic human melanoma M21 and M21L cells (kind donation

from Dr Amin Hajitou, Imperial College London, UK) were grown

in DMEM media, supplemented with 10 % fetal calf serum, 2 mM

L-glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin

(Invitrogen, Paisley, Refrewshire, UK). M21L is a FACS sorted

αvβ3 integrin deficient cell line derived from parental M21 cells [5].

MDA-MB-231-luc-D3H2LN (MDA-MB-231) (Xenogen, Alame-

da, CA, USA) were grown in RPMI 1640 media, supplemented

with 10 % fetal calf serum, 2 mML-glutamine, 100 U ml−1

penicillin and 100 μg ml−1 streptomycin (Invitrogen, Paisley,

Refrewshire, UK). All cells were maintained at 37 °C in a

humidified atmosphere containing 5 % CO2.

Flow Cytometry Analysis

Flow cytometry was performed using standard techniques [17]. For

detailed methodology, see Supplementary Methods in the Electron-

ic Supplementary Material (ESM).

Western Blot

Western blot on whole-cell lysate and plasma membrane fractions

of cells was performed using standard techniques [18]. For detailed

methodology, see Supplementary Methods.

Radiochemistry

The commercially available precursor, DOTA-cyclic RGDfK dimer

(DOTA-[c(RGDfK)]2), was radiolabeled with 68GaCl3 using a

method outlined in Supplementary Methods.

In Vitro 68Ga-DOTA-[c(RGDfK)]2 Uptake

Tracer uptake in cell lines was characterized using a method

outlined in Supplementary Methods.

In Vivo Tumor Models

All animal experiments were performed by licensed investigators in

accordance with the United Kingdom Home Office Guidance on

the Operation of the Animal (Scientific Procedures) Act 1986 and

in keeping with the published guidelines for the welfare and use of

animals in cancer research [19]. Female BALB/c nude mice (aged

6–8 weeks; Harlan) were used. M21 and MDA-MB-231 cells were

injected subcutaneously on the back of mice (5×106 cells in 100 μl

sterile PBS). M21L cells (5×106) were also injected subcutaneous-

ly on the back of mice along with Matrigel at a 1:1 ratio in a total

volume of 100 μl (BD Biosciences, Rockville, MD USA). Animals

were used when the xenografts reached ∼100 mm3. Tumor

dimensions were measured continuously using a caliper and tumor

volumes were calculated by the equation: volume=(π/6)×a×b×c,

where a, b, and c represent three orthogonal axes of the tumor.

PET Imaging Studies and Image Analysis

Dynamic 68Ga-DOTA-[c(RGDfK)]2 imaging scans were carried

out on a dedicated small animal PET scanner (Siemens Inveon PET

module, Siemens Medical Solutions USA, Inc., Malvern, PA,

USA) following a bolus i.v. injection in tumor-bearing mice of

∼3.7 MBq [20]. Dynamic scans were acquired in list-mode format

over 60 min. The acquired data were then sorted into 0.5–mm

sinogram bins and 19 time frames for image reconstruction (4×

15 s, 4×60 s, and 11×300 s), which was done by 2D-ordered

subset expectation maximization (OSEM) reconstruction. The

Siemens Inveon Research Workplace software was used for

visualization of radiotracer uptake in the tumor; 30 to 60 min

cumulative images of the dynamic data were employed to define

three-dimensional (3D) regions of interest (ROIs). For each animal,

ROIs encompassing the heart (to derive the arterial input function)

and the tumor were manually drawn on the summed PET image

and used to compute time–activity curves (TACs). For the

computation of the heart TACs, data were sorted into 25 time

frames for image reconstruction (8×5 s, 1×20 s, 4×40 s, 1×80 s,

and 11×300 s) to better capture the peak of the TACs. Both tumor

and heart TACs were normalized to injected dose, measured by a

VDC-304 dose calibrator (Veenstra Instruments, Joure, The

Netherlands), and expressed as percentage injected dose per

milliliter of tissue (%ID/ml).

Tracer uptake values were first assessed using three semi-

quantitative parameters. The ratio of uptake between tumor and

heart at 60 min (tumor/heart), the area under the curve of the tumor

TAC from 0 to 60 min (AUC0–60), and the normalized uptake of

radiotracer at 60 min in the tumor (%ID/ml60 or NUV60) were

compared across the groups. Kinetic analysis of the data was also

performed. To this aim, a standard two-tissue reversible compart-

ment model was employed to fit each tumor TAC with the

corresponding image-derived blood TAC as input function to

estimate the kinetic parameters K1 (milliliters per cubic centimeter

per minute), k2 (per minute) k3 (per minute), and k4 (per minute).

I.S. Alam et al.: Radiolabeled RGD Tracer Kinetics Annotates Differential αvβ3

The volume of distribution (VT) and the binding potential (BPND)

were then computed for each animal as VT=K1/k2(1+k3/k4) and

BPND=k3/k4, as previously reported for the RGD peptide

[18F]fluciclactide [14]. These values, derived from modeling, were

used to assess physiological differences in receptor expression

between the three tumor models, analogous to the semi-quantitative

parameters.

