In Vivo MicroCT Imaging Characteristics of a Long-Acting
Blood-Pool Agent in Normal and Tumor-Bearing Mice
GN Ton1,2, BY Durkee1,MA Longino1, JP Weichert1
1University of Wisconsin-Madison,WI and 2Alerion Biomedical Inc., San Diego, CA

Results and Discussion

High-resolution microCT systems capable of sub 20-micron isotropic spatial
resolution provide an attractive new approach for noninvasively studying models ofhuman diseases in live small animals.1,2 However, relatively long acquisition times
preclude the use of conventional water-soluble contrast media for imaging
pathological conditions associated with vascular abnormalities. Utilizing the abilityof PEG chains to modify surfaces of biological macromolecules, we have developed
a long-acting blood-pool contrast agent (BP) based on our hepatocyte-selective CT
contrast agent platform for vascular microCT imaging applications.3,4Macromolecular structures of BP and ITG particles are shown in Fig 1.
Incorporation of PEG moieties into the phospholipid monolayer shell of the ITG
vehicle interferes with the association of Apo-E, which is believed to be essential forhepatocyte recognition. Delaying the sequestration of ITG by liver cells results inprolonged vascular residence time. The liver (ITG) and vascular (BP)contrast
agents are now known as FenestraTM LC and VC, respectively.
The overall goal of thisproject is to evaluate the invivo imaging efficacy ofFenestra
VC
asamacromolecular blood poolselective and microCT
compatible contrast agent topotentially monitor tumor
angiogenesis in tumorbearing mice. In addition,
tissue intensity-time profilesof the agent were examined
in normal mice for directcomparison.

LCVC

Normalized Intensity (HU/Voxel)

BCDLCoronalview

Axialview

TT

T

Poor soft tissue contrast is clearly shown in the non-contrast coronal image (A).
The superior quality of vascular contrast enhancement achieved after 10 min post
injection of Fenestra VC is readily apparent in Fig 4(B-D).

AFig 2. Normalized Vascular Intensity-Time Profilesof Fenestra VC in normal and C51-bearing mice
IVC_Balb/c
IVC_Balb/c-C51

600

400200020A

Contrast enhanced at t=10 minFig 1. Structure of Fenestra LC and VC

The blood-pool contrast agent (Fenestra VC) was obtained from ART
Advanced Research Technologies Inc. (Montreal, Canada) . The finale iodine
concentration and mean particle size of this lipid emulsion were 50 mg I/mL and 180nm, respectively. Mice were placed on a soft diet at least 1 day prior to the initiationof the study to minimize streak artifacts produced by rodent chow. Fenestra VC wasadministered as a single IV (tail vein) bolus dose (120 mL/kg bw) into normal ortumor-bearing mice. Anesthetized mice were scanned using a Siemens microCAT IIsystem (70 kVp, 500 µA, 200 msec, 720 steps) prior to and within 10 minutes ofinjection.
Images were reconstructed (Feldkamp filtered back projection algorithms inreal-time) and subsequently displayed and analyzed using Amira 3-D visualization
software (V3.1). CT values of volumetric ROIs in the inferior vena cava (IVC) andliver were normalized to Hounsfield Units (HU). Normalized signal intensity(HU/voxel) was obtained from the difference of enhanced SI and the baseline value.

10Non-contrast

Fig 5. Fenestra VC-enhanced microCT high density surface-rendered 3-Dimages obtained in an anesthetized Balb/c mouse bearing C51 xenograft
following a single iv dose of 20 mL/kg.

Materials and Methods

0Fig 4. Fenestra VC-enhanced microCT vascular images obtained following iv
injection (20 mL/kg) into an anesthetized Balb/c mouse bearing a colon
carcinoma C51 xenograft.

