European Radiology
© Springer-Verlag 2004
10.1007/s00330-004-2517-8
В исследовании, проведенном немецкими специалистами, сравнивается
эффективность метода трансартериальной химиоэмболизации в качестве монотерапии и
эффективность его же в сочетании с другими видами терапии. Исследование проводилось
на 50 крысах, которым была субкапсулярно привита гепатома Морриса, после чего
животные были разделены на несколько групп по видам терапии опухоли.
Теги: Печень - Гепатоцеллюлярная карцинома - трансартериальная химиоэмболизация –
Германия – 2004.
Experimental
Transarterial chemoembolization alone and in
combination with other therapies: a study in an
animal HCC model
A. Maataoui1 , J. Qian1, D. Vossoughi1, M. F. Khan1, E. Oppermann2, W. O. Bechstein2
and T. J. Vogl1
(1) Institute for Diagnostic and Interventional Radiology, Johann Wolfgang Goethe University,
Frankfurt/Main, Germany
(2) Department of Visceral Surgery, Johann Wolfgang Goethe University, Frankfurt/Main, Germany
A. Maataoui
Email: adel.maataoui@gmx.de
Phone: +49-69-63017277
Fax: +49-69-63017258
Received: 26 May 2004 Revised: 27 August 2004 Accepted: 2 September 2004 Published online:
4 December 2004
Abstract The purpose of this study is to compare transarterial chemoembolization (TACE)
alone and in combination with other therapies in an animal model. Subcapsular implantation of a
solid Morris hepatoma 3924A in the liver was carried out in 50 male ACI rats (day 0). Tumor
volume (V1) was measured by MRI (day 13). After laparotomy and retrograde placement of a
catheter into the gastroduodenal artery (day 14), the following protocols of the interventional
procedure were applied: TACE (mitomycin C + lipiodol) + immunotherapy (group A: TNF +
IL-2, group B: OK-432 + IL-2); TACE + antiangiogenesis therapy (group C: TNP-470, group D:
endostatin); TACE alone in group E (control group). Tumor volume (V2) was assessed by MRI
and the mean ratio of x (V2/V1) was calculated. Data were analyzed using Dunnett s t test
(comparing therapeutic groups with the control group) and the Student-Newman-Keuls test
(comparing significant therapeutic groups). Multivariate analysis showed a significant reduction
in the tumor growth rate (P<0.05) in groups B (x=6.53) and C (x=4.01) compared to the mean
ratio of the control group E (x=9.14). Significant results were observed in group C (P<0.05) in
comparison with the other therapeutic groups. TACE combined with immunotherapy (OK-432)
and antiangiogenesis therapy (TNP-470) retards tumor growth compared with TACE alone in an
HCC animal model.
Keywords Liver - Hepatocellular carcinoma - Transarterial chemoembolization
The first two authors contributed equally to this publication.
Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide,
responsible for an estimated 1 million deaths annually and carrying a poor prognosis due to its
rapid infiltrating growth and complicating liver cirrhosis [1].
To date, surgical approaches including liver resection and liver transplantation are regarded as
potentially curative treatments for HCC, particularly in patients with small and non-invasive
tumors [2]. However, only a small minority of patients are suitable for surgical therapy due to
multicentric tumors, extrahepatic metastases, early vascular invasion, shortage of donor organs,
high complication rates and comorbidities [3–5].
Local methods of tumor ablation, which include transarterial chemoembolization (TACE),
percutaneous ethanol injection (PEI), radiofrequency ablation (RFA) [6–8], microwave
coagulation therapy (MCT) and laser-induced thermotherapy (LITT), are promising additional
tumor therapies, especially in patients with limited liver function and unresectable or multifocal
tumors [9]. Since TACE was introduced as a palliative treatment in patients with unresectable
HCC, it has become one of the most common forms of interventional therapies [10–12]. TACE
has been shown to reduce systemic toxicity, increase local effects and thus improve the
therapeutic results [11]. Its therapeutic effect is limited by the lack of appropriate and reliable
embolic agents in those patients when the tumor is infiltrative in nature or hypovascular [13].