Biodistribution and In Vivo Tracer Metabolism

Blood, plasma, and tissues were acquired for the biodistribution

study. In addition, to verify the integrity of the radiotracer in vivo,

tissues and blood were analyzed for the presence of radiolabeled

metabolites (see Supplementary Methods for detailed protocols).

Immunohistochemistry

For immunohistochemical staining of tumor αvβ3 integrin expres-

sion, formalin-fixed paraffin embedded tumor slices (of 5 μm

thickness) were stained with rabbit anti-β3 integrin antibody (Cell

Signalling Technology, Danvers, MA, USA; 1:100).

Blood vessels were detected from serial sections stained with

a rabbit antibody to endothelial cell marker; platelet endothelial

cell adhesion molecule (PECAM)/CD31 (Millipore, MA, USA).

After PBS wash and secondary antibody application, anti-rabbit

and avidin–biotin complex (ABC) chromogen were applied

sequentially for 10 min each separated by a PBS wash. Tissue

sections were counterstained with hematoxylin (Harris’ Austra-

lian Biostain, Victoria, Australia) and mounted. Microvessel

density (MVD) was assessed from CD31 stained sections using

a method originally described by Weidner et al. [21] (described in

Supplementary Methods).

Statistics

Data were expressed as mean±standard error of the mean (SEM),

unless otherwise shown. The significance of comparison between

two data sets was determined using Student’s t test (Prism v5.0

software for windows, GraphPad Software, San Diego, CA, USA).

Differences between groups were considered significant if p≤0.05.

Results

Differential Expression of αvβ3 Integrin in CellsConfirmed by Flow Cytometry and Western Blot

M21, M21L and MDA-MB-231 cells, which vary in the

levels of αvβ3 integrin expressed, were chosen to test the

ability of 68Ga-DOTA-[c(RGDfK)]2 peptide to detect differ-

ences in receptor density in vivo. To ascertain cell intrinsic

αvβ3 integrin expression in the three cell lines, flow

cytometry analysis was performed using a FITC-labeled

αvβ3 integrin specific antibody.

The cell lines were shown to express αvβ3 integrin

receptor in the order M21999MDA-MB-2319M21L

(Fig. 1a). The greatest level of staining with the αvβ3

integrin specific antibody was observed with the M21 cells

that exhibited a 23-fold increase in fluorescence in stained

vs. unstained samples (Fig. 1b). The M21L cells showed

significantly less staining than M21 cells (pG0.001) at 1.6-

fold. This difference between the two isogenic cell lines is to

be expected as the M21 cells are known to express high

levels of αvβ3 integrin while the M21L lack expression [5,

22]. MDA-MB-231 cells showed a threefold increase in

staining in comparison to their unstained counterparts. These

cells have αvβ3 integrin levels that are significantly greater

than in M21L cells and significantly lower than in the M21

cell line (pG0.001) (Fig. 1b).

Lysates from the three cell lines were also analyzed by

western blot to define αvβ3 integrin protein levels. An

antibody specific for the β3 integrin subunit was used since

the αvβ3 integrin heterodimer specific antibody was not

compatible with the western blot assay. Whole cell lysates

analyzed showed high β3 integrin expression in both

melanoma cell lines (Fig. 1c) in contrast to the differences

in expression detected by flow cytometry (the latter

experiment probed specifically for cell surface αvβ3integrin). Three distinct bands corresponding to the β3subunit bands were observed for the M21 line corresponding

to 97, 110, and 130 kDa whereas the 130 kDa band is less

prominent for the M21L cells. MDA-MB-231 cells surpris-

ingly showed the lowest expression of total β3 integrin.

In contrast to the total β3 protein, western blot analysis of

the plasma membrane fraction of the cell lines showed much

greater discrimination in β3 integrin expression between the

two melanoma cell lines with M21 cells showing high

membrane β3 integrin expression (Fig. 1d). MDA-MB-231

cells this time showed greater plasma membrane expression

of β3 integrin than the M21L cells, consistent with the flow

cytometry analysis. These results reflect the fact that M21L

cells lack the αv gene, thus, the expressed β3 subunit is

unable to dimerize with an αv chain and remains cytosolic

rather than as part of a functional heterodimer at the plasma

membrane [22]. Although endogenous levels of total β3integrin appeared not to differ in the melanoma cells

(Fig. 1c), the plasma membrane fraction of the subunit

detected here as a marker for αvβ3 integrin, differs greatly

and western blot analysis of the membrane fraction was in

the order M21999MDA-MB-2319M21L (Fig. 1d). Flow

cytometry and western blot analysis, therefore, supported

selection of the models expressing varying levels of αvβ3integrins.

