Early Detection of Liver Tumors in Mice using MicroCT
and a Hepatocyte-selective Contrast Agent
Jamey Weichert,1 Sharon Weber,2 Chen Qi,2 Ben Durkee,1 Jude Longo,1 Fred Lee, Jr1

University of Wisconsin Departments of Radiology1 and Surgery2Introduction
A major impediment in cancer research is theinability to accurately detect and monitor tumor growth inmouse models. Due to the recent advances in genetic
manipulation, these models have become increasinglyimportant. MicroCT scanners1,2 capable of sub 50-micron
spatial resolutions have recently been introduced thatafford potential to more effectively monitor tumor growthand development in small animal models. Relatively long
image acquisition times, however, preclude the use ofconventional water-soluble contrast agents for tumordetection. The goal of this report is to evaluate ITG, a new
hepatocyte-selective CT contrast agent that resides in theliver for several hours, for its ability to detect very small
tumors in mice.
Targeting the Apo-E Receptor
Our strategy for the site-specific delivery of imaging
agents to the liver is based on a biochemical approach,whereby naturally occurring compounds known to be
stored or metabolized in the liver server as carriers for theradiologic moiety. Once absorbed by the intestinal tract,
triglycerides are are incorporated into chylomicrons which
are subsequently secreted into and transported through thethoracic duct until they reach the circulation. Following a
rapid transfer of surface bound apoprotein C-II fromcirculating HDL to the chylomicrons (Fig 1)chylomicrons, now associated with apo C-II, are thenacted upon by lipoprotein lipase in peripheral tissues
including adipose, muscle and lung. After the naturaltriglyceride is removed from the core of the chylomicron
the resulting triglyceride depleted chylomicron, know as achylomicron remnant, rapidly acquires apo-E from theplasma.
Once associated with apo-E, the small
chylomicron remnants (<200 nm) enter the space of Disseand gain access to hepatocytes. Once associated withsurface bound Apo-E receptors on hepatocytes, theremnants undergo endocytosis thought to be mediated byhepatic triglyceride lipase. This process is quite efficientin humans as the plasma half-life for clearance ofchylomicrons and remnants following a meal is about 15
minutes. Humans are capable of processing up to 100
grams of triglyceride in this manner daily. Hepatocytetargeted delivery based on the natural chylomicronremnant/Apo-E receptor uptake pathway thus represents
an attractive biomimetic mechanism for delivering
lipophilic agents to the liver. Polyiodinated triglycerides
were viewed as appropriate liver-specific carrier
molecules for the design and synthesis of thesehepatocyte-selective CT imaging agents.
Project Aim. The goal of this project was to evaluate theefficacy of a new hepatocyte-selective CT contrast agent,
1,3-bis [7-(3-amino-2,4,6-triiodophenyl) heptanoyl]-2oleoyl-glycerol (ITG, Fig. 2) as a liver-selective, andmicroCT compatible, contrast agent for the early detectionof liver tumors in both a xenograft and a transgenic mousemodel.

AB
48

Chylomicron

LPL

C
E
B,E-receptor*Weichert email address: jpweichert@facstaff.wisc.edu

ECCMR

A

C

Capillaryendothelium

HDL
Hepatocyte

Results and Discussion

Fatty acid-albumincomplexes

Fig 1. Chylomicron Remnant Delivery SystemNH2

Fig 2: ITG StructureIIOO
CH3(CH2)7CH=CH(CH2)7CO

FattyAcids

TGFα is a mitogen for epithelial cells and binds to theEGF receptor; unregulated expression of TGFα results intumor formation. In male CD1 mice expressing thetransgene TGFα under the control of the zinc-inducible
metallothionine 1 (MT1) promoter, 75-80% develop HCC
after 12 months of age. However, when the alkylating
agent diethylnitrosamine (DEN), a chemical carcinogen,is used to induce tumor growth at 15 days of life, 90% ofmice develop HCC by 6 months of age. On histologic
examination, these tumors consist of well differentiatedhepatocellular carcinomas of a solid pattern. Because thetumors arise spontaneously, we utilize these animals as asuitable model for preclinical studies.

