Gene Electrotransfer of Plasmid-Encoding IL-12 Recruits the M1 Macrophages and Antigen-Presenting Cells Inducing the Eradication of Aggressive B16F10 Murine Melanoma

Abstract

Cancer immunotherapy is currently one of the leading approaches in cancer treatment. Gene electrotransfer of plasmids encoding interleukin 12 (IL-12) into the cells leads to the production of IL-12, which drives immune cell polarization to an antitumoral response. One of the cell types that shows great promise in targeting tumor cells under the influence of IL-12 cytokine milieu is that of macrophages. Therefore, the aim of this study was to evaluate gene electrotransfer of antibiotic resistance-free plasmid DNA-encoding murine IL-12 (mIL-12) in mice bearing aggressive B16F10 murine melanoma. IL-12 electrotransfer resulted in the complete long-term eradication of the tumors. Serum mIL-12 and murine interferon γ (mIFNγ) were increased after IL-12 gene electrotransfer. Further on, hematoxylin and eosin (HE) staining showed increased infiltration of immune cells that lasted from day 4 until day 14. Immunohistochemistry (IHC) staining of F4/80, MHCII, and CD11c showed higher positive staining in the IL-12 gene electrotransfer group than in the control groups. Immune cell infiltration into the tumors and the high density of MHCII- and CD11c-positive cells suggest an antitumor polarization of macrophages and the presence of antigen-presenting cells that contributes to the important antitumor effectiveness of IL-12.

Figure 1

Construction of plasmid pORF-mIL-12-ORT by standard cloning methods and operator-repressor titration (ORT) technology. AmpR: ampicillin resistance gene; CM: chloramphenicol resistance gene; Kan: kanamycin resistance gene.

Figure 2

(a) Tumor growth of B16F10 tumors in C57/Bl6 after IL-12 GET compared to that in the control animal groups. The experiments were performed independently twice. (b) ELISA assay for the detection of mIL-12 in the LPS group (positive control) on days 1, 4, and 8 and in the pORF-mIL-12-ORT + EP group on days 1, 4, 8, and 14. (c) Detection of mIFNγ in the LPS and pORF-mIL-12-ORT + EP groups on days 1, 4, 8, and 14 using an ELISA assay.

Figure 3

Representative images of hematoxylin/eosin staining of all the experimental groups (negative control (only skin) with no specific day and H₂O, H₂O + EP, pControl + EP, LPS (positive control), and pORF-mIL-12-ORT + EP) on day 1, day 4, day 8, and day 14 (11). A semiquantitative scoring system for immunopositive cells was used: (+) low, (++) moderate, and (+++) high positivity. Various histological characteristics of the figures are marked: immune cell infiltration (→), blood vessels (▲), melanin (→), necrosis (●), and epithelium damage (◊). The images were taken under 20x magnification (numerical aperture 0.85).

Figure 4

Immunohistochemical staining of F4/80-positive cells of all the experimental groups (negative control, H₂O, H₂O + EP, LPS (positive control), pControl + EP, and pORF-mIL-12-ORT + EP) on day 1, day 4, day 8, and day 14 (11). A semiquantitative scoring system for immunopositive cells was used: (+) low, (++) moderate, and (+++) high positivity. The negative images of all groups are not related to a specific day. The images were taken under 20x magnification (numerical aperture 0.85).

Figure 5

Immunohistochemical staining of MHCII-positive cells of all the experimental groups (negative control, H₂O, H₂O + EP, LPS (positive control), pControl + EP, and pORF-mIL-12-ORT + EP) on day 1, day 4, day 8, and day 14 (11). A semiquantitative scoring system for immunopositive cells was used: (+) low, (++) moderate, and (+++) high positivity. The negative images of all groups are not related to a specific day. The images were taken under 20x magnification (numerical aperture 0.85).

Figure 6

Immunohistochemical staining of CD11c-positive cells of all the experimental groups (negative control, H₂O, H₂O + EP, LPS (positive control), pControl + EP, and pORF-mIL-12-ORT + EP) on day 1, day 4, day 8, and day 14 (11). A semiquantitative scoring system for immunopositive cells was used: (+) low, (++) moderate, and (+++) high positivity. The negative images of all groups are not related to a specific day. The images were taken under 20x magnification (numerical aperture 0.85).