Cell uptake of 68Ga-DOTA-[c(RGDfK)]2 was then

assessed in the three cell lines. Significantly higher 68Ga-

DOTA-[c(RGDfK)]2 uptake was detected in M21 (0.44 %

total radioactivity/mg protein) compared to MDA-MB-231

cells (0.17 % total radioactivity/mg protein; pG0.0001) and

M21L cells (0.12 % total radioactivity/mg protein; pG0.001)

(Fig. 1e). The relative magnitude of tracer uptake followed

the same order as the expression of αvβ3 as detected by flow

cytometry and the western blot of the plasma membrane

fraction of cells. Uptake was significantly reduced in M21


and MDA-MB-231 cell lines by 54 and 43 %, respectively,

following blocking with cold ligand showing specificity of

the uptake (pG0.001). M21L cells demonstrated no signif-

icant reduction in tracer retention following blocking

indicating that any retention observed was likely a result of

non-specific binding.

PET Imaging and Kinetic Modeling

The highest tumor localization was observed in the M21

xenograft characterized by a heterogenous pattern of tracer

uptake (Fig. 2a). In contrast, uptake was low and relatively

homogeneous in the M21L xenograft. Tumor TACs showed

that higher uptake was indeed observed in the M21 tumor

group while M21L tumors showed the lowest uptake

(Fig. 2b). 68Ga-DOTA-[c(RGDfK)]2 uptake was reversible

decreasing in the order M21≥MDA-MB-2319M21L, with

statistically significant differences (pG0.05) between M21

and M21L tumors (Fig. 2c).

VT values (Fig. 3a), were significantly higher in M21

tumors than in M21L tumors (58.3 % higher, p=0.0022).

The MDA-MB-231 tumor group also showed significantly

higher VT than M21L tumors (58.1 % higher, p=0.0012).

Notably, VT was similar between the M21 and MDA-MB-

231 groups. BPND (Fig. 3b), was also significantly higher in

M21 tumors (1.81±0.70) and MDA-MB-231 tumors (1.00±

0.13) compared to M21L tumors (0.44±0.19), (pG0.05).

Although, due to large within-group variations of BPND,statistically significant differences were not obtained be-

tween the M21 and MDA-MB-231 groups; these results

suggest that, as expected, BPND was more suitable than VT

for differentiating across these two groups due to its

independence of the delivery component.

PET data were also corroborated by ex vivo gamma

counting of excised tissues (Fig. S1 in ESM). The

Fig. 1. Analysis of αvβ3 integrin expression in M21, M21L and MDA-MB-231 cells. a–b Analysis of αvβ3 integrin expression by

flow cytometry. a Histograms of fluorescein isothiocyanate (FITC) (Exc, λ=490; Em, λ=530 nm) fluorescence intensities in

unstained cells (blue peaks) and the subsequent increase in fluorescence upon staining with the αvβ3 specific antibody

conjugated to FITC (red peaks). The largest shift upon staining is observed with M21 cells, followed by MDA-MB-231 cells while

M21L cells show the smallest shift. b Median fluorescence intensities (MFI) corrected to autofluorescence levels of unstained

cells in the FITC channel (y-axis). Data shown are MFI±SEM (n=3, samples run as duplicates). The cell lines show significant

differences in levels of staining with the αvβ3 specific antibody (***pG0.001). Western blot analysis of β3 integrin expression in c

whole cell and d plasma membrane protein fractions of M21, M21L, and MDA-MB-231 cell lysates. β-actin and Na-K ATPase

were used as loading controls. e68Ga-DOTA-[c(RGDfK)]2 uptake in the three different cell lines and in the presence of a

blocking RGD monomer. Data shown are mean uptake values±SEM normalized to protein (n=3, samples run in triplicate). Cells

show significant differences in tracer uptake (**pG0.01, ***pG0.001) and uptake is significantly reduced in M21 and MDA-MB-

231 cells in the presence of blocking agent (***pG0.001).


percentage injected dose/gram of tissue (%ID/g) values in

the tumor at 60 min were 3.93±0.34, 2.82±0.30 and 1.37±

0.21 for M21, MDA-MB-231 and M21L tumors, respec-

tively. The M21 group had a mean %ID/g value 1.4-fold

higher than in MDA-MB-231 tumors (pG0.05) and 2.9-fold

higher than in M21L tumors (pG0.001). MDA-MB-231

tumors also showed significantly higher uptake than M21L

tumors (pG0.01). While differences in tracer uptake between

the three different xenograft models were seen, what is clear

is that these differences were smaller in magnitude compared

to the cell intrinsic differences in αvβ3 integrin expression

and cellular radiotracer uptake (Fig. 1).

