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Year : 2020  |  Volume : 16  |  Issue : 7  |  Page : 1703-1709

Transcatheter arterial chemoembolization (TACE) with iRGD peptide in rabbit VX2 liver tumor

1 Department of Radiology, Key Laboratory of Diagnostic Imaging and Interventional Radiology of Liaoning Province, First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China
2 Department of Radiology, Qilu Hospital of Shandong University, Jinan, Shandong, China

Date of Submission23-Sep-2020
Date of Acceptance24-Nov-2020
Date of Web Publication9-Feb-2021

Correspondence Address:
Ke Xu
Department of Radiology, Key Laboratory of Diagnostic Imaging and Interventional Radiology of Liaoning Province, First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1411_20

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 > Abstract 

Purpose: Transcatheter arterial chemoembolization (TACE) is the first-line therapy for unresectable hepatocellular carcinoma (HCC). However, its therapeutic effects are hampered by the poor distribution of anticancer drugs in tumors. iRGD, a novel tumor-penetrating peptide, enhances the penetration distance and therapeutic efficacy of anticancer drugs. Herein, we evaluated the therapeutic effects of iRGD coupled with TACE in the rabbit VX2 liver tumor model.
Subjects and Methods: This study had two stages: tumor permeability assay and anticancer efficacy evaluation. In the tumor permeability assay, we coadministered TACE with either iRGD + lipiodol-doxorubicin emulsion (LDE) or LDE in the rabbit VX2 liver tumor model. We evaluated the doxorubicin (DOX) distribution at predetermined times by immunofluorescence microscopy. To evaluate anticancer efficacy, we administered saline, LDE, or iRGD + LDE to tumor-grafted rabbits. We measured tumor volume using magnetic resonance scanning. We quantified the expression levels of Bax, Bcl-2, and cleaved caspase-3 using Western blot (WB) analysis and determined the apoptosis rate in tumor cells using transferase-mediated dUTP nick-end labeling assay.
Results: The iRGD + LDE infusion significantly increased the DOX concentration and DOX penetration in tumors compared with the LDE infusion (P < 0.05). The antitumor efficacy of the iRGD + LDE in tumor inhibition was higher than that of the other treatments (P < 0.05). Besides, iRGD + LDE induced more apoptosis (P < 0.05).
Conclusions: We demonstrated that iRGD coadministered with TACE is effective against HCC.

Keywords: Doxorubicin, hepatocellular carcinoma, iRGD peptide, transhepatic arterial chemoembolization, VX2

How to cite this article:
Liu X, Xie Y, Qi X, Xu K. Transcatheter arterial chemoembolization (TACE) with iRGD peptide in rabbit VX2 liver tumor. J Can Res Ther 2020;16:1703-9

How to cite this URL:
Liu X, Xie Y, Qi X, Xu K. Transcatheter arterial chemoembolization (TACE) with iRGD peptide in rabbit VX2 liver tumor. J Can Res Ther [serial online] 2020 [cited 2021 Mar 5];16:1703-9. Available from: https://www.cancerjournal.net/text.asp?2020/16/7/1703/308761

 > Introduction Top

Hepatocellular carcinoma (HCC) causes 782,000 deaths annually.[1] Around 70% of HCC patients are unamenable to surgical resection.[2] Transcatheter hepatic arterial chemoembolization (TACE) is recommended for unresectable HCC.[3] It consists in injecting a lipiodol-doxorubicin emulsion (LDE) into the tumor-feeding arteries.[4] However, additional chemotherapeutic agents do not improve TACE over transcatheter arterial embolization.[5],[6],[7] The poor distribution of anticancer drugs in tumors can hamper their effects.[8],[9],[10] The new peptide iRGD, containing a tumor-homing motif (RGD) and a tissue-penetrating motif (CendR) (CRGDK/RGPD/EC), could overcome this challenge.[11] iRGD binds to the neuropilin-1 (NRP-1) receptor, which tumors express, and facilitates the penetration of drugs.[12],[13] iRGD is effective against liver cancer.[14],[15] We hypothesized that iRGD could enhance TACE. We thus coadministered iRGD and LDE to the rabbit VX2 liver cancer model using TACE. We evaluated the distribution of doxorubicin (DOX) in VX2 rabbit liver cancer and the antitumor effect of coadministered iRGD and LDE.

