Nuclear Medicine and Biology 33 (2006) 963 – 969
www.elsevier.com/locate/nucmedbio

Radioiodinated VEGF to image tumor angiogenesis in aLS180 tumor xenograft model
Mitsuyoshi Yoshimotoa,4, Seigo Kinuyab, Atsuhiro Kawashimac, Ryuichi Nishiid,e,Kunihiko Yokoyamab, Keiichi Kawaia,ebaDivision of Health Sciences, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-0942, Japan
Department of Biotracer Medicine, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-8640, Japanc
Kanazawa Medical Center, Ishikawa 920-8650, Japand
Department of Radiology, Fujimoto Hayasuzu Hospital, Miyazaki 885-0055, Japane
Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193, Japan
Received 28 April 2006; received in revised form 9 August 2006; accepted 10 August 2006

Abstract
Introduction: Angiogenesis is essential for tumor growth or metastasis. A method involving noninvasive detection of angiogenic activity invivo would provide diagnostic information regarding antiangiogenic therapy targeting vascular endothelial cells as well as important insight
into the role of vascular endothelial growth factor (VEGF) and its receptor (flt-1 and KDR) system in tumor biology. We evaluatedradioiodinated VEGF121, which displays high binding affinity for KDR, and VEGF165, which possesses high binding affinity for flt-1 andlow affinity for KDR, as angiogenesis imaging agents using the LS180 tumor xenograft model.
Methods: VEGF121 and VEGF165 were labeled with 125I by the chloramine-T method. Biodistribution was observed in an LS180 humancolon cancer xenograft model. Additionally, autoradiographic imaging and immunohistochemical staining of tumors were performed with
125
I-VEGF121.
Results: 125I-VEGF121 and 125I-VEGF165 exhibited strong, continuous uptake by tumors and the uterus, an organ characterized byangiogenesis. 125I-VEGF121 uptake in tumors was twofold higher than that of 125I-VEGF165 (9.12F98 and 4.79F1.08 %ID/g at 2 h,respectively). 125I-VEGF121 displayed higher tumor to nontumor (T/N) ratios in most normal organs in comparison with 125I-VEGF165. 125IVEGF121 accumulation in tumors decreased with increasing tumor volume. Autoradiographic and immunohistochemical analyses confirmedthat the difference in 125I-VEGF121 tumor accumulation correlated with degree of tumor vascularity.
Conclusion: Radioiodinated VEGF121 is a promising tracer for noninvasive delineation of angiogenesis in vivo.
D 2006 Elsevier Inc. All rights reserved.
Keywords: Tumor imaging; Angiogenesis; VEGF; VEGF receptors

1. Introduction
Tumors require angiogenesis in order to grow beyond thesize of 1–2 mm in diameter, a condition in which nutrientsand oxygen are not adequately supplied via passive
diffusion through existing blood vessels [1]. Furthermore,
the metastatic property of tumors strongly correlates withtumor angiogenesis [2,3]. In particular, the balance between
various activators and inhibitors closely associated withoverexpression of vascular endothelial growth factor(VEGF) and its receptors serves to regulate angiogenesis.
4 Corresponding author. Tel.: +81 76 265 2539; fax: +81 76 234 4366.
E-mail address: myoshi@mhs.mp.kanazawa-u.ac.jp (M. Yoshimoto).
0969-8051/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2006.08.006

It is well known that VEGF plays a pivotal role inangiogenesis — proliferation and migration of endothelial
cells. Five VEGF isoforms, produced by alternative splicingof responsible genes, have been identified. VEGF121 andVEGF165 are the predominant isoforms [4]. Endothelial
cells express two VEGF receptors (VEGFRs): flt-1(K d = 26F6 pM for VEGF165) and KDR (K d = 80F13 pM
for VEGF121, K d = 2070F520 pM for VEGF165) [5]. These
receptors are up-regulated in endothelial cells in tumor
tissue [6,7].
Numerous compounds possessing an antiangiogenic
profile have been investigated [8–10]. Conventional antitumor agents act directly on tumor cells. In contrast, mostantiangiogenic agents primarily target endothelial cells,