30

40

Post Injection Time (Hours)

50

Imaging data fromboth normal and C51 tumorbearing mice exhibitedcomparable vascular contrast
enhancement profiles (Fig2). Signal intensities in theIVC remained above 400
HU after 4 hours. Unlike
water-soluble contrast agents
which rapidly diffuse into
extravascular spaces, theblood-pool contrast agent
remained in the circulation
for several hours, affording
susbtained vascular contrast
enhancement.

IVCAATTV

B

13.12 mm

Combined 3-D surface renderingand coronal slice image display
excellent abdominal vascular rendering
including the inferior vena cava (IVC),abdominal aorta (AA), hepatic vessels
as well as tumor (T) feeder vessels(TV). Fenestra VC-induced contrast
enhancement (Fig 5A) permitted thequantitative characterization of thetumor vascular network. Enlarged andwell-developed extratumoral feeder
vessels are evident as a direct result ofangiogenesis. The diameters of themajor tumor feeder vessels shown in
Fig 5A could be measured accurately
and were in the range of 0.60-0.79 mm.
It should be noted that the image was
acquired at 62x62x73 µm3 voxel
resolution. Thus Fenestra VC is clearly
capable of providing superior anatomic
vascular imaging at or in excess of thelimits of spatial resolution of the
microCT II. A microCT segmentation
image of the same mouse ( shown in
Fig 5A) allows tumor volume to bemeasured accurately in real time,
providing substantial flexibility tomonitor tumor development as well as
response to therapy. The exceptional
tolerance profile of Fenestra VC,
permits the administration of a second
dose (20 mL/kg bw) within 2 weeks tononinvasively monitor progression oftumor growth with respect to tumor
volumeandtumor
vascularcharacterization in the same mouse.
MicroCT images obtained 10 min
following injection of the second doseis shown in Fig 6.

Liver uptake, as measured
by an increase in the signal
intensity, was faster in C51bearing mice than in normal
Balb/c mice at 24 hours post
injection (Fig3). Kinetic
profiles of the two groups
clearly demonstrated a direct
correlation between the blood
clearance and liver uptake ofFenestra VC. The contrast
agent was well tolerated in thetested mice when given as asingle iv injection of 20 mL/kg
bw, followed by microCT
imaging at various timeintervals
accordingthespecified protocol.

Fig 3. Normalized Liver Intensity-Time Profilesof Fenestra VC in normal and C51-bearing mice
Normalized Intensity (HU/Voxel)

Introduction

500
Liver SI_Balb/cLiver SI_Balb/c-C51

400300200100001020304050

Post Injection Time (Hours)

Fig 6. Fenestra VC-enhanced microCT high density surface-rendered 3-Dimages obtained from the same mouse (shown in Fig 5) 10 min following a
2nd iv injection of VC (20 mL/kg) 2 weeks after the 1st dose.
BAIVC

18.16 mm

TVCombined 3-D surface rendering and coronal slice demonstrate the major
tumor feeder vessels (TV) visible in Fig 6. grew significantly longer and larger
in diameter in comparison to the measurements obtained following the 1st
injection of Fenestra VC. Tumor size increased substantially within the 2-weekperiod. Utilizing the superior vascular enhancement capacity of Fenestra VC inconjunction with microCT, quantitative characterization of tumor progession
associated with angiogenesis in the same tumor-bearing mouse was possible.

Conclusions
Owing to its macromolecular nature, Fenestra VC offers the capability ofproviding superior vascular imaging which may prove extremely useful forassessing progression of tumor growth and monitoring tumor response to therapy atthe vascular level including anti-angiogenic agents.

References[1] Paulus MJ, et. al, Neoplasisa (2000) 2:62-70.
[2] Paulus MJ, et. al, Lab Animals (2001) 30:1-10.
[3] Weichert JP, et.al, Academic Radiol. (1998) 5:S16-19.
[4] Weichert JP, et.al, Radiology, (2000) 216:865-871.
Supported in part by the UWCCC, UW-Radiology, and Alerion Biomedical Inc.
gn.ton@hosp.wisc.edu, Poster #283