 > Subjects and Methods Top

Rabbit VX2 liver tumor model

The study was conducted in strict accordance with the “Guide for the Care and Use of Laboratory Animals,” 8th edition (International Publication No: 978-0-309-15400-0) and approved by the Institutional Animal Care and Use Committee of the China Medical University (CMU) Animal Care and Use Committee.

Adult male New Zealand white rabbits were purchased from the experimental animal center of the CMU. Thirty-eight rabbits (weight: 2.5–3 kg, 2.7 ± 0.2 kg, aged 5 months) were used to establish the rabbit VX2 liver tumor model. In all the experiments, animals were anesthetized by intramuscular injection of pentobarbital sodium (25 mg/kg) (Shanghai Chemical Reagent Co., Shanghai, China). The VX2 liver tumors were implanted as previously described.[16] The VX2 tumors were injected into the hind limb of a donor rabbit. The tumors were harvested when they reached 1 cm in diameter [Figure 1]a. The excised fresh tumor samples were cut into small cubes (~1 mm3). The tumor fragments were then pushed into the liver lobe, percutaneously, through an 18-G needle under ultrasound guidance [Figure 1]b. Typically, the tumors were suitable for experiment within 2 weeks of implantation.[17] Finally, the tumors were observed using a clinical 3.0-T magnetic resonance imaging (MRI) scanner (GE Healthcare, Milwaukee, WI, USA).[18]
Figure 1: The preparation before transcatheter arterial chemoembolization. (a) VX2 tumors were inoculated in the hind limbs of rabbits. (b) The VX2 tumor fragments were pushed percutaneously into the liver by ultrasound guidance. (c) The right femoral artery was catheterized with a sheath

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Transhepatic arterial catheterization

TACE was performed after the successful implantation of the VX2 liver tumor. Concisely, the right femoral artery was exposed by an incision under anesthesia and was later punctured using an 18-G puncture needle. It was then catheterized with a 5-F vascular sheath (Terumo, Tokyo, Japan), as depicted in [Figure 1]c, followed by the retrograde insertion of a 2.7-F microcatheter (Terumo, Tokyo, Japan) into the common hepatic artery under monitoring by X-ray fluoroscopy. The iodixanol, a contrast agent (Jiangsu Hengrui Medicine Co., Ltd., Jiangsu, China), was injected manually (3 ml) at a speed of 0.5 ml/s. The location and staining of the VX2 liver cancer were confirmed using a digital subtraction angiography machine (Artis zee ceiling-mounted system, Siemens, Erlangen, Germany). The infusion was then hand-injected into the VX2 carcinoma-feeding artery as per the group-specific protocol. Finally, we removed the catheter, ligated the right femoral artery, and sutured the wounds. Rabbits were later aroused and allowed to recover, returned to their cages, and followed up daily until they were sacrificed.

Tumor permeability assay

First, the LDE was prepared by dissolving 3 mg/kg DOX per 0.1 ml of contrast agent, and this solution was later mixed with 0.2 ml lipiodol.[9],[15],[19] As per the drug regimen, the animals were divided into two groups (n = 10 in each group): the iRGD + LDE group (1 mg/kg iRGD [GL Biochem Co., Ltd., Shanghai, China] + 0.3 ml LDE) and the LDE group (0.3 ml LDE).[20] The solution was hand-injected gradually into the hepatic artery. Rabbits (five per group) were sacrificed at 10 min and 4 h (five rabbits per group and time point) under an overdose of anesthesia.[9],[15] The tumors were then harvested and sectioned using a cryostat. These tumor sections were incubated first with the primary antibody, CD31 (1:20 dilution; Dako; Palo Alto, California, USA), which is a vasculature marker. The sections were then incubated with fluorescein isothiocyanate-conjugated secondary antibody for 2 h. The nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride, and imaging was performed using confocal microscopy (model FV1,000-IX-81, Olympus, Tokyo, Japan). The DOX fluorescence spots and microvessel density of the tumors were evaluated using Image-Pro Plus (IPP) software (Media Cybernetics, Warrendale, Pennsylvania, USA) where a minimum of five microscopic fields were randomly visualized. The penetration distance between the vessels was evaluated using IPP software. These methods have been previously described.[9],[15],[21]