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M. Yoshimoto et al. / Nuclear Medicine and Biology 33 (2006) 963 – 969

which enables their application to various types oftumors. Most antiangiogenic agents target the VEGF–
VEGFR system, which inhibits proliferation of endothelial
cells [11–14].
Despite its invasive nature, conventional angiography
has been the standard imaging tool for evaluation of tumor
vascularity. Less invasive approaches such as computed
tomographic angiography and, more recently, three-dimensional magnetic resonance angiography can effectively
demonstrate the vasculature. These modalities, however,merely delineate preexisting vasculature; they do notdisclose the dynamic changes in blood vessels (endothelial
cells) attributable to antiangiogenic therapy. Positron emission tomography and single photon emission computed
tomography may enable lesional characterization in the
management of cancer patients. Positron tracers such as
2-18F-fluoro-2-deoxyglucose (18F-FDG) and 3V-deoxy3V- 18 F-fluorothymidine ( 18 F-FLT) would afford data
corresponding to tumor therapeutic responses. The major
action of antiangiogenic therapy is the induction of
dormancy of tumors, which may alter glycolysis or DNA
synthesis in tumors. However, alternations in these cellularfunctions in antiangiogenic therapy occur secondarily,
following the primary effects of antiangiogenic agents onendothelial cells. So-called perfusion agents, e.g., 201Tl,
99m
Tc-sestamibi and 99mTc-tetrofosmin, would also beinadequate, as factors other than tissue perfusion substantially affect the distribution of these tracers. For instance,
99m
Tc-sestamibi accumulation in brain tumors is independent of tumor vascularization [15]. Neo-angiogenesis intumors is strongly correlated with patient prognosis;
consequently, a method enabling noninvasive estimationof angiogenesis in vivo would surely provide beneficial
clinical information. Furthermore, such information may
permit selection of those patients most likely to benefit fromantiangiogenic therapy as well as facilitate monitoring of
its efficacy.
In order to develop noninvasive, specific techniques for
estimation of tumor vascularity, this investigation utilized
VEGF121 and VEGF165 radiolabeled with 125I. The specificity and binding ability of VEGF121 and VEGF165 toreceptors have been well established with 125I-VEGF121 and
125
I-VEGF165 in biochemical field. These compounds were
evaluated as candidates for angiogenesis imaging using ahuman cancer xenograft model.

25 Ag of chloramine-T in 20 mM phosphate buffer (pH 7.2)
in a final volume of 110 Al. Iodination was allowed toproceed at room temperature for 1 min. The reaction was
terminated upon the addition of 5 Al of sodium metabisulfite(5 mg/ml). 125I-VEGF121 and 125I-VEGF165 were purified
on a gel filtration (Sephadex G-25) column (Amersham
Biosciences, Uppsala, Sweden) and on a Heparin-Sepharosecolumn (Amersham Biosciences), respectively. 125 IVEGF121 and 125I-VEGF165 displayed specific activities of
13.0–18.5 and 7.2–8.0 GBq/mg, respectively.
2.2. Cell culture
LS180 human colorectal adenocarcinoma cell line waspurchased from Dai-Nippon Seiyaku Co. Ltd. (Japan) andgrown in Eagle’s minimal essential medium (Sigma, St.
Louis, MO) containing 2 mM l-glutamine, nonessential
amino acids and 10% FBS. Cells were cultured in a 5%
CO2-humidified atmosphere at 378C.
2.3. Serum stability
Mouse serum (50 Al) was mixed with 18.5 kBq of 125IVEGF121 and 125I-VEGF165, respectively, and was incubated at 378C. After incubation for 0, 1, 2 and 4 h,trichloroacetic acid (TCA) was added to the samples.
Precipitates were filtrated on glass filters, and the filters
were washed with 5% TCA. Radioactivity in precipitates at
0 h was used as control. Percent precipitability of control
was calculated.
2.4. Biodistribution of 125I-VEGF
Animal studies were performed in compliance withguidelines for the care and use of laboratory animals of
Kanazawa University. Biodistribution studies were conducted in nude mice bearing LS180 tumor. Balb/c nu/nu
female mice, 5–6 weeks of age (Japan SLC, Inc., Shizuoka,
Japan), were xenografted subcutaneously in the left thighwith 5106 LS180 cells. LS180 displays considerable
VEGF production and no VEGF receptors; moreover, thisgrowth is suppressed by an anti-VEGF antibody in vivo[17]. Biodistribution was examined at 7, 10 and 14 days after
implantation in order to include various tumor sizes.
Mice were injected via the tail vein with 55.5 kBq/100 Al(saline containing 1% bovine serum albumin) 125I-VEGF121
or 125I-VEGF165. Mice were sacrificed at 0.5, 1, 2, 4 and24 h postinjection, and tissue samples were excised. Tissue
samples were weighed and radioactivity was measured witha g-counter (ARC-1000M, Aloka, Tokyo, Japan). Uptake in

