Hepatocellular carcinoma (HCC) often presents as a diffuse or multifocal
tumor making it difficult to control by surgery or radiation. Radio-inducible herpes simplex virus
thymidine kinase (HSV-TK) gene therapy has been shown to enhance local
tumor control after
radiation therapy (RT), while limiting the expression of the transgene in the irradiated
tumor tissues. To prevent liver
tumor recurrence and control systemic disease while limiting the potential bystander toxicity of HSV-TK
therapy, we proposed to stimulate endogenous dendritic cell (DC) proliferation with systemic adenovirus
Flt3 ligand (Adeno-Flt3L) gene therapy, followed by primary
tumor radiation therapy combined with a radio-inducible HSV-TK gene therapy. We hypothesized that adenovirus-expressing Flt3L gene therapy will stimulate DC proliferation, allowing the upregulated DCs to locally harness
tumor antigens released from HSV-TK/RT-treated HCC cells, thereby converting irradiated
tumors to an autologous in situ
tumor vaccine in mice with primary liver
tumors. To test this hypothesis, an expression vector of HSV-TK was constructed under the control of a radio-inducible promoter early-growth response (Egr-TK) and a recombinant adenovirus-expressing human Flt3L was constructed. The Adeno-Flt3L [10(9) plaque forming units (pfu)] was administered intravenously on days 1 and 8 after
radiation therapy. The murine
hepatoma cell line (BNL1ME) was stably transfected by Egr-TK or Egr-Null (encoding no therapeutic gene). Palpable
tumors in BALB/c mice were treated with a localized dose of 25 Gy of radiation followed by
ganciclovir (GCV, 100