Antitumor effect of iRGD + lipiodol-doxorubicin emulsion

First, the LDE was prepared by dissolving 1 mg DOX per 0.1 ml of contrast agent, and this solution was later mixed with 0.2 ml lipiodol.[15],[19] The animals were divided into three groups (n = 6 per group) according to the injected regimen: the iRGD + LDE group (1 mg/kg iRGD + 0.3 ml LDE), the LDE group (0.3 ml LDE), and the control group (0.3 ml normal saline). The solution was hand-injected gradually into the hepatic artery. The tumor volume was evaluated by unenhanced MRI with a 3.0-T MRI using a knee coil 10 days later, and the parameters of the T2-weighted image (T2WI) sequence were set to repetition time: 5.7, echo time: 1.6, slice thickness: 2 mm, and NEX:1.[18],[22] The tumor volume was calculated using the following ellipsoid volume formula: Where a represents the maximum diameter of the tumor and b represents the minimum diameter of the tumor perpendicular to a. After scanning, all rabbits were sacrificed, and the tumors were harvested. Then, each tumor sample was divided into two parts: one part was frozen in liquid nitrogen for a WB analysis of apoptosis-related proteins and the other part was fixed in 10% neutral-buffered formalin for the transferase-mediated dUTP nick-end labeling (TUNEL) assay.

Western blotting

WB analysis was carried out using the following primary antibodies: anti-Bax (1:1,000 dilution, Proteintech, Chicago, IL, USA), anti-Bcl-2 (1:1000 dilution, Proteintech, Chicago, IL, USA), and anti-cleaved caspase-3 (1:1000 dilution, Abcam, Cambridge, UK) antibodies. Besides, the β-actin antibody (Abcam) was used as an internal reference. Proteins were extracted using a radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Nanjing, China) and quantified using a BCA protein assay kit. Proteins were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and later transferred to polyvinylidene difluoride membranes. The membranes were blocked using 5% bovine serum albumin and incubated with the primary antibody at 4°C overnight. Then, they were thoroughly washed three times with tris-buffered saline with tween-20 (TBST) and later incubated with the peroxidase-conjugated secondary antibody for 2 h at room temperature. Anti-β-actin was used as an internal reference. After washing the membranes with TBST three times, the blots were developed using a chemiluminescence substrate solution. The protein bands were visualized using the Odyssey (LI-COR, USA). The density values were calculated using Image J software and normalized to that of β-actin.

Transferase-mediated dUTP nick-end labeling assay

To conduct the TUNEL assay, tumor paraffin sections were prepared as described above, and a TUNEL assay kit (Roche, Mannheim, Germany) was used following the manufacturer's instruction. To count the TUNEL-positive cells, five microscopic fields were randomly selected from each tumor sample. The apoptotic index for each field was calculated as the number of TUNEL-positive cells per 100 randomly selected cells.

Statistical analysis

All results are presented as the mean ± standard deviation. The groups were compared using the Student's t-test or one-way analysis of variance followed by least significant difference t-test. All statistical analyses were performed using SPSS version 22.0 statistical software (IBM, Armonk, NY, USA), and P < 0.05 represented statistical significance.

 > Results Top

Magnetic resonance imaging and digital subtraction angiography imaging of the VX2 liver cancer

Rapid growth and hypervascularity are the characteristic features of the VX2 tumor, a leporine anaplastic squamous cell carcinoma. Like human HCC, rabbit hepatic VX2 carcinoma is majorly pedicled with the hepatic artery. These features make rabbit hepatic VX2 carcinoma an ideal model for interventional radiologists working on preclinical investigations of HCC.[9],[23] Herein, 14.5 ± 1.5 days after VX2 tumor implantation, we detected a high T2WI signal in the VX2 hepatic tumor [Figure 2]a.[24] Angiography of the hepatic artery located in the left lobe of the liver demonstrated that the tumor was a round mass with rich hypervascularity [Figure 2]b. X-ray fluoroscopic images depicted an apparent lipiodol deposition in the tumor area after TACE [Figure 2]c.
Figure 2: Magnetic resonance imaging and digital subtraction angiography images of a rabbit liver tumor. (a) Representative axial T2-weighted images. (b) Angiography of the hepatic artery showed a round tumor with rich hypervascularity tumor staining. (c) Lipiodol was appropriately deposited in the tumor after transcatheter arterial chemoembolization

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Tumor-penetrating ability of iRGD