Fig. 2. PET image analysis of 68Ga-DOTA-[c(RGDfK)]2. a Representative axial 3D-OSEM PET-CT images of the tracer in M21,

M21L and MDA-MB-231 tumor bearing mice, showing 30–60 min summed activity. The tumor margins, as identified by CT, are

outlined in red. The heart, observed as a hot spot in the center of each subject is indicated (H). b Mean±SEM tumor time

activity curves (TACs) for the three different groups obtained from 60 min dynamic PET imaging. Mean±SEM blood TAC from

the M21 group is also shown with a more rapid clearance profile than observed in tumors. c Uptake ratio for tumor relative to

heart at 60 min (tumor/heart), area under the curve (AUC 0–60 min) taken from tumor TACs and the decay corrected normalized

uptake values for tumors at 60 min (NUV60). Data points represent mean±SEM, n=6 mice per group, *pG0.05, **pG0.01.

Fig. 3. Kinetic parameters of 68Ga-DOTA-[c(RGDfK)]2 in M21, M21L and MDA-MB-231 tumors. a Volume of distribution (VT,

milliliter per cubic centimeter) and b binding potential (BPND, unitless) of the tracer in the different tumor types (n=6, data are

expressed as mean±SEM, *pG0.05; **pG0.01). VT reflects the tissue-to-plasma concentration and BPND represents the

combined effect of binding affinity and receptor expression.


68Ga-DOTA-[c(RGDfK)]2 Does not UndergoSystemic Metabolism

Representative radio-chromatograms (see Supplementary

Methods) of 68Ga-DOTA-[c(RGDfK)]2 derived from liver,

kidney and plasma show a single peak (RT 6.5 min) similar

to that of the dose solution (standard) (Fig. S2 in ESM). This

confirmed that the tracer does not undergo systemic

metabolism, thus, metabolite correction of the PET data

was not required for kinetic modeling. Furthermore, it can be

assumed that the total activity observed in the TACs

(Fig. 2b) represents the intact tracer.

Analysis of αvβ3 Integrin Levels and Mean VesselDensity In Vivo

To ascertain whether differences in VT and BPND values

between the tumor groups were due to variations in receptor

density and not merely tracer delivery, tumor αvβ3 integrin

expression and microvessel density (MVD) were determined

and compared to the imaging data.

A heterogeneous pattern of αvβ3 integrin staining on

immunohistochemistry was observed within M21 tumor

tissue with clusters of highly positive β3 integrin cells

(Fig. 4a). Furthermore, the staining was clearly concentrated

on the plasma membrane of cells (Fig. 4b). Within the M21

tumor group, a variation in the magnitude of β3 integrin

expression was observed; some tumor samples analyzed

showed much lower β3 integrin staining (Fig. S3c and S3d in

ESM) and a lack of distinct β3 integrin positive clusters as

typically observed (Fig. 4a, b). M21L tumor sections

showed the weakest staining for β3 integrin overall

(Fig. 4c). Moreover, staining of cells appeared cytosolic

and diffusely present throughout the cells rather than clear

localization at the cell membrane (Fig. 4d). MDA-MB-231

tumor cells showed overall higher levels of staining than

observed in M21L tumors but overall lower levels of

staining than M21 tumor sections (Fig. 4e). Rather than

being localized to tumor cell complexes as observed in the

M21 tumors, here the staining was similar in pattern to the

CD31 staining of these tumors and appeared to be associated

with the vasculature (Fig. 4f).

The above data led us to hypothesize that the inability of the

imaging variables VT and BPND to discriminate between M21

and MDA-MB-231 in spite of the large differences in cell

intrinsic αvβ3 integrin expression was due to high vessel

expression and neoangiogenesis in the latter. In support of this

hypothesis, we determined the MVD with an anti-CD31

antibody (Fig. 5). CD31, which is constitutively expressed on

vascular endothelial cells, is widely used as a pan-endothelial

cell marker to demonstrate the presence of endothelial cells in

histological tissue sections (Fig. 5a and b). Vascularization

expressed as MVD was similar between the two melanoma

xenografts (10.3 and 8.4 microvessels/×400 field in the M21

and M21L groups, respectively) (Fig. 5c). Vessels were

distributed homogeneously across the tumor sections for both

groups. These data suggest that the higher VT values in

M21 tumors vs. M21L tumors is likely to be due to

higher expression of αvβ3 levels in the former as

confirmed by histology (Fig. 4). MDA-MB-231 tumors were

by contrast significantly more vascularized than the melanoma

tumors, with a MVD of 30.4 microvessels/×400 field (Fig. 5b).

The high VT for this tumor group, which is comparable

to that of the M21 group (Fig. 3a), is therefore likely to be due

to high vessel expression of αvβ3 in the MDA-MB-231

xenograft (Fig. 4f).

