Transcatheter arterial embolization combined with hypoxia-replicative oncolytic adenovirus perfusion enhances the therapeutic effect of hepatic carcinoma

Abstract

Purpose: Transcatheter arterial embolization or transcatheter arterial chemoembolization has become a critical therapy for unresectable hepatocarcinoma. Although hypoxia caused by embolization can induce apoptosis and necrosis of the majority of tumor cells, a small proportion of cells can survive with hypoxia and chemotherapy resistance. HIF-1α induced by hypoxia is the key factor rendering surviving tumor cells invasive and metastatic properties. Thus, we generated a synthetic hypoxia-replicative oncolytic adenovirus (HYAD) expecting to further eliminate the surviving tumor cells, which expressed HIF-1α.

Materials and methods: In our study, we detected protein expression, proliferation, apoptosis, and necrosis of hepatic tumor cell line when infected with HYAD under hypoxia and normoxia. And we constructed VX2 hepatic cancer rabbit models to explore the therapeutic effect of transcatheter arterial embolization combined with HYAD perfusion under digital subtraction angiography. Inhibition of tumor growth and invasion was detected by histopathological examination and contrast-enhanced CT scan.

Results: Experiments in vitro verified that HYAD expressed and replicated along with HIF-1α expression or hypoxia. Compared with wild adenovirus type 5 (WT), HYAD expressed much more under hypoxia, which was the main principle of HYAD killing surviving tumor cells posttransarterial embolization. In vivo experiment of VX2 models, HYAD perfusion combined with polyvinyl alcohol (PVA) embolization achieved the highest expression quantity and the longest expression duration compared with simple HYAD perfusion, WT perfusion combined with PVA embolization, and simple WT perfusion. Because adenovirus expression protein E1A had the properties of promoting apoptosis, inhibiting invasion, and inhibiting metastasis, HYAD perfusion combined with PVA embolization group efficiently repressed tumor growth and intrahepatic metastases compared to other processing groups.

Conclusion: HYAD can overcome the hypoxic tumor microenvironment postembolization and target the surviving tumor cells with specificity. In turn, HYAD perfusion combined with PVA embolization can bring out the best effect in each other.

Keywords: hepatocarcinoma; hypoxia; oncolytic adenovirus; transcatheter arterial embolization.

Figure 1

Standard curve of RT-PCR.

Figure 2

Expression of HepG2 infected with HYAD vs WT under normoxia and hypoxia. Notes: (A) Western blot showed the expression of E1A and HIF-1α: 1) represented no virus infection under normoxia,2) no virus under hypoxia; 3) HYAD under normoxia; 4) HYAD under hypoxia; 5) WT under normoxia; and 6) WT under hypoxia. (B) Semiquantitative analysis of Western blot showed under hypoxia, the amount of HIF-1α was 14.4-, 3.4-, and 1.9-fold higher in control, HYAD, and WT than that under normoxia, respectively (***P<0.001). The amount of E1A in HYAD under hypoxia was 5.0 folds (**P<0.01) that in HYAD under normoxia and 3.6 folds (*P<0.05) that in WT under hypoxia. (C) Absolute quantification of E1A in RT-PCR revealed that after infected for 24 hours, the translation of E1A in HYAD group (at MOI 10) under hypoxia was 17.2-fold (*P<0.05) higher than that in HYAD under normoxia and 17.9-fold (*P<0.05) higher than that in WT under hypoxia. Abbreviations: HYAD, hypoxia-replicative oncolytic adenovirus; MOI, multiplicity of infection; WT, wild adenovirus type 5.

Figure 3

proliferation of HepG2 and LO2 in control, HYAD, and WT group after being infected for 24, 48, 72, and 96 hours at MOI 1, 10, and 100, respectively. Notes: CCK-8 kit was used to detect the proliferation and the absorbance was measured at 450 nm. Cells were harvested at the indicated time. Data are the mean ± SD of three independent experiments. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: CCK-8, cell-counting kit-8; HYAD, hypoxia-replicative oncolytic adenovirus; MOI, multiplicity of infection; WT, wild adenovirus type 5.