To determine the ability of iRGD to promote DOX penetration in the rabbit VX2 liver tumors, we evaluated the microvessel density by immunohistochemical staining and the DOX penetration by quantifying the DOX fluorescence spots. Microvessels appeared as green fluorescence and were distributed within the tumor [Figure 3]a. We observed no apparent difference in microvessel density between the LDE and iRGD + LDE groups at 10 min (31.80 ± 0.94 and 31.07 ± 1.28) and 4 h (32.20 ± 1.86 and 32.20 ± 2.15). DOX appeared as red fluorescence and was distributed around tumor blood vessels [Figure 3]a. At 10 min, the total DOX fluorescence spots were similar (1116.53 ± 144.14 and 1234.00 ± 171.05) in both the groups [Figure 3]b. After 4 h, the DOX fluorescence spots of the two groups increased significantly [2439.20 ± 422.04 vs. 5353.20 ± 588.26; P < 0.05; [Figure 3]b]. Moreover, DOX penetration was similar (34.53 ± 3.80 vs. 34.60 ± 5.70) in the LDE and iRGD + LDE groups at 10 min [Figure 3]c. Conversely, after 4 h, the drug penetration was significantly higher in the iRGD + LDE group than in the LDE group [70.33 ± 6.76 vs. 155.13 ± 11.52; P < 0.05; [Figure 3]c].
Figure 3: Immunofluorescence image of the tumor section. (a) Immunofluorescence image of doxorubicin and vessel staining in tumor (×200). (b) Statistical analyses of the sum of doxorubicin fluorescence spots and (c) the doxorubicin penetration distance (*P < 0.05, **P < 0.01, ***P < 0.001)

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Tumor volume evaluation by magnetic resonance imaging

We performed transhepatic arterial catheterization of the tumor-bearing rabbits to validate the therapeutic effect of the coadministration of iRGD and LDE. We evaluated the mean tumor volume at baseline and 10 days after drug administration by MRI [Figure 4]a. At baseline, the tumor volumes of the three groups were similar [Figure 4]b. On the 10th day, the mean tumor volume of the iRGD + LDE group (152.82 ± 52.19 mm3) was significantly lower than those of the LDE group (1245.22 ± 166.43 mm3) and the control group (3719.65 ± 420.06 mm3) [Figure 4]c. Thus, the iRGD + LDE treatment showed higher mitigation of tumor progression.
Figure 4: Altered tumor volume after transcatheter arterial chemoembolization. (a) T2-weighted images of the tumor at day 0 and day 10. (b and c) Tumor volume changes with different treatments at different times (*P < 0.05, **P < 0.01, ***P < 0.001)

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Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 expression

Chemotherapeutic drugs induce apoptosis in tumorigenic cells. Bax, Bcl-2, and cleaved caspase-3 are proteins associated with apoptosis.[25] The WB assay showed that the experimental groups had higher levels of Bax and cleaved caspase-3 and lower levels of Bcl-2 than the control group [Figure 5]a. The Bax expression in the iRGD + LDE and LDE groups was significantly higher than that of the control group (P < 0.05) [Figure 5]b. Conversely, the Bcl-2 expression was significantly lower in the iRGD + LDE and LDE groups than in the control group (P < 0.001) [Figure 5]c. However, the cleaved caspase-3 levels were significantly higher in the iRGD + LDE and LDE groups than in the control group (P < 0.001) [Figure 5]d. Furthermore, in the iRGD + LDE group, the Bax and cleaved caspase-3 levels were significantly higher, and the Bcl-2 level was significantly lower than in the LDE group (P ≤ 0.001).
Figure 5: Western blot analysis. (a) The Western blot bands of Bax, Bcl-2, and cleaved caspase-3 protein. (b-d) Quantitative analysis of protein expression and its statistical evaluation (*P < 0.05, ** P < 0.01, ***P < 0.001)

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Transferase-mediated dUTP nick-end labeling assay

To confirm the enhanced therapeutic efficacy of LDE and iRGD coadministration, we detected tumor apoptosis via a TUNEL assay. We observed a higher apoptosis rate in the tumor cells of the iRGD + LDE group than in the tumor cells from the other two groups [Figure 6]a. The apoptosis rate was higher in the iRGD + LDE group than in the LDE group [P < 0.01; [Figure 6]b].
Figure 6: Transferase-mediated dUTP nick-end labeling assay. (a) Transferase-mediated dUTP nick-end labeling assay of the tumor tissue from different groups after transcatheter arterial chemoembolization (×200). (b) The apoptosis rate of tumor cells after different treatments (*P < 0.05, **P < 0.01, ***P < 0.001)

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 > Discussion Top

In this study, we evaluated the efficacy of the tumor-penetrating peptide iRGD coupled with TACE therapy on VX2 rabbit liver cancer using TACE therapy. We used a DOX permeation assay and tumor treatment response experiments. To confirm the anticancer effects of iRGD with TACE, we performed WB and TUNEL assays. Our data demonstrated that iRGD promotes DOX penetration and enhances the therapeutic effects of TACE in the rabbit VX2 liver tumor model.

