Generation of α Gal-enhanced bifunctional tumor vaccine

Abstract

Hepatocellular carcinoma (HCC) is a common malignant tumor with poor prognosis and high mortality. In this study, we demonstrated a novel vaccine targeting HCC and tumor neovascular endothelial cells by fusing recombinant MHCC97H cells expressing porcine α-1,3-galactose epitopes (αGal) and endorphin extracellular domains (END) with dendritic cells (DCs) from healthy volunteers. END⁺/Gal⁺-MHCC97H/DC fusion cells induced cytotoxic T lymphocytes (CTLs) and secretion of interferon-gamma (IFN-γ). CTLs targeted cells expressing αGal and END and tumor angiogenesis. The fused cell vaccine can effectively inhibit tumor growth and prolong the survival time of human hepatoma mice, indicating the high clinical potential of this new cell based vaccine.

Keywords: Cytotoxic T lymphocytes; Dendritic cells; Endoglin; Fusion cells; Hepatocellular carcinoma; Immunotherapy; Tumor neovascular endothelial cells; αGal.

Graphical abstract

Figure 1

Generation of END⁺-MHCC97H, Gal⁺-MHCC97H and END⁺/Gal⁺-MHCC97H cells. (A) Immunofluorescence was used to detect the expression of END, α1,3 GT, and α1,3 GT-END in MHCC97H cells. END⁺-MHCC97H cells were bound by FITC-conjugated anti-human END antibody; Gal⁺-MHCC97H cells were stained by Alexa Fluor 647-conjugated isolectin GS-IB4 lectin; END⁺/Gal⁺-MHCC97H cells were labeled by both fluorescent dye. MHCC97H cells were used as negative controls. Red: GS-IB4; Green: anti-human END antibody; Blue: nucleus. (Original magnification, 400 ×; Scale bar, 50 μm). (B) Expression of α1,3 GT and END in MHCC97H, MHCC97H (pLVX-Puro), END⁺-MHCC97H, Gal⁺-MHCC97H and END⁺/Gal⁺-MHCC97H cells were assessed by flow cytometry. Quantitative analysis for immunofluorescence (C) and flow cytometry (D). ∗∗∗∗P < 0.0001, ns stands for no difference. Data are representative of at least three individual experiments and are presented as mean ± SD.

Figure 2

Detection of cell surface markers and production of IL-12p70 by five types of MHCC97H/DC fusion cells. Flow cytometry was used to assess the expression of cell surface markers, including CD83, CD86, HLA-ABC, HLA-DR, END and αGal. Flow cytometry analysis of CD83, CD86, HLA-ABC, and HLA-DR (A) and quantitative analysis (B). (C) Flow cytometry analysis of END as well as αGal. (D) Quantitative analysis. (E) ELISA was used to assay levels of IL-12p70 secreted after 48 h culture. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns stands for no difference. Data show mean ± SD and individual values from three independent experiments.

Figure 3

Cytotoxicity of T cells against ⁵¹Cr-labeled target cells after induction by different types of MHCC97H/DC fusion cells, at different ratios of effector cells (T cells) to target cells (E:T). Target cells were (A) 293T, (B) A549, (C) HUVEC, (D) MHCC97H, (E) END⁺-MHCC97H, (F) Gal⁺-MHCC97H, and (G) END⁺/Gal⁺-MHCC97H/DC. (H) Cytotoxicity of T cells stimulated by END⁺/Gal⁺-MHCC97H/DC fusion cells was compared for other indicated target cells. In all cases, the ratio of T cells to target cells was 30:1. Measurement of IFN-γ-producing T cells following 7-day co-culture of with different types of MHCC97H/DC fusion cells. (I) Representative ELISPOT results. (J) Average numbers of IFN-γ-producing T cells per sample of 3 × 10⁵ T cells. Spot numbers of Gal⁺-MHCC97H/DC and END⁺/Gal⁺-MHCC97H/DC fusion cells were significantly greater than that of other fusion cell types. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns stands for no difference. Data are representative of at least three individual experiments and are presented as mean ± SD.

Figure 4

Antitumor effects of T cells induced by different types of MHCC97H/DC fusion cells. (A) Mice injected with MHCC97H cells (2 × 10⁶ cells/mouse on Day 0) were treated with stimulated T cells once a week (from Day 5 to Day 33). (B) Tumor volume was measured every 5 days from Day 5 to Day 35. Tumors were significantly smaller in mice treated with T cells primed by END⁺/Gal⁺-MHCC97H/DC fusion cells than in mice treated with T cells primed by other types of fusion cells. ∗∗∗P < 0.001 (by Day 35). (C) Kaplan–Meier survival curves of hepatoma-bearing nude mice (n = 15 in each group). Curves were compared using the log-rank test. ∗∗∗∗P < 0.0001.

Figure 5

Mice had been injected with PBS or T cells stimulated by one of the following types of fusion cells: MHCC97H/DC, MHCC97H (pLVX-Puro)/DC, END⁺-MHCC97H/DC, Gal⁺-MHCC97H/DC, END⁺/Gal⁺-MHCC97H/DC. T cells stimulated by END⁺/Gal⁺-MHCC97H/DC fusion cells increased tumor cell apoptosis and inhibited their proliferation and microvessel formation in hepatoma-bearing nude mice. Cells apoptosis in tumor tissues of mice that received different treatments were detected by TUNEL. (A) TUNEL staining in the tumor tissues of mice that received different treatments (Original magnification, 200 ×; scale bar, 100 μm). (B) The mean and standard deviation of TUNEL staining in each group. (C) PCNA staining in tumor tissues of mice that received different treatments (Original magnification, 400 ×; scale bar, 50 μm). (D) PCNA-positive cells were counted in five randomly selected fields of tumor thin sections. Quantitative analysis for average numbers of positive cells. (E) Expression of CD31 in tumor tissues of mice that received different treatments (Original magnification, 200 ×; scale bar, 100 μm). (F) CD31-positive microvessels were counted in five randomly selected fields of tumor thin sections. Quantitative analysis for average numbers of microvessels. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Data are representative of at least three individual experiments and are presented as mean ± SD.