Discussion

A key question in integrin imaging research is to what extent

neovessel integrin expression contributes to radiolabeled

RGD binding and thus the utility of RGD probe imaging as

readout of the angiogenic phenotype. It has previously been

shown that the interpretation of the RGD PET signal can be

complex as αvβ3 integrin can be expressed on both tumor

cells and endothelial cells of tumor neovasculature [23]. The

Fig. 4. Immunohistochemical staining of β3 integrin expres-

sion. Total magnification: ×100 (left column, a, c, e) and ×600

(right column, b, d and f) in representative M21 (top row),

M21L (middle row) and MDA-MB-231 tumors (bottom row). A

cluster of positively stained cells in the M21 tumor is

indicated by the black arrow (a) and also shown at high

magnification (b). Scale bar represents 100 μm.


expression of αvβ3 integrin as a genuine marker of

angiogenesis is only applicable to specific tumor types

where its expression is restricted to vasculature [10]. In this

manuscript, we demonstrate that radioligand binding kinetics

of PET data can be used to discriminate tumors with

different αvβ3 integrin expression in vivo (in a blood

volume-independent manner). The studies also support the

preconception that vessel fraction of αvβ3 integrin substan-

tially skews radiolabeled RGD binding in keeping with their

role as angiogenesis imaging agents and impacts on the

kinetic parameters of binding.

The isogenic lines, the M21 and M21L human melanoma

cells, which express high and low levels of αvβ3 integrins,

respectively, were chosen for the purpose of this study. The

differences in the αvβ3 integrin expression, contributes to the

rather different phenotypes of the two cell lines; M21 cells

are adherent while M21L cells are a suspension cell line.

Despite these fundamental differences, the advantage of

using isogenic models to investigate the effect of receptor

density on tracer uptake is that other sources of variability

that may be introduced when comparing non-isogenic

models can be minimized. In addition to the two isogenic

models, the human breast cancer MDA-MB-231 model was

also included in this study because of its intermediate

integrin expression.

The lack of the αv gene in M21L cells leads to the

retention of the β3 integrin unit in the cytoplasm [5]. This is

clearly shown by the western blot for total β3 integrin where

we observed similar levels between the two isogenic

melanoma lines versus the flow cytometry data probing

specifically for the αvβ3 integrin on the surface of the cells

along with the western blot of β3 integrin from the isolated

plasma membrane fraction of cells. MDA-MB-231 cells

showed staining with fluorescent αvβ3 integrin-specific

antibody, an order of magnitude lower than the M21 cells,

yet significantly higher than M21L cells. Of relevance for

non-invasive imaging, 68Ga-DOTA-[c(RGDfK)]2 tracer up-

take studies corroborated the expression profile seen.

Blocking of tracer binding upon incubation with a cold

competing peptide further suggested specificity in tracer

binding observed in the M21 and MDA-MB-231 cells. This

blocking effect was, however, incomplete, likely due to the

fact that the cold competitor used was a monomer RGD,

which has lower affinity for αvβ3 integrin than the 68Ga-

labeled dimer. Lack of any blocking in the M21L cells

suggested any retention of the tracer in the cell line was

likely non-specific. Having confirmed the choice of three

models differing greatly in αvβ3 integrin expression, similar

differences were expected in vivo.

In vivo, the 68Ga-DOTA-[c(RGDfK)]2 tracer showed

greater uptake in the M21 integrin positive tumors compared

to MDA-MB-231 and M21L tumors as shown by the time

versus radioactivity curves and biodistribution data. Despite

large differences in cellular levels of αvβ3 integrin observed

between the M21 and MDA-MB-231 cell lines in vitro, the

tracer uptake and kinetic parameters obtained from the in

vivo PET studies did not reflect the same dramatic

differences in receptor density. We found that this could be

Fig. 5. Immunohistochemical examination of CD31 expression on a representative a M21 and b MDA-MB-231 tumor section

(total magnification ×400). CD31-positive staining on vessels is indicated by the black arrows. c Microvessel density (MVD)

evaluation in the tumor groups. Mean vessel density was 10.1±0.8, 8.4±1.02, and 30.6±2.6 microvessels/×400 field in the

M21, M21L, and MDA-MB-231 tumors respectively (n=4), MVD was counted on four separate field of views for each tumor

tissue section. Data are expressed as mean±SEM. There is no significant difference in vessel density between the M21 and

M21L groups but both show significantly fewer vessels than MDA-MB-231 tumors (**pG0.01). Scale bar represents 100 μm.


attributed to integrin expression resulting from vasculature

that is independent from cell intrinsic expression. Although

MDA-MB-231 cells show much lower expression of αvβ3integrin than the M21 cells in vitro, in vivo this distinction is

made less clear because of the angiogenic nature of MDA-

MB-231 tumors (Fig. 5b). Given the significantly higher

MVD in the MDA-MB-231 tumors, the relatively high

integrin levels in the MDA-MB-231 tumors are likely to

originate from this vessel component. It is well documented

that αvβ3 integrin is not only restricted to a variety of tumor

cells but its high expression also occurs on the surface of

activated endothelial cells present in newly formed blood

vessels during angiogenesis [13, 24]. It has been demon-

strated previously that αvβ3 expression is weak in quiescent

normal vessels but is a marker of tumor associated

angiogenic blood vessels [25] and that there is a strong

correlation specifically between αvβ3 integrin positive MVD

and RGD tracer uptake [23]. In a study by Zhang et al.,

similar observations, that integrin expression on MDA-MB-

435 tumor cells grown in culture did not reflect the levels of

integrin in tumor tissue of the model and that that the αvβ3integrin expressed on neovasculature tended to be much

