Virus-Like Particles Presenting the FGF-2 Protein or Identified Antigenic Peptides Promoted Antitumor Immune Responses in Mice

Abstract

Background: Fibroblast growth factor (FGF)-2 is overexpressed in various tumor tissues. It affects tumor cell proliferation, invasion and survival, promotes tumor angiogenesis and is tightly involved in the development of systemic and local immunosuppressive tumor mechanisms.

Purpose: This study aimed to develop an effective vaccine against FGF-2 and to investigate the effects of anti-FGF-2 immunization on tumor growth and antitumor immune responses.

Methods: A set of thirteen synthesized overlapping peptides covering all possible linear B-cell epitopes of murine FGF-2 and a recombinant FGF-2 protein were conjugated to virus-like particles (VLPs) of recombinant hepatitis B core antigen (HBcAg). The VLPs were immunized through a preventive or therapeutic strategy in a TC-1 or 4T1 grafted tumor model.

Results: Immunization with FGF-2 peptides or full-length protein-coupled VLPs produced FGF-2-specific antibodies with a high titer. Peptide 12, which is located in the heparin-binding site of FGF-2, or protein-conjugated VLPs presented the most significant effects on the suppression of TC-1 tumor growth. The levels of IFN-γ-expressing splenocytes and serum IFN-γ were significantly elevated; further, the immune effector cells CD8⁺ IFN-γ⁺ cytotoxic T lymphocytes (CTLs) and CD4⁺ IFN-γ⁺ Th1 cells were significantly increased, whereas the immunosuppressive cells CD4⁺ CD25⁺ FOXP3⁺ Treg cells and Gr-1⁺ CD11b⁺ myeloid-derived suppressor cells (MDSCs) were decreased in the immunized mice. In addition, VLP immunization significantly suppressed tumor vascularization and promoted tumor cell apoptosis. In mice bearing 4T1 breast tumor, preventive immunization with FGF-2-conjugated VLPs suppressed tumor growth and lung metastasis, and increased effector cell responses.

Conclusion: Active immunization against FGF-2 is a new possible strategy for tumor immunotherapy.

Keywords: VLP; anticytokine active immunization; fibroblast growth factor-2; immune response; tumor; virus-like particle.

Figure 1

Immunization with FGF-2 peptide- or protein-conjugated VLPs elicited specific antibody responses. A set of overlapping peptides was designed to cover all possible linear B cell epitopes of the mouse FGF-2 molecule. The mice were immunized subcutaneously with FGF-2 protein- or peptide-conjugated HBcAg VLPs three times at two-week intervals. (A) Amino acid sequences of the FGF-2 protein and synthetic peptides; (B) IgG responses induced at one week after the second immunization; (C) IgG responses induced at one week after the third immunization (n = 5/group).

Figure 2

Preventive immunization with FGF-2 protein- or peptide-conjugated VLPs significantly inhibits the growth of TC-1 graft tumors. A graft tumor model of TC-1 cells was employed, and FGF-2-specific IgG responses were induced before the inoculation of tumor cells. (A) The protocol applied in the mouse model; (B) the expression level of FGF-2 in tumor tissues; (C) tumor growth dynamically monitored twice a week. *Represents a comparison with the HBcAg control (^ns no significant, *p < 0.05, **p < 0.01, and ***p < 0.001; n = 5/group); (D) representative pictures of the size of tumor masses; (E) statistical analyses of tumor weight.

Figure 3

Therapeutic immunization with FGF-2 protein- or peptide 12-conjugated VLPs suppresses the growth of established tumors. Effective IgG antibody responses were induced after the tumor size reached a diameter of 5–6 mm, and the carrier or PBS was used instead of FGF-2 protein- or peptide 12-conjugated VLPs as the controls. (A) The protocol applied in the mouse model; (B) the FGF-2-specific IgG responses from the third immunization; (C) tumor growth was dynamically monitored once a week (***p < 0.001; n = 5/group).

Figure 4

Preventive immunization with FGF-2 protein or peptide 12-conjugated VLPs promotes the responses of antitumor cellular immune responses. The isolated splenocytes were cultured and stimulated with the antigenic peptide E7_49–57, and then ELISPOT and flow cytometry analyses were performed. (A) Representative picture of ELISPOT showing the responses of IFN-γ⁺-expressing lymphocytes; (B) statistical analyses on the results of ELISPOT; (C) the IFN-γ level in serum detected by ELISA; (D) percentage of CD8⁺ IFN-γ⁺ lymphocytes in splenocytes (right) and representative flow cytometry dot plots for each group (left); (E) percentage of CD4⁺ IFN-γ⁺ lymphocytes in splenocytes (right) and representative dot plots (left) (*p < 0.05, **p < 0.01, and ***p < 0.001; n = 5/group).

Figure 5

Preventive immunization with FGF-2 protein- or peptide 12-conjugated VLPs decreases the level of immunosuppressive cells. (A) Percentage of CD25⁺ Foxp3⁺ cells in CD4⁺ splenocytes (right) and representative dot plots (left); (B) percentage of Gr-1⁺ CD11b⁺ MDSCs in isolated splenocytes (right) and representative dot plots (left) (*p < 0.05, and ***p < 0.001; n = 5/group).

Figure 6

Preventive immunization with FGF-2 protein- or peptide 12-conjugated VLPs decreases vascular formation in tumor tissue and promotes tumor cell apoptosis. (A) Representative pictures for anti-CD31-stained tumor tissue sections analyzed by an immunofluorescence assay (40×, bar=50um) (left) and statistical analyses on the area of blood vessels expressing CD31 with ImageJ software (right); (B) Representative pictures for anti-caspase 3-stained tumor tissue sections analyzed by an immunofluorescence assay (40×, bar=50um) (left) and statistical analyses on the caspase 3-positive area with ImageJ software (right) (**p＜0.01, and ***p＜0.001; n=5/group).

Figure 7

Preventive immunization with FGF-2 protein-conjugated VLPs suppresses the primary tumor growth and lung metastasis in the 4T1 model. (A) Dynamic monitoring of the changes in tumor size; (B) tumor weight; (C) lung nodule number; (D) representative images of lung metastatic nodules stained with Indian ink; (E) IFN-γ-expressing splenocytes detected by ELISPOT; (F) the percentage of CD8⁺IFN-γ⁺ lymphocytes in splenocytes (*p < 0.05, and **p < 0.01; n = 5/group).