In the past years, locoregional immunotherapy and antiangiogenesis therapy for treating
unresectable HCC have been reported with encouraging results, especially for inhibiting
intrahepatic metastases and the recurrence of HCC [14–16]. Such adjuvant treatments in
conjunction with TACE have the potential to enhance the therapeutic effect of TACE alone.
However, there have been few randomized experimental studies to assess the value of these
combined therapies in an animal model of HCC up to now. Thus, the purpose of our study was to
compare TACE alone and in combination with other therapies in an animal HCC model.
Material and methods
Animal model
Morris hepatoma 3924A, a rapidly growing, poorly differentiated hepatocellular carcinoma, was
used in ACI rats in this study. The hepatoma specimen was obtained from the German Cancer
Research Center (DKFZ; Heidelberg, Germany).
Fifty inbred male ACI rats (Harlan Winkelmann; Borchen, Germany) weighing 220–260 g were
used. The animals were kept under conventional conditions at a temperature of 22±2°C, a
relative humidity of 55±10% and a dark/light rhythm of 12 h, and they were fed standard
laboratory chow and tap water ad libitum. All of the experiments on animals were approved by
the German government and the institutional animal research review board.
Agents
Locoregional immunotherapeutic agents
TNF (tumor necrosis factor) is known to have various biological activities as a main
inflammatory mediator and major initiator of a cytokine network, but most clinical attempts to
use TNF as an antitumor drug in phase I trials have failed to date. A dose of 7.5 g TNF
(Sigma-Aldrich Chemical GmbH, Munich, Germany) was dissolved in 0.5 ml 0.9% NaCl
solution 20 min before application.
OK-432 (Picibanil), an antitumor reagent prepared from Streptococcus pyogenes (Chugai
Pharmaceutical Co., Tokyo, Japan) was administered at a dose of 0.05 mg OK-432, which was
dissolved in 0.5 ml 0.9% NaCl solution 20 min before application.
IL-2 (Interleukin-2) has been known to be a powerful drug for treating cancer by various
immunological mediations. A dose of 5×103IU IL-2 (Sigma-Aldrich Chemical GmbH, Munich,
Germany) was dissolved in 0.5 ml 0.9% NaCl solution 20 min before application.
Antiangiogenesis agents
Endostatin is a cleavage product of collagen XVIII that inhibits tumor angiogenesis and growth
in mice. A dose of 0.8 mg endostatin (Calbiochem Company, Darmstadt, Germany) was
suspended in 0.5 ml 0.9% NaCl solution plus 0.05 ml 10% ethanol 10 min before administration.
TNP-470 (AGM-1470), a synthetic analog of fumagillin, a natural product of Aspergillus
fumigatus, can selectively inhibit endothelial proliferation, with a subsequent antitumor effect. A
dose of 5.0 mg TNP-470 (Takeda Chemical Company, Osaka, Japan) was suspended in 1.0 ml
citrate-phosphate buffer (pH 6.2) 10 min before administration.
Anesthesia
The animals were anesthetized with intraperitoneal injection of ketamine hydrochloride,
100 mg·kg–1 (Ketanest, Parke-Davis, Heidelberg, Germany) and xylazinhydrochloride,
15 mg·kg–1 (Rompun, Bayer, Heidelberg, Germany) in all interventional and imaging
procedures.
Tumor implantation (day 0)
The technique for tumor implantation was basically similar to that described by Yang et al. [17]
with minor modifications [18]. The Morris hepatoma 3924A tumor tissue, recovered from a rat 2
weeks after subcutaneous implantation (corresponding to 5×106 tumor cells), was cut into small
cubes about 2 mm3.
A small subcapsular incision on the left lateral lobe of the liver was made in the recipient ACI
rats under anesthesia. The tumor fragment was gently placed into the pocket with a small cotton
swab on the liver surface, and the abdominal wall was then closed.