higher than that on the tumor cells of this particular breast

cancer model, were made [16]. Pasqualini et al. have also

shown that the αvβ3 integrin expressed within the tumor

tends to be concentrated on the apical surface of vasculature

and has a more potent contribution to ligand binding

presumably because it is more accessible to the circulating

ligand without requiring diffusion into the tissue [26]. The

high VT and BPND in MDA-MB-231 tumors are, therefore,

likely due to high expression of the vascular component of

αvβ3 integrin within the tumor contributing to the uptake of

the RGD tracer. VT in this model became comparable to that

of the M21 tumor group despite cell intrinsic differences in

target expression.

Quantification of αvβ3 expression by SDS-PAGE was not

feasible because antibodies like LM609 used in the flow

cytometry study, requires the heterodimeric feature in its

intact form, which is lost during sample preparation for

SDS-PAGE. The less specific β3 integrin subunit (also

present in αIIβ3 expressed on platelets) was used instead

[27]. The staining observed in the tumor sections in this

case, however, is clearly present on cells. Furthermore,

because the β3 expressed in the M21L model does not

manifest as part of a functional αvβ3 heterodimer, the

antibody used here overestimated the αvβ3 content in this

particular model. We were able to overcome this in vitro

through the western blot analysis of the plasma membrane

fraction of cell lysates and in vivo through the analysis of the

spatial pattern of staining on the tumor sections. The

observation that the M21 tumors exhibit a high level of

αvβ3 integrin expression on tumor cells relative to tumor

endothelial cells is in keeping with previous clinical studies

in melanoma tumors with the tracer 18F-Galacto-RGD [23].

The αvβ3 integrin receptor binds to RGD peptides in its

activated state where the receptor adopts an open

conformation [28]. Although we have concentrated on

characterizing overall expression levels of αvβ3 integrin to

corroborate the PET data, the levels of activated receptor in

the different models is what would ultimately determine the

binding of the tracer. Activated levels of the receptor as a

percentage of global levels is much more challenging to

characterize in vivo. Dumont et al. have shown, using 64Cu-

DOTA-RGD, that levels of activated αvβ3 integrin could

change even where absolute levels of the receptor remained

unchanged [29]. Although this was in the context of

response to treatment with Src family kinase inhibitor

Dasatinib and restricted to the tumor cells, it demonstrates

that there are limitations when comparing overall levels of

the receptor between models. In the MDA-MB-231 model,

the activation status of the high vessel integrin component

irrespective of the overall lower levels of αvβ3 integrin than

in the M21 model could contribute to tracer binding.

Mathematical approaches were used in this study to

provide physiologic appreciation of RGD binding. Despite

smaller than expected differences between the models in

vivo due to vessel formation, there were significant differ-

ences in VT and BPND between the two melanoma tumor

models that could be clearly attributed to the differences in

αvβ3 integrin expressed by tumor cells. Overall, M21 tumors

exhibited higher BPND values than the MDA-MB-231 group

and expressed higher levels of β3 integrin but due to large

variations in the data this difference was not statistically

different. We propose that the lack of significant difference

between the two models can be attributed to inter-group

variations (tumor heterogeneity) in receptor expression in

the M21 group (Fig. S3), as well as the contribution of high

vessel density in the breast model. In this regard, BPND is a

more suitable parameter than VT for differentiating between

these two models as it is independent of the delivery

component. The studies support the preconception that vessel

fraction of αvβ3 integrin substantially skews radiolabeled RGD

binding. BPND is strictly dependent on the estimated arterial

input function and hence also likely to suffer from partial

volume effects and spill over errors which could compromise

accuracy. Arterial blood sampling remains the ideal method for

deriving the arterial input function, although it is not practical

in small-animal imaging.

Integrin expression by tumor and vascular endothelial

cells, therefore, both contribute to the signal measured

during integrin imaging which makes the interpretation of

tracer uptake and its kinetic parameters more complex [13].

Conclusion

Kinetic parameters, VT and BPND, can be used to discrim-

inate between tumors with different levels of αvβ3 integrin in

vivo. High MVD increases blood-to-tissue partitioning and

ligand-receptor interaction, hence VT and BPND, consistent

with expression of αvβ3 integrin on vascular endothelial

cells. This makes it more challenging to distinguish tumors

based on the integrin status of tumor cells alone. The αvβ3


integrin vasculature component should therefore be consid-

ered as a major contributor to overall radiotracer uptake

when investigating integrin receptor status of tumor models

and interpreting PET data for the uptake of RGD tracers.

