Establishment and evaluation of cell and animal models expressing BORIS subfamily 2 variant

Abstract

Background: The possibility of a cancer vaccine aimed at stimulating or mobilizing the body's immune system to control and kill tumor cells is emerging as a potential new strategy for tumor immunological therapy. CCCTC-binding Factor Like (CTCFL)/Brother of the Regulator of Imprinted Sites (BORIS), a cancer-testis antigen (CTA), has 23 mRNA splice variants classified into six subfamilies (sfs) and potentially encodes 17 distinct polypeptides. Based on our previous long-term research on hepatocellular carcinoma (HCC), we were particularly interested in whether BORIS sf2 could be a promising candidate for immunotherapy targeting liver cancer cells. Therefore, in this study, we aimed to construct an animal model to study the immunogenicity of human BORIS sf2 in murine hepatoma cells.

Methods: We established a hepatoma cell line expressing human BORIS sf2/C68 by inserting the sequences into a lentiviral vector pLVX-EF1α-IRES-Puro carrying the puromycin resistance gene. We achieved the stabilized expression of BORIS sf2/C68 in the oncogenic Hepa1c1c7 cells through lentivirus-mediated approach. The Hepa1c1c7 cells expressing the BORIS sf2/C68 (5×10⁶/mouse) were inoculated subcutaneously into 6-week-old C57BL/6 mice to induce the formation of tumors.

Results: In the tumor formation experiment, the murine hepatoma cells expressing human BORIS sf2/C68 showed progressive growth in C57BL/6 mice. The animal model we constructed could be used to study the in vivo immunogenicity of the human BORIS sf2 in murine hepatoma cells.

Conclusions: The animals bearing BORIS sf2/C68-positive tumors may serve as an animal model for studying the therapeutic potency and safety of HCC vaccine directed at the CT-antigen BORIS sf2.

Keywords: BORIS sf2; Brother of the Regulator of Imprinted Sites (BORIS); animal model; cell model; murine hepatoma cell.

Figure 1

Detection of the green fluorescence during lentiviral packaging and infection. (A) The sectional sequence map for plasmid pCS-CG. (B) The packaging efficiency of 293LTV cells (4×) for Lenti-pCS-CG and Lenti-pCS-CG C2/A4. (C) The infection efficiency of Lenti-pCS-CG and Lenti-pCS-CG C2/A4 to the Hepa1-6 cell line (10×). (D) The infection efficiency of Lenti-pCS-CG and Lenti-pCS-CG C2/A4 to the A549 cell line (10×).

Figure 2

The experimental analyses of the weak fluorescence emitted by EGFP in the fusion protein. (A) The integration efficiency of the EGFP and BORIS sf2 genes in the genomes of Hepa1-6 and A549 cell lines infected by Lenti-pCS-CG or Lenti-pCS-CG sf2 evaluated by PCR. (B) The transcriptional level of the EGFP and BORIS sf2 genes in A549 and Hepa1-6 cell lines infected by Lenti-pCS-CG or Lenti-pCS-CG sf2 determined by RT-PCR. (C) The EGFP protein expression in lentivirus-infected Hepa1-6 cells and A549 cells determined by western blotting. β-actin is also analyzed as an internal control. (D) The predicted spatial conformation of the BORIS sf2/EGFP fusion protein (yellow), the reported spatial structure of free EGFP (red, PDB:2Y0G), and the merging of these two three-dimensional structures. The results are shown as the means ± SD. P<0.05 is considered statistically significant. RT-PCR, reverse transcriptase-polymerase chain reaction; ns, no significance.

Figure 3

Analysis of autophagy and unfolded protein response in cells. (A) The fluorescence intensity of lentivirus-infected Hepa1-6 cell line and A549 cell line with/without 3-MA treatment (10×). The mean fluorescence intensity was quantified by Image J. Left: Hepa1-6 cell line; Right: A549 cell line. (B) The protein expression of EGFP and BORIS sf2-EGFP fusion protein in lentivirus-infected Hepa1-6 and A549 cell lines with/without 3-MA treatment was determined by western blotting. The β-actin was also analyzed as an internal control. (C) RT-PCR analysis of unfolded protein response genes. ***, P<0.001. RT-PCR, reverse transcriptase-polymerase chain reaction.

Figure 4

The expression of BORIS sf2/C68 in the murine hepatoma cell line Hepa1c1c7. (A) The growth status of lentivirus-infected Hepa1c1c7 cells and Hepa1-6 cells under an inverted fluorescence microscope. Left: the Hepa1c1c7 cell line (10×); Right: the Hepa1-6 cell line (10×). (B) The mRNA expression of BORIS sf2/C68 in the Hepa1c1c7 cells was detected by RT-PCR. RT-PCR, reverse transcriptase-polymerase chain reaction.

Figure 5

The oncogenicity of BORIS sf2/C68-expressing Hepa1c1c7 cells. Six-week-old female C57BL/6 mice (n=12) are subcutaneously injected with 5×10⁶ Hepa1c1c7 cells. The mice are monitored for tumor growth by periodic palpation and inspection.