Interventional therapy (day 14)
A PE-10 polyethylene catheter (inner diameter 0.28 mm, outer diameter 0.61 mm; Wenzel;
Heidelberg, Germany) was used for catheterization under laparotomy. By using a binocular
operative microscope (M651, Leica; Wetzlar, Germany), the catheter was inserted retrogradely
into the gastroduodenal artery and pushed forward to the common hepatic artery. Due to small
hepatic artery branches no superselective embolization was possible. To ensure that the main
agent flow was directed into the left hepatic artery, the right hepatic artery was always
compressed manually. The mentioned agents were injected through the catheter to the hepatic
artery by sandwich technique (subsequent injection of mitomycin—biotherapy—lipiodol) within
20 min. The placement of the catheter in the hepatic artery during the injection of the agents
could be regarded as central embolization and prevented a reflux of the agents to the common
hepatic artery and the coeliac trunk. Fifty rats were randomly assigned to one of the five groups
(A–E), each comprising ten animals. All animals underwent transarterial chemoembolization
(TACE), applying 0.1 mg mitomycin (Medac, Hamburg, Germany) and 0.1 ml lipiodol (Byk
Gulden, Konstanz, Germany). In groups A and B, intraarterial immunotherapy was combined
using TNF + IL-2 (group A) and OK-432 + IL-2 (group B). Antiangiogenesis therapy was
combined using TNP-470 (group C) and endostatin (group D). The ten animals assigned to the
control group E were treated only with TACE.
MR imaging (day 13; day 27) and analysis
Of the different imaging modalities for the examination of hepatocellular carcinomas [19–23],
we decided to use magnetic resonance imaging. All studies were performed with a 1.5-T Sonata
superconducting system (Siemens; Erlangen, Germany) using a wrist coil before (day 13) and
after therapy (day 28). T1-weighted (SE: TR/TE, 460/15 ms) and T2-weighted (TSE: TR/TE,
3,170/99 ms) transverse images with a section thickness of 2 mm and 184×256 matrix were
acquired. The images were interpreted regarding tumor size, tumor necrosis and the development
of intrahepatic, metastatic lesions by two radiologists. There was no gap between sections, and
no contrast medium was administered. The tumor volume was determined and evaluated in T2weighted image according to the following formula [24]:
Statistical analysis
The mean growth ratio of V2/V1 was analyzed by using Dunnett s t test (SAS-Software, USA),
for comparing the effect of each therapeutic group with the control group. Comparisons between
significant therapeutic groups were performed using the Student-Neuman-Keuls (SAS-Software,
USA), test. P <0.05 was accepted as significant.
Results
Tumor implantation
The rate of tumor implantation reached 100%. None of the animals died during implantation or
interventional therapy. A total of 50 individual HCC tumors were seen with unenhanced MR
imaging in the liver of 50 rats (100%) before treatment. The tumors grew in 50 of 50 rats (100%)
after interventional treatment.
MR imaging and analysis
Due to the slice thickness of 2 mm, taking account of the small tumor volume, the presented
imaging technique faces limitations. The differentiation of the tumor from the surrounding liver
tissue was judged as sufficient by the interpreting radiologists from different signal intensities.
The mean ratio of V2/V1 was 7.30 in group A, 6.53 in group B, 4.01 in group C, 7.78 in group D
and 9.14 in group E. Compared to the control group E (TACE alone), groups B (TACE + OK432 + IL-2) and C (TACE + TNP-470) showed a significant decrease in the tumor growth ratio
(P<0.05) using multivariate analysis (Dunnett s t test), while groups A (TACE + TNF + IL-2)
and D (TACE + endostatin) did not (P>0.05). Significant results were observed in group C
(P<0.05) in comparison with the other therapeutic groups using the Student-Newman–Keuls test.
Intrahepatic metastasis occurred in one of ten rats in groups A and D and two of ten rats in group
E (Fig. 1). Hypodense areas in the T2-weighted images, which are probably presented by
intratumoral necrosis, occurred in one of ten rats in groups A, B, D and E, and two of ten rats in
group C (Fig. 2) (Table 1).