Acknowledgments. This work was funded by Cancer Research UK-Engineering and Physical Sciences Research Council grant C2536/A10337. E.O.A’s laboratory receives core funding from the UK MedicalResearch Council (MC US A652 0030).

Conflict of Interest. The authors declare no conflicts of interest.

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1

Radiolabeled RGD tracer kinetics annotates differential αvβ3 integrin expression linked

to cell intrinsic and vessel expression

Israt S. Alam, Timothy H. Witney, Giampaolo Tomasi, Laurence Carroll, Frazer J. Twyman

Quang-Dé Nguyen and Eric O. Aboagye.

Comprehensive Cancer Imaging Centre, Faculty of Medicine, Imperial College London,

London W12 0NN, UK.

Correspondence to: Eric Aboagye; e-mail: [email protected]

Electronic Supplementary Materials, Molecular Imaging and Biology:

Materials and Methods

1) Flow cytometry

Cell pellets (0.5 x 106) were washed in ice-cold phosphate buffered saline (PBS) with 1%

BSA and resuspended in 100µl of the same buffer containing anti-Integrin αvβ3 antibody

clone LM609 conjugated to fluorescein isothiocyanate (FITC; Millipore, M.A., U.S.A.). Cells

were incubated with the antibody for 30 minutes at 4°C. Cells were subsequently washed

twice, resuspended in PBS and kept briefly on ice and then analyzed in an LSRII flow

cytometer (BD Biosciences, Rockville, MD USA), with 10, 000 events/analysis recorded.

Data were analyzed using FlowJo software Tree Star, Inc. Ashland, OR, U.S.A).

2) Western blot

For whole-cell lysate preparation, cells were harvested and lysed in RIPA buffer (Thermo

Fisher Scientific Inc., Rockford, IL, USA). Plasma membrane proteins were fractionated

using a membrane protein extraction kit from Biovision (Mountain View, CA) [18]. The final

protein pellet was resuspended in 0.5% SDS in PBS. Whole cell and plasma membrane

proteins were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride

2

membranes using a standard western blotting protocol. Membranes were probed using a

rabbit anti-β3 integrin antibody (Cell Signalling Technology, Danvers, Massachusetts, USA;

1:1000). Rabbit anti-actin antibody (Sigma-Aldrich Co. Ltd, Poole, Dorset, UK; 1:5000) was

used as loading control and a peroxidase-conjugated donkey anti-rabbit IgG antibody (Santa

Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:2000) as the secondary antibody.

Proteins were visualized using the Amersham ECL kit (GE Healthcare, Chalfont St Giles,

Bucks, UK). Blots were scanned (Bio-Rad GS-800 Calibrated Densitometer; Bio-Rad,

Hercules, CA, USA) and signal quantification was performed by densitometry using scanning

analysis software (Quantity One; Bio-Rad).

3) Radiochemistry

In this study we radiolabeled DOTA-cyclic RGDfK dimer (DOTA-[c(RGDfK)]2), with

68GaCl3 eluted from a TiO2-based 370 MBq

68Ge/

68Ga generator (Eckert & Ziegler Isotope

Products IGG100, Berlin, Germany) using 0.1 M HCl (Sigma-Aldrich). 10ml of acid was

used to elute the generator, with the first 1.5ml bolus containing the majority of the activity

collected and used for labelling. DOTA-[c(RGDfK)] dimer acetate was purchased from ABX

advanced biochemical compounds GmbH (Radeberg, Germany). 30µl (1mg/ml H2O) of

DOTA-[c(RGDfK)]2 dimer acetate was added to 120µl sodium acetate (160mg/ml) in a

reactor vessel. Then, 1.5 ml of 68

GaCl3 (315-370 MBq) was added. The reaction was heated at

100oC for 420 seconds and subsequently trapped on tC18 cartridge (pre-conditioned with 2ml

ethanol and then 2ml water). The cartridge was washed with water (5 ml), before the labelled

compound was eluted (in 100 µl fractions) with 25% ethanol in sterile PBS (v/v).

3

4) In vitro cell uptake of 68

Ga-DOTA-[c(RGDfK)]2

Cells (3 x 105) were plated into 6-well plates the night prior to analysis. On the day of the

experiment, fresh growth medium, containing 40 µCi 68

Ga-DOTA-[c(RGDfK)]2, was added

to individual wells. Cell uptake was measured following incubation at 37°C in a humidified

atmosphere of 5% CO2 for 30 minutes. Plates were subsequently placed on ice, washed 3

times with ice-cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific Inc., Rockford,

IL, USA; 0.5 ml, 10 min). For the M21L suspension cell line, the cells were washed by

spinning down in Eppendorf tubes for 3 minutes at 600g. Cell lysates were transferred to

counting tubes and decay-corrected radioactivity was determined on a gamma counter (Cobra

II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK). Aliquots were snap-

frozen and used for protein determination following radioactive decay according to a BCA

96-well plate assay (Thermo Fisher Scientific Inc., Rockford, IL, USA). Data were expressed

as percent of total radioactivity per mg protein. For blocking studies, cells were incubated

with a cold monomer RGD (10mM; Sigma-Aldrich) for 30 minutes prior to addition of

radioactivity and for the duration of the uptake time course.