2. Materials and methods
2.1. Radioiodination of VEGF
Radioiodination of human recombinant VEGF121 (R&D
Systems, Minneapolis, MN) and VEGF165 (PeproTech EC,
London, UK) was performed via the chloramine-T method
described previously [5,16]. Briefly, 1.0 Ag of VEGF121 or
VEGF165 was labeled with approximately 18.5 MBq of
Na125I (Amersham Pharmacia, Buckinghamshire, UK) andTable 1
Serum stability of 125I-VEGF121 and 125I-VEGF165 evaluated by TCA
precipitability measurements
Incubation time
125

I-VEGF121
125
I-VEGF165

1h

2h

4h

93.8F2.0
97.3F2.2

92.5F1.2
96.8F0.9

94.9F2.6
97.5F0.7

The stability was expressed as the percent precipitant of control (n = 2–3).

M. Yoshimoto et al. / Nuclear Medicine and Biology 33 (2006) 963 – 969

965

Table 2
Biodistribution of 125I-VEGF121 in LS180 tumor-bearing mice (n = 4)
Organ

Tumor
Blood
Brain
Liver
UterusHeart
Lung
StomachKidneyPancreas
SpleenMuscleBoneIntestine

Time after injection
0.5 h

1h

2h

4h

24 h

5.94F0.41
40.32F2.45
0.88F0.13
7.16F0.62
6.62F2.39
9.36F1.59
18.88F3.11
4.49F0.39
14.91F0.60
1.97F0.20
6.77F0.89
1.27F0.33
3.32F0.66
3.40F0.69

8.43F0.58
38.69F4.34
0.89F0.17
6.86F1.15
11.24F3.70
9.43F1.04
18.54F2.26
7.32F0.94
14.16F0.78
3.27F0.53
6.46F0.34
1.33F0.06
4.19F1.22
3.93F0.74

9.12F0.98
32.03F0.59
0.70F0.08
5.49F0.44
13.76F2.19
8.39F0.41
13.31F2.05
8.81F1.87
11.46F0.78
2.30F0.73
5.90F1.36
2.48F0.86
4.90F1.05
3.84F0.36

9.02F1.59
24.90F2.99
0.44F0.03
5.21F0.57
15.58F4.13
6.48F1.05
14.11F1.62
10.85F2.18
9.21F1.04
2.91F0.15
4.53F0.79
1.74F0.44
2.76F0.18
3.10F0.19

2.55F0.28
7.19F0.57
0.16F0.02
1.44F0.11
3.72F0.88
2.09F0.18
3.56F0.37
4.23F1.22
3.00F0.14
1.17F0.21
1.67F0.11
0.90F0.10
1.12F0.28
1.08F0.13

Radioactivity in organs was expressed as %ID/g tissue (meanFS.D.).
Tumor volume used in this study was less than 200 mm3.

organs was expressed as percent injected dose per gram(%ID/g) of tissue.
2.5. Autoradiography of125

I-VEGF121 in tumor

125

I-VEGF121 displayed higher tumor to nontumor (T/N)
ratio in comparison to 125I-VEGF165; as a result, intratumoral distribution of 125I-VEGF121 was analyzed autoradiographically (ARG). Mice were injected via the tailvein with 740–999 kBq/100 Al. Tumors were excised frommice at 4 h postinjection. Specimens were embedded in
OCT compound (Sakura Finetechnical Co. Ltd., Tokyo,
Japan) and frozen in dry ice–acetone. Frozen sections
were cut to 20-Am thickness with a cryostat and mounted
on glass slides. Glass slides were placed on an imaging
plate (BAS-SR 2025, Fuji Photo Film Co. Ltd., Tokyo,
Japan); subsequently, the exposed plate was scanned witha bio-imaging analyzer (BAS-5000, Fuji Photo Film) to
detect radioactivity.