Fig. 1 Images in an ACI rat with a solid HCC in group E. a Pretherapeutic unenhanced T2-weighted axial
MR imaging with TSE sequence (3,170/99). The hyperintense lesion with a size of 0.49×0.46 mm2
(arrow) is well discernible from the surrounding liver tissue. b Post-therapeutic unenhanced T2-weighted
axial MR imaging with TSE sequence (3,170/99). The tumor with a size of 0.96×0.96 mm2 has a rapid
growth compared with that before therapy. It also shows an inhomogeneous hyperintense area (arrow)
corresponding to the intrahepatic metastasis
Fig. 2 Images in an ACI rat with a solid HCC in group C. a Pretherapeutic unenhanced T2-weighted axial
MR imaging with TSE sequence (3,170/99). The hyperintense lesion with a size of 0.41×0.40 mm2
(arrow) is well discernible from the surrounding liver tissue. b Post-therapeutic unenhanced T2-weighted
axial MR imaging with TSE sequence (3,170/99). It shows the hyperintense lesion (0.73×0.71 mm2;
arrow) with central hypointense area corresponding to the intratumoral necrosis
Table 1 The tumor volume rate (V2/V1) in different groups (n=50)
Rat
no.
1
Group A
(TNF )
5.98
Group B (OK- Group C (TNP- Group D
432)
470)
(endostatin)
8.79
3.55
8.22
Group E
(control)
5.63
2
9.84a b
7.33
7.72
9.51
4.38
b
3
7.97
5.24
5.61
7.14
10.51b
4
6.77
5.59
3.01
5.49
7.74
5
9.96
6.06
3.87
6.29
8.64
Rat
no.
Group A
(TNF )
Group B (OK- Group C (TNP- Group D
432)
470)
(endostatin)
Group E
(control)
6
5.38
7.81b
5.75b
9.67
8.20
7
8.49
5.37
3.67
8.98
8.37
ab
8
6.02
5.47
3.72
10.01
8.54a
9
7.31
4.78
3.28
6.88
11.53
10
5.28
8.84
3.30
7.42
12.72a
aTumor
with intrahepatic metastasis.
necrosis.
bIntratumoral
Discussion
Since TACE was introduced as a palliative treatment in patients with unresectable HCC, it has
become one of the most common forms of interventional therapies [10–12]. TACE with iodized
oil has been shown to result in regression of HCC and reduction of systemic toxicity, thereby
improving the therapeutic effects [11]. However, it remains questionable whether the therapies
prolong patients overall survival [25].
It is well known that an improvement in the overall therapeutic effects of liver malignancies
depends on the combined therapies [26–28]. However, few experimental or clinic reports have
been published about the combination of TACE with biotherapy. Therefore, our present study
was performed to determine the value of combining TACE with targeting locoregional
immunotherapy and antiangiogenesis therapy versus TACE alone by MRI in the rat model of
Morris hepatoma 3924A. This model was demonstrated as similar to hepatocellular carcinoma in
humans and was superior to the tumor models of Walker-256 or VX-2 [29].
OK-432 can augment the antitumor effect of mitomycin, because OK-432 itself has a direct
cytotoxic and cytostatic activity against tumor cells. Chemotherapy can also increase the
susceptibility of tumor cells to cytotoxic effector s cells, including lymphocytes, macrophages
and neutrophils activated by OK-432 through direct damage or modulation of surface antigens
by chemotherapy [30]. In addition, mitomycin can eliminate the suppressor cells or suppressor
factor in the blood or effusion, resulting in augmented anticancer activity of OK-432-activated
immunopotentiating cells, especially T-cells [31, 32]. It was also proven by histological
examination that transarterial immunoembolization (TIE) seems to be more effective than
conventional TAE for the therapy of extracapsular invasion and prevention of intrahepatic
metastases in humans [14, 33].
Okuno et al. found that hepatic infusion of IL-2 can increase the permeability of the hepatic
endothelium, resulting in an increase of hepatic extraction of antitumor agents [34]. This effect
may improve the delivery of antitumor drugs to the liver, thereby reducing the systemic drugassociated toxicity [35]. Our approach of immuno-chemoembolization was based on the abovementioned reports showing the value of TIE and locoregional immunochemotherapy. The results
showed a significant reduction of the tumor growth rate by OK-432 plus TACE compared with
TACE alone. No intrahepatic metastasis was observed. In our experiments, OK-432 seems to
exert its more potent antitumor effects in TACE.