5) Biodistribution study

For the biodistribution study, mice were maintained under anesthesia following their PET

scan and sacrificed by exsanguination via cardiac puncture at 60 min post radiotracer

injection to obtain blood, plasma, urine, heart, lung, liver, kidney, muscle and tumour. Tissue

radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter, Packard

Biosciences Co, Pangbourne, UK) and decay corrected. Data were expressed as percent

injected dose per gram of tissue.

4

6) In vivo tracer metabolism

Briefly, female BALB/c mice under general anaesthesia (2.5% isofluorane; non-recovery

anaesthesia) were administered a bolus i.v. injection of 68

Ga-DOTA-[c(RGDfK)]2 (~ 3.7

MBq) and sacrificed by exsanguination via cardiac puncture at 30 min post radiotracer

injection. Aliquots of heparinized blood were rapidly centrifuged (14000 g, 5 min, 4oC) to

obtain plasma. Plasma samples were subsequently snap-frozen in liquid nitrogen and kept on

dry ice prior to analysis. Kidney and liver samples were immediately snap-frozen on dry ice.

To process the samples for HPLC analysis, samples were thawed and kept at 4°C

immediately before use. Ice cold methanol (1.5 ml) was added to the ice cold plasma (200 µl)

and the resulting suspension centrifuged (14000 g; 4°C; 3 min). The supernatant was then

decanted and evaporated to dryness on a rotary evaporator (bath temperature, 40°C), then

resuspended in HPLC mobile phase (Solvent A: acetonitrile/water/ethanol/acetic acid/1.0 M

ammonium acetate/0.1 M sodium phosphate [800/127/68/2/3/10]; 1.1 ml). Samples were

filtered through a hydrophilic syringe filter (0.2 µm filter; Millex PTFE filter, Millipore,

MA., USA) and the sample (∼1ml) then injected via a 1 ml sample loop onto the HPLC for

analysis.

Tissues were homogenized in ice-cold methanol (1.5 ml) using an Ultra-Turrax T-25

homogenizer (IKA Werke GmbH and Co. KG, Staufen, Germany) and subsequently treated

as per plasma samples. Samples were analyzed on an Agilent 1100 series HPLC system

(Agilent Technologies, Santa Clara, CA, USA), configured as described above, using the

method of [23]. A Gemini C18 HPLC column (Phenomenex, CA, USA.; 4.6 x 150 mm)

stationary phase and a mobile phase comprising of Solvent A (water/0.1% TFA) and Solvent

B (acetonitrile/0.1% TFA) delivered at a flow rate of 1 ml/min were used for analyte

5

separation. The gradient was set as follows: 95% A for 2 mins; 95% to 5% A in 10 min; 5%

A for 2 mins; 5% to 95% A in 2 min.

7) Calculating microvessel density

Microvessel density (MVD) was assessed from CD31 stained sections using a method

originally described by Weidner et al [21]. Briefly, MVD was measured by microscopic

examination of most vascularized areas of the tumour. Areas of greatest staining were

primarily assessed at a low magnification of x100 (x10 objective lens and x10 ocular lens).

After selection of representative zones, MVD was counted at a higher magnification of x400

(x40 objective lens and x10 ocular lens). A single microvessel was defined as a discrete

cluster of cells positive for CD31 staining, with no requirement for the presence of a lumen.

Mean vessel density represents the mean number of vessels counted on four separate

microscopic fields performed at x400 magnification.

6

Results

Fig. S1. Biodistribution of 68

Ga-DOTA-[c(RGDfK)]2 in M21, M21L and MDA-MB-231

tumour bearing mice 60 minutes post injection of tracer. SI, small intestine; LI, large

intestine. High activity in the urine and kidney is suggestive of renal excretion of the

radiotracer. (Data points represent mean ± SEM, n = 6, *p<0.05, **p<0.01, ***p<0.001).

7

Fig. S2. Representative radio-chromatograms of 68

Ga-DOTA-[c(RGDfK)]2 standard and of

plasma, kidney and liver samples taken from mice 30 minutes post-administration of the

tracer. Graphs depict activity in counts per second (CPS) against time. The dimeric RGD

tracer is metabolically stable in these samples (n=3).

8

Fig. S3. Immunohistochemical staining of β3 integrin expression in two different M21

tumours showing high (a and b) and low (c and d) levels of staining. Total magnification:

x200 (left column, a and c) and x600 (right column, b and d). Scale bar represents 100 µm

and 50 µm at x200 and x600 magnification respectively.

Documents

Radiolabeled RGD Tracer Kinetics Annotates Differential αvβ3 Integrin Expression Linked to Cell Intrinsic and Vessel Expression