CIK cell-based delivery of recombinant adenovirus KGHV500 carrying the anti-p21Ras scFv gene enhances the anti-tumor effect and safety in lung cancer

Abstract

Purpose: Adenovirus (Ads) is one of the most popular vectors used in gene therapy for the treatment of cancer. However, systemic therapy is limited by circulating antiviral antibodies and poor viral delivery in vivo. In this study, we used cytokine-induced killer (CIK) cells as delivery vehicles of Ads KGHV500 carrying the anti-p21Ras scFv gene to treat Ras gene-related lung cancer and investigate the anti-tumor effect in vitro and in vivo.

Methods: The human lung cancer cell line A549 was employed to investigate the anti-tumor activity of recombinant Ads KGHV500 harboring the anti-p21Ras scFv gene using MTT, wound healing, transwell invasion, and apoptosis assays in vitro. Next, CIK cells were used as delivery vehicles to deliver KGHV500 carrying the anti-p21Ras scFv gene to treat A549-transplanted tumors in nude mice, and viral replication, p21Ras scFv expression, and the therapeutic efficacy were assessed.

Results: In vitro studies showed that KGHV500 had potent anti-tumor activity. In addition, in vivo, this combination therapy significantly inhibited the growth of lung cancer xenografts compared with mice treated with KGHV500 alone. KGHV500 and anti-p21Ras scFv were observed in tumor tissue, but were nearly undetectable in normal tissues.

Conclusions: The co-delivery of anti-p21Ras scFv by CIK cells and KGHV500 could increase the anti-tumor effect and safety, and possess considerable advantages for the treatment of Ras-related cancer.

Keywords: Adenovirus; Anti-p21Ras scFv; CIK cells; Carrier; Lung cancer; Therapy.

Fig. 1

KGHV500-infected A549 cells and CIK cells. a CD46 is a receptor of recombinant adenovirus that mediates the combination of KGHV500 and target cells. IHC analysis showed that CD46 is highly expressed on the surface of A549 cells (× 400). b A549 cells were treated with KGHV500 at different MOIs for 48 h. The green fluorescence signals were detected by microscopy (× 100); an MOI of 100 was the best infection efficiency. c Many virus particles were scattered throughout the cytoplasm and nucleus. d IHC analysis showed that CIK cell markers (CD3, CD46, and CD56) were expressed on the surface of CIK cells, and hexon expression in KGHV500 was detected in CIK cells (× 200)

Fig. 2

Anti-tumor biological activities of KGHV500 in vitro. a MTT assay to measure cell killing in A549 cells treated with KGHV500 and KGHV400. The absorbance values in the KGHV500 group were lower than that in the control group. b, c Cell migration was observed at 12 h and 24 h after scratching and infection by KGHV500 and KGHV400 (× 100). The migration areas of the KGHV500 group were smaller than those of the KGHV400 and PBS groups. d, e Transwell assays observed the inhibition of invasiveness. Fixed and stained cells of the membrane were counted. The number of invading A549 cells in the KGHV500 group was much lower than that of the other two groups. f, g Apoptotic tumor cells of different groups were detected by TUNEL staining (green fluorescence), and nuclei were detected by DAPI staining (blue fluorescence) (× 1000). The number of apoptotic cells in the KGHV500 group was markedly greater than that in the control group. All experiments were performed in triplicate. All the data are represented as the means ± standard deviation (SD) of three experiments. *P < 0.05; **P < 0.01

Fig. 3

Anti-tumor activity against human lung cancer A549 cell line xenografts in nude mice. a After treatment, the tumor volume was measured every 3 days, and the therapeutic effects were compared by drawing growth curves. The tumor volumes of the CIK + KGHV500 group were much smaller than those of the other groups. The data points are expressed as the means ± SD of the tumor volumes. b, c The changing trends of the grayscale value in d were evaluated and depicted. d KGHV500 and anti-p21Ras scFv expression in the tumors of the CIK + KGHV500 and KGHV500 groups was detected by WB for 7 days. Hexon protein expression in CIK + KGHV500 increased with time, and the increasing trend was higher than that in the KGHV500 group. In addition, scFv was continuously expressed in tumors and increased gradually, and CIK + KGHB500 had a higher scFv content. e Detection of scFv expression in various tissues at day 34. scFv expression could only be detected in tumor and spleen tissues in the CIK + KGHV500 group and in all tissues except the brain in the KGHV500 group. *P < 0.05; **P < 0.01

Fig. 4

Distribution of KGHV500 in vivo. Hexon expression in various tissues at day 7 was detected by IHC to track the virus. In the CIK + KGHV500 group, hexon expression was detected in tumor and spleen tissues. Because of the blood–brain barrier, hexon could not be detected in brain tissue of the KGHV500 group (× 40). *P < 0.05; **P < 0.01

Fig. 5

Detection of the apoptosis-inducing capacity of CIK + KGHV500 in vivo. a The percentage of apoptotic cells in CIK + KGHV500 was more than that in the other groups. b The expression level of apoptotic genes was analyzed by the qRT-PCR. In the CIK + KGHV500 group, the expression of the proapoptotic genes caspase-3, caspase-7 and p53 was increased and that of the anti-apoptotic genes Bcl-2 and survivin was decreased. *P < 0.05; **P < 0.01