Tumor progression-related transmembrane protein aspartate-β-hydroxylase is a target for immunotherapy of hepatocellular carcinoma

Background & Aims

Hepatocellular carcinoma (HCC) has a poor survival rate due to recurrent intrahepatic metastases and lack of effective adjuvant therapy. Aspartate-β-hydroxylase (ASPH) is an attractive cellular target since it is a highly conserved transmembrane protein overexpressed in both murine and human HCC tumors, and promotes a malignant phenotype as characterized by enhanced tumor cell migration and invasion.

Methods

Dendritic cells (DCs), expanded and isolated from the spleen, were incubated with a cytokine cocktail to optimize IL-12 secretion and co-stimulatory molecule expression, then subsequently loaded with ASPH protein for immunization. Mice were injected with syngeneic BNL HCC tumor cells followed by subcutaneous inoculation with 5–10×10⁵ ASPH loaded DCs using a prophylactic and therapeutic experimental approach. Tumor infiltrating lymphocytes (TILs) were characterized, and their role in producing anti-tumor effects determined. The immunogenicity of ASPH protein with respect to activating antigen specific CD4+ T cells derived from human peripheral blood mononuclear cells (PBMCs) was also explored.

Results

We found that immunotherapy with ASPH-loaded DCs suppressed and delayed established HCC and tumor growth when administered prophylactically. Ex-vivo re-stimulation experiments and in vivo depletion studies demonstrated that both CD4+ and CD8+ cells contributed to anti-tumor effects. Using PBMCs derived from healthy volunteers and HCC patients, we showed that ASPH stimulation led to significant development of antigen-specific CD4+ T-cells.

Conclusions

Immunization with ASPH-loaded DCs has substantial anti-tumor effects which could reduce the risk of HCC recurrence.

Abbreviations: HCC, hepatocellular carcinoma, ASPH, aspartate-β-hydroxylase, DCs, dendritic cells, TILs, tumor infiltrating lymphocytes, PBMCs, peripheral blood mononuclear cells, HBV, hepatitis B, HCV, hepatitis C, CTLs, cytotoxic T lymphocytes, TAAs, tumor-associated antigens, APCs, antigen-presenting cells, TCR, T cell receptor, NK, natural killer cells, CTLA-4, cytotoxic T-lymphocyte antigen-4, HBSS, Hank’s buffered salt solution, DMEM, Dulbecco’s modified eagle medium, H&E, Haematoxylin and Eosin

Keywords: Hepatocellular carcinoma, Immunotherapy, Dendritic cells, Aspartate-β-hydroxylase, CD4+ cells