TNF , another biological response modifier, has a variety of biological activities in vitro, such
as cytotoxic activity to cancer cells, damage to endothelial cells and enhancement of fibroblast
proliferation [36]. Watanabe et al. found that the intraarterial infusion of TNF /lipiodol
emulsion may produce prominent antitumor effects in rabbits, possibly due to the higher
concentration and prolonged retention of TNF in the tumors, which causes damage to the
endothelium of the tumor vessels [36]. In the experimental study of Yang et al., it was indicated
that tumor growth is significantly retarded by continuous hepatic infusion of TNF plus ligation
of the hepatic artery in ACI rats [37]. In this respect the placement of a catheter in the hepatic
artery for a substantial period could be regarded as central embolization of the tumor vessel. Our
study design was limited by the long-term placement of the catheter in the hepatic artery, and for
this reason, a lower total dose of TNF combined with IL-2 was transarterially injected in the
liver tumor in comparison with the study of Yang et al. This could be an explanation why the
single combined therapy of TNF + TACE did not induce a significant reduction in the tumor
growth.
Antiangiogenic agents have low toxicity due to their selective effect on tumor vasculature; their
combination with anticancer agents may potentiate their anticancer effects [38–40]. For the best
clinical results, antiangiogenic therapy should be used in combination with other adjuvant
therapies [41–43].
TNP-470 inhibits proliferation and migration of endothelial cells and capillary tube formation at
cytostatic but not cytotoxic concentrations. It is believed that ischaemic hypoxia and necrosis
induced by TACE stimulates angiogenesis in the residual viable HCC [44]. Combination
treatment in animals showed that TNP-470 potentiates the anticancer effects of some cytotoxic
and biological agents [45], but the terminal plasma half-life of TNP-470 is short, and the drug is
rapidly cleared from the circulation after a single 1-h infusion [46]. It was reported that TAE
combined with TNP-470 may enhance the anticancer effect of TAE alone in the treatment of
HCC without severe side effects on the liver or body weight gain [47, 48]. Our results indicated
that the tumor growth ratio is markedly reduced by combined transarterial administration of
TNP-470 and TACE compared with TACE alone (P<0.05). Its antitumor effect is stronger than
that of the immunotherapy group (OK-432 + TACE and TNF + TACE).
Endostatin, which was used as an angiogenesis inhibitor in our study, is a new kind of potent
antiangiogenic factor [49]. Endostatin was shown to inhibit VEGF-induced endothelial cell
migration in vitro and to have antitumor activity in vivo, without any apparent sign of toxicity
[50]. It has also been reported that endostatin can inhibit the growth of transplantable HCC in
nude mice by 52.2% [51], and the addition of endostatin to conventional chemotherapy enhances
the antitumor effect in a murine model of early colorectal liver metastasis [52]. Our present
results indicated that the selective delivery therapy of endostatin in conjunction with TACE has
no significant advantage in stemming tumor growth compared with TACE alone. Intrahepatic
metastases (10%) occurred in this group. We hypothesized that the availability of this therapy
relates to the higher doses of citrate-phosphate buffer solution (1.0 ml), in which 0.8 mg
endostatin can be dissolved. As a result, more mitomycin and lipiodol could perhaps be washed
out from the tumor, and thus the antitumor effect was influenced.
In summary, an encouraging result (significant reduction of tumor growth rate combining TACE
with OK-432 and TNP-470 compared to TACE alone) was obtained by combining different
locoregional therapies with TACE for treating HCC in rats in comparison with TACE alone and
may prove valuable to human application as a therapeutic approach for the treatment of HCC.
However, detailed therapeutic mechanisms, therapeutic indications, monitoring and validation of
these combined therapies still remain unclear and require more randomized animal experimental
studies.
Practical application
Locoregional therapies including immune and antiangiogenesis therapy combined with TACE
demonstrated an obvious reduction in tumor growth compared with TACE alone. These results
may represent a first approach to an effective modulation of transarterial chemoembolization in
the treatment of HCC for further clinical studies.
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