Figure 4

Apoptosis and necrosis of HepG2 and LO2 cells infected with HYAD and WT under normoxia and hypoxia at 96 hours postinfection. Notes: (A) Flow cytometry analysis was applied to detect the apoptosis and necrosis with HYAD and WT at MOI 10. (B) The percentages of apoptosis and necrosis of all the cells were presented in the graphs. Data are the mean ± SD of three independent experiments. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: HYAD, hypoxia-replicative oncolytic adenovirus; MOI, multiplicity of infection; WT, wild adenovirus type 5; 7-AAD, 7-aminoactinomycin D.

Figure 5

DSA of VX2 liver tumor rabbit model. Notes: After the tumor matured, in treatment groups requiring PVA embolization, under fluoroscopic guidance, about 0.5 mL PVA particles in 100–300 µm size of mixture were injected into the blood supplying arteries of tumor in each rabbit, and the embolization ended as antegrade blood flow was completely static. One week later, groups A and B took a second injection of HYAD with 1×10⁹ PFU (1 mL PBS dilution). At first intervention in groups A and B, contrast dyeing of neoplasm was obvious after injecting contrast agent (marked by white arrow). One week later, because of PVA embolization, contrast dyeing of neoplasm could not be seen in group A but could be seen in group B. Abbreviations: DSA, digital subtraction angiography; HYAD, hypoxia-replicative oncolytic adenovirus; HYAD+PVA, HYAD combined with PVA particle embolization; PVA, polyvinyl alcohol.

Figure 6

Immunofluorescent staining of HIF-1α and E1A at days 1, 3, and 7 post the first interventional manipulation. Notes: (A–D) Double immunofluorescent labeling of HIF-1α and E1A in HYAD perfusion combined with PVA embolization, simple HYAD perfusion, WT perfusion combined with PVA embolization, and simple WT perfusion groups, respectively. (E) Immunofluorescent staining of HIF-1α in PVA embolization and PBS perfusion (control). (F) Semiquantitative analysis of the staining by integrated optical density demonstrating the expression levels in different groups. Each data point indicates mean ± SD. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: HYAD, hypoxia-replicative oncolytic adenovirus; HYAD+PVA, HYAD combined with PVA particle embolization; PVA, polyvinyl alcohol; WT, wild adenovirus type 5 perfusion; WT+PVA, WT combined with PVA particle embolization.

Figure 7

TUNEL assay and tyrosine kinase Axl staining. Notes: (A) Rabbits were sacrificed, and the tumors were collected on the days 1, 3, and 7 after the first interventional manipulation. TUNEL and H&E staining on day 3 and Axl staining at days 1, 3, and 7 in each group were performed. (B) Semiquantitative analysis of the staining by integrated OD demonstrating the expression levels in different groups. Each data point indicates mean ± SD. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: HYAD, hypoxia-replicative oncolytic adenovirus; HYAD+PVA, HYAD combined with PVA particle embolization; PVA, polyvinyl alcohol.

Figure 8

Tumor volumes of primary and metastatic lesions detected by contrast-enhanced CT scan. Notes: (A) Tumors of primary and metastases were illustrated at 3 weeks post the first interventional manipulation (primary tumor marked with blue arrow, metastases marked with red arrow). (B) Before interventional operation and at 1, 2, and 3 weeks post the first interventional manipulation, the volumes of primary and metastatic lesions were detected by contrast-enhanced CT scan. Each data point indicates mean ± SD. Abbreviations: HYAD, hypoxia-replicative oncolytic adenovirus; HYAD+PVA, HYAD combined with PVA particle embolization; PVA, polyvinyl alcohol.

Figure 9

Correlation analysis of expression proteins HIF-1α and E1A. Notes: (A) Scatter plots were shown of groups A, B, C, and D at days 1, 3, and 7 postmanipulation, and the Pearson’s correlation coefficient (Pearson’s-Rr) of each time point was analyzed, respectively. (B) Colocalization was illustrated as overlapping circles. Red circles represent HIF-1α, green circles represent E1A, and yellow areas represent the colocalization of HIF-1α and E1A. Rcoloc was represented in terms of percentages. Abbreviations: HYAD, hypoxia-replicative oncolytic adenovirus; HYAD+PVA, HYAD combined with PVA particle embolization; PVA, polyvinyl alcohol; Rcoloc, relation of colocalization; WT, wild adenovirus type 5 perfusion; WT+PVA, WT combined with PVA particle embolization.