2.6. Immunohistochemistry by CD34 in tumors
Frozen sections adjacent to the ARG specimens weresliced to 5-Am thickness and mounted on silanized slides.
The sections were air-dried and fixed in cold acetone for5 min. Subsequently, sections were rinsed in PBS to remove
the OCT compound. Endogenous peroxidase activity was
blocked with 0.3% H2O2, and nonspecific binding was
blocked with 10% normal rabbit serum. After washing in
PBS, rat antimouse CD34 monoclonal antibody (BDpharmingen, La Jolla, CA), diluted 1:20, was applied tothe sections and incubated at room temperature (RT) for
30 min. Next, after a PBS wash, biotinylated rabbit antirat
IgG antibody (Vector Laboratory, Burlingame, CA), diluted
1:200, was applied to the sections and incubated at RT for
30 min. After washing again in PBS, streptavidine (DakoCytomation, Glostrup, Denmark), diluted 1:300, was applied
to the sections, incubated at RT for 30 min, and washed again
in PBS. The sections were then developed with diamino-

Table 3
Biodistribution of 125I-VEGF165 in LS180 tumor-bearing mice (n = 3–5)
Organ

Tumor
Blood
Brain
Liver
UterusHeart
Lung
StomachKidneyPancreas
SpleenMuscleBoneIntestine

Time after injection
0.5 h

1h

2h

4h

24 h

2.06F0.40
1.88F0.25
3.17F0.22
16.15F2.05
6.24F0.47
14.36F0.44
31.30F7.56
12.65F1.34
25.19F1.37
10.07F1.30
17.80F2.39
4.68F2.78
2.55F0.10
4.13F0.16

3.09F0.72
3.33F0.46
2.53F0.14
7.71F0.23
4.93F1.02
9.93F1.18
20.32F2.95
17.96F6.90
18.64F1.35
9.43F1.16
9.00F1.62
3.71F1.31
2.57F0.35
3.87F0.74

4.79F1.08
5.15F0.55
1.88F0.51
5.72F0.95
5.24F0.95
6.59F0.95
15.71F1.53
32.79F10.06
12.45F1.71
6.82F0.68
6.78F1.75
3.91F0.76
2.28F0.17
4.15F0.36

1.84F0.08
2.48F0.45
0.76F0.06
4.26F0.05
2.56F0.40
2.96F0.48
7.84F1.68
23.93F8.71
8.36F0.46
3.68F0.56
4.14F0.46
1.66F0.08
1.36F0.29
2.53F0.51

0.17F0.03
0.61F0.52
0.31F0.01
2.58F0.31
0.63F0.30
1.31F0.11
4.12F0.37
1.11F0.19
6.67F1.38
1.00F0.13
2.53F0.54
0.50F0.04
0.25F0.05
0.43F0.05

Radioactivity in organs was expressed as %ID/g tissue (meanFS.D.).
Tumor volume used in this study was less than 220 mm3.

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M. Yoshimoto et al. / Nuclear Medicine and Biology 33 (2006) 963 – 969

benzidine (DakoCytomation) and counterstained with hematoxylin (Merck, Darmstadt, Germany). Additional slices
were also examined using hematoxylin–eosin staining.
3. Results
3.1. Serum stability
Stability study showed no decrease in precipitability of
I-VEGF121 and 125I-VEGF165 in mouse serum (Table 1).
In serum, no obvious deiodination of 125I-VEGF121 and
125
I-VEGF165 was found.
125

3.2. Biodistribution and tumor accumulation of 125I-VEGF
Tables 2 and 3 summarize the results of biodistribution
studies of 125I-VEGF121 and 125I-VEGF165 in LS180bearing mice, respectively. Accumulation of 125I-VEGF121
in tumors gradually increased with time, reaching a peak
(9.12 %ID/g) at 2 h postinjection; subsequently, 125IVEGF121 accumulation remained unchanged through 4 hpostinjection. The uterus demonstrated a 125I-VEGF121 accumulation pattern similar to that of tumors.
125
I-VEGF165 exhibited higher accumulation in normal
tissues, with the exception of blood, than 125I-VEGF121;

Fig. 1. Tumor to nontumor ratios in LS180 tumor-bearing mice at 0.5, 1, 2,
4 and 24 h after injection of 125I-VEGF121 (A) and 125I-VEGF165 (B).
Columns, mean; bars, SD (n = 3–5).

Fig. 2. Relationship between 125I-VEGF121 accumulation and tumor
volume. Data correspond to 125I-VEGF121 accumulation at 4 h postinjection.
LS180-xenografted mice were examined at 7, 10 and 17 days after
implantation. Tumor volume was calculated as p/6(r 1r 2r 3); (r 1:length; r 2: width; r 3: depth).

however, 125I-VEGF165 was cleared more rapidly than 125IVEGF121, particularly in liver, heart, lung, kidney andspleen. 125I-VEGF121 accumulation in tumors was two- andfivefold greater than that of 125I-VEGF165 at 2 and 4 hpostinjection, respectively.