iRGD is a newly identified peptide. Similar to conventional RGD peptides, it enhances the uptake of therapeutic agents, but it can carry ten times more drug into the tumor.[11] In the last decade, several studies have successfully applied RGD and iRGD on rabbit VX2 liver cancer.[14],[15],[26],[27] In 2007, Grace Hu et al. developed RGD conjugated[11] in nanoparticles with a higher uptake by the rabbit VX2 tumor than the nonconjugated control after intravenous injection.[27] The uptake of RGD-conjugated hollow gold nanoparticles by the rabbit VX2 tumor model was also higher than that of the nonconjugated hollow gold nanoparticles.[14] A previous study evaluated the iRGD-conjugated and DOX-loaded ZrO2 nanoparticles with lipiodol treatment in the rabbit VX2 liver tumor model using the TACE technique.[15] This is the first study that validated the efficacy of iRGD-LDE coadministration on rabbit VX2 hepatoma xenografts using the TACE.

Xie et al. reported significant differences in DOX fluorescence spots and penetration distance between the iRGD-conjugated DOX nanoparticles and the nonconjugated DOX nanoparticles on the rabbit with VX2 liver cancer using TACE strategy.[15] Nevertheless, we validated the effect of DOX and iRGD coadministration instead of covalently coupled iRGD. The iRGD peptide augmented the efficacy of iRGD-conjugated drugs and coadministered drugs, but the latter mode was more effective.[20],[28] The coadministration strategy does not rely on the available number of NRP-1 receptors. It activates bulk transfer through the bystander, which is more practical and less expensive than the conjugation process.[28],[29],[30] We chose the observation time based on a previous report,[9],[15] which detected DOX in tumor cells within 10 min,[31] and showed that its complete release from lipiodol takes < 4 h after transarterial infusion.[32] Although we analyzed the effect of iRGD only at two time points, our results are consistent with previous reports, showing that the coadministration of iRGD enhanced the penetration of anticancer drugs.[12],[20],[33]

DOX is a first-line chemotherapeutic drug for TACE of HCC.[34],[35] However, it is effective only when it penetrates the deeper tumor tissue, and only then can it kill the maximum number of tumor cells. Although TACE enhances the DOX distribution in liver cancer compared to systemic chemotherapy, the extent of distribution is unsatisfactory and requires further improvement.[8],[36] In the current study, using the DOX permeability assay, we demonstrated that coadministering iRGD with LDE significantly increased the DOX accumulation in the tumor tissue after transcatheter intra-arterial infusion. Besides, we demonstrated that co-injecting mixed iRGD and LDE into the feeding artery of liver cancer significantly reduced the tumor volume compared with LDE alone. These outcomes confirm previous reports which have shown that the coadministration of an unconjugated iRGD peptide could promote the penetration of anticarcinogen agents into the tumor parenchyma, thereby increasing the apoptosis rate of the tumor cells.[12],[20],[33] LDE induces HCC apoptosis through the expression of Bax, cleaved caspase-3, and Bcl-2.[37],[38] The WB analysis revealed that the pro-apoptotic Bax and cleaved caspase-3 protein expression was upregulated and the anti-apoptotic Bcl-2 protein expression was downregulated in the iRGD + LDE group. Besides, the TUNEL assay confirmed that the iRGD + LDE group had the highest apoptotic rate. These outcomes proved that iRGD-LDE coadministration increases the therapeutic efficacy of TACE in HCC treatment.

There were several limitations to this study. First, the sample size was small. Second, the mechanism, dosing, and administration schedules of iRGD coupled with TACE need to be optimized to attain the best therapeutic outcome. Third, we selected only two time points, namely 10 min and 4 h post-DOX infusion, based on previous studies. However, the DOX penetration in the tumor cells is a dynamic process, and thus, more time points need to be explored. This is only a preliminary exploration, and reaching a final clinical application requires more investigation.

 > Conclusions Top

This research is the first reported transcatheter iRGD injection combined with TACE to improve drug penetration for liver cancer treatment. Furthermore, transcatheter iRGD injection enhanced drug distribution. Moreover, the combined use of iRGD and TACE increased apoptosis rates and decreased tumor growth rates, resulting in better outcomes.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (Beijing, China) (Grant number 81,630,053).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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