OC ( )
6OC ( )6OIIIINH2Materials and Methods
ITG (Fig 2) was synthesized and formulated into a
synthetic chylomicron remnant microemulsion according
to our standard methods.3,4 The final ITG preparation
was tested consisting of 20% total lipid and administeredas a single IV (tail vein) bolus dose (0.2 ml/20g bw) intoBalb/C mice bearing CT-26 colon adenocarcinoma(5x105 cells/50µl) xenograft implants in the liver oralternatively into transgenic TGFα hepatoma bearing
mice. Anesthetized mice were scanned 4 hours post
injection using an ImTek microCT scanner (43 KVP,410 µA, 390 steps) prior to and at three-day intervals for
up to 21 days following tumor cell implantation in thexenograftmodel.
Imageswerereconstructed(256x256x256 voxels/Shepp-Logan filter/no beamhardening correction/back projection over an appropriate
sub volume) and subsequently displayed and analyzedusing Amira 3-D visualization software (V2.3). Uponcompletion of final scanning, animals were sacrificed
and radiologic-pathologic correlation performed.
Transgenic Mouse HCC Model. The development of
spontaneous hepatocellular cancer in transgenic miceover expressing the TGFα gene has been extensivelyevaluated and is an extremely promising animal model
for study of this disease.5

Although image quality is excellent for high contrast
structures like bone, differentiation of low contrast soft
tissue structures is difficult with non contrast-enhanced
microCT. Long-acting organ-selective contrast agents
however, will improve detection of tumors in these organs
significantly.
Liver uptake of ITG in mice followed a delayed
pharmacodynamic pattern relative to other animal models
examined. In mice, optimal liver enhancement occurs
around 3-4 hours, whereas in other species including rat
and dog optimal opacification occurs around 45-60
minutes following injection.
Utilizing ITG, a hepatocyte-selective contrast agent
for microCT, it has been possible to consistently detectand accurately localize liver tumors less than 300 microns
in diameter in both xenograft and spontaneous transgenic
mouse models (Figs 3-5). Using this approach we havebeen able to accurately monitor tumor growth and it is also
likely that it will be possible to monitor tumor regression
in response to therapy. Moreover, utilizing this approach
we have recently performed microCT- guided small needle
biopsies of liver tumors in live mice thus enabling thepotential to follow tumor histology serially over extended
periods of time.

Ax178

Sag 88TTCor 118

Cor 95TT

Fig 4. MicroCT images obtained in a transgenic hepatomabearing mouse 4 hours post ITG administration. Axial, sagital,and coronal views presented. Note viable hepatocytes adjacent tohepatoma cells in Cor 95 and 118. (Tumor = T).

Fig 5. ITG-enhanced microCT 3D image of an anesthetized HCCbearing TGFαα mouse showing the segmentally rendered hepatoma within
the axial slice and it relative location within the surface renderedskeleton.

Conclusions
This is the first time ITG or any CT contrast agent(to our knowledge) has been evaluated in live mouse liver
tumor models. ITG, a hepatocyte-selective CT contrast
agent afforded superior tumor edge detection capability.
Due to its biomimetic nature, we feel that ITG offers thecapability of providing not only superior anatomiccapability, but also functional information capable ofnearing or exceeding the spatial resolution of the CT
scanners themselves.

References
Fig 3. ITG-enhanced microCT image of CT-26 tumor implants in [1] Paulus MJ, et al., Lab Animal (2001) 30:36-45.
mouse liver 6 days post inoculation. Tumors (arrows) are clearly[2] Paulus MJ, et al., Neoplasia (2000) 2:62-70.
visible as low attenuation areas in both the coronal (left) and axial[3] Weichert JP, et al., Radiology (2000) 216:865-871.
(right) views.

[4] Weichert JP, et al., J Med Chem (1995) 38:636-646.
[5] Lee GH, et al., Cancer Research (1992) 52:5162-5170.