Fig. 3. Autoradiography image of intratumoral distribution at 4 h postinjection of 125I-VEGF121. Images were obtained from LS180 tumor tissues
displaying volumes of (A) 101.9 mm3 and (B) 361.9 mm3. Tissue slice
photos (C) and (D) correspond to ARG images (A) and (B), respectively (T:tumor; M: muscle).

M. Yoshimoto et al. / Nuclear Medicine and Biology 33 (2006) 963 – 969

967

125

high localization of I-VEGF121 was evident in portions of
the muscle area in large tumors (361.9 mm3). Fibrosis was
induced at regions of high 125I-VEGF121 localization intumoral area. Homogeneous accumulation of 125I-VEGF121
was observed in the tumor area, with the exception ofareas of fibrosis.
Fig. 4A and B indicates microvessel staining of tumor
tissues by CD34 in Fig. 3A and B, respectively. As shown in
Fig. 4A, abundant vasculature was observed in small
tumors. On the other hand, poor vasculature was apparent
in large tumors (Fig. 4B).
4. Discussion

Fig. 4. Vascular staining with an anti-CD34 mAb in LS180 tumor. Images(A) and (B) are representative areas of Fig. 3A and B, respectively. Bar
indicates 100 ı̀m in (A).

Fig. 1A and B displays T/N ratios of 125I-VEGF121 and
I-VEGF165, respectively. Tumor-to-nontumor ratios of125
I-VEGF121 were markedly higher than those of 125IVEGF165, with the exception of blood. Tumor-to-blood ratioof 125I-VEGF121 increased to 4 h postinjection, although theratio was strikingly low. In contrast, the tumor-to-blood ratioof 125I-VEGF165 was maximal at 30 min; subsequently, it
decreased with time.
125

3.3. Relationship between tumor volume and 125I-VEGF121
accumulation in tumors
Fig. 2 illustrates the relationship between tumor volume(mm3) and accumulation in tumors. 125I-VEGF121 exhibiteda tendency toward diminishing accumulation in tumor withincreasing tumor volume.
3.4. Autoradiography and histopathological findings
Fig. 3 displays intratumoral distribution of 125I-VEGF121.
I-VEGF121 demonstrated overall high localization insmall tumors (101.9 mm3) (Fig. 3A). As shown in Fig. 3B,

125

Neo-angiogenesis is a common characteristic of all
proliferating and metastatic tumors. Development of novel
drugs possessing anti-angiogenic profile is a major area ofinterest in clinical oncology. In vivo detection of angiogenesis clearly affords valuable information in terms ofmonitoring the efficacy of anti-angiogenic therapy. The
VEGF–VEGFR system would be the most suitable imaging
target for this purpose as this system plays a key role inangiogenesis in tumors. Positron-labeled monoclonal antibody (VG76e) that binds human VEGF has been developedin order to measure VEGF expression in tumor tissues [18].
However, substances for imaging VEGFR density would be
required to monitor therapeutic responses to anti-angiogenic
agents given that most agents act as inhibitors of VEGFR.
Our findings provide a rationale for VEGFR imaging intumors utilizing radiolabeled VEGF.
125
I-VEGF121 and 125I-VEGF165 displayed similarities
and differences in terms of in vivo behavior in tumorxenografted mice. High initial uptake was observed in thelung and heart, probably due to abundant vascularity inthese organs. Based on the molecular sizes, 125I-VEGF121
and 125I-VEGF165 were filtered by the kidney, resulting inearly large accumulation in that tissue. In addition, radioiodinated metabolites may also contribute to the accumulation in kidney. In contrast, prominent 125I-VEGF165
accumulation in the stomach in comparison to 125I-VEGF121
suggests that the former was more prone to deiodination
than the latter. Furthermore, differences in tissue distribution
between these two compounds were observed in various
other organs, including the brain and spleen, which was
likely attributable to the difference corresponding to bindingsites recognized by each tracer: VEGF121 binds only to
KDR with high affinity [5,19], whereas VEGF165 binds toflt-1 with higher affinity but possesses lower affinity for
KDR [5,20]. KDR mRNA is primarily expressed in thekidney, heart, spleen and lung [21], whereas flt-1 mRNA isexpressed in the brain, heart, liver, kidney and lung [22]. In
kidney, heart and lung, which express both VEGFRs, 125IVEGF165 uptake was greater than that of 125I-VEGF121.
Although the precise reasons regarding the different
accumulation in these organs remain unclear, previous
reports have suggested a possible mechanism for this968

M. Yoshimoto et al. / Nuclear Medicine and Biology 33 (2006) 963 – 969

phenomenon: the difference in the expression between flt-1
and KDR on endothelial cells in adult tissues. That is,endothelial cells in adult tissues express flt-1 mRNA in amanner similar to those in embryonic tissues [23], whereas
KDR is down-regulated on endothelial cells in adult tissues
relative to corresponding cells in fetal tissues [24]. Thesefindings indicate that VEGF121 would be the more
appropriate targeting molecule of VEGFRs in terms oflow background activities.
The superiority of 125I-VEGF121 was also suggested byits greater tumor accumulation as compared to 125IVEGF165. We cannot exclude the possibility that theradioactivity in blood contributes to the radioactivity intumor especially at early time points. However, the factthat tumor-to-blood ratios gradually increased through 4 haffords evidence of specific binding to KDR. Similarity of
uptake profile of 125I-VEGF121 in tumor to that of the uterus
where angiogenesis occurs physiologically also indicated its
specific uptake by the receptors. Therefore, the difference in
expression amount and/or binding affinity between flt-1 andKDR is an essential factor with respect to determination oftargeting level in tumors between 125I-VEGF121 and 125IVEGF165. In addition, the up-regulation of VEGFRs may
affect the binding affinity of 125I-VEGF121 to flt-1 [25],resulting in high accumulation of 125I-VEGF121 in tumors.
In the clinical setting, 123I-VEGF165 scintigraphy has
successfully delineated gastrointestinal tumors and theirmetastases in lung and liver despite the high background
activity in these organs [26]. Our present results suggest that123
I-VEGF121 would lead to improved detection ability interms of abdominal lesions relative to 123I-VEGF165 consequent to higher T/N ratios expected with 125I-VEGF121.
125
I-VEGF121 uptake is greater in tumors characterized
by abundant vascularity than in tumors displaying poor
vascularity. Greater expression of KDR in metastatic tumors
with larger vessel counts in comparison to nonmetastatic
tumors with smaller vessel counts has been documented[27]. Although we could not accurately measure microvessel density due to the well-developed nature of thesinusoidal vascular network in this experimental tumor
model, this previous report supports our findings.
Microvessel density is correlated with invasion andmetastasis, which suggests that this parameter may serve
as a powerful prognostic marker [2,3]. In addition,
antiangiogenic therapy leads to diminished microvessel
density [11,28]. Therefore, radioiodinated VEGF may be
useful for tumor diagnosis and monitoring of anti-angiogenic therapy.
Angiostatin and endostatin are endogenous angiogenesis
inhibitors and are specific inhibitors of endothelial cellproliferation. The potential of 123/125I-angiostatin and 99mTcEC-endostatin as markers for evaluating angiogenic activity
has been demonstrated [29,30]. tumor-to-nontumor ratios
for liver and muscle, which are comparable, are even higher
for 125I-VEGF121 in comparison with these compounds: theratio of tumor to liver and muscle is 1.74 and 5.49 for 125I-

VEGF121, 0.69 and 3.54 for 123/125I-angiostatin and 0.16
and 6.1 for 99mTc-EC-endostatin, respectively. Moreover,125
I-VEGF121 produced a higher tumor-to-intestine ratio of
2.92 relative to that of 123/125I-angiostatin and 99mTc-ECendostatin. These findings are suggestive of the feasibilityof radioiodinated VEGF121 as a targeting agent.
A possible critical problem associated with radioiodinated VEGF121 involves prolonged elevated radioactivity inthe circulation. A plausible explanation for this phenomenon
corresponds to the binding of VEGF121 to endothelial
progenitor cells and/or KDR-positive cells in the circulation.
Endothelial progenitor cells that express KDR originate in
the bone marrow and circulate to tissues where these cells
contribute to angiogenesis [31,32]. This obstacle may beovercome by co-injection of unlabeled VEGF121, which
would suppress binding of radioiodinated VEGF121 to
KDR, leading to increased availability of free radioiodinated
VEGF121; thus, accumulation of radioiodinated VEGF121 intumors would be enhanced in a manner similar to thatobserved with 125I-insuline-like growth factor (IGF)-I
binding to IGF-binding proteins [33].

5. Conclusion
Diagnosis of angiogenic activity in tumors provides
beneficial information concerning the efficacy of antiangiogenic therapy and risk of distant metastasis. In thisreport, we confirmed that radioiodinated VEGF121 is apromising tracer for this purpose. In terms of VEGFRs,
KDR is a superior target in comparison to flt-1.

Acknowledgment
This study was supported by a Grant-in-Aid for Young
Scientists (B) [KAKENHI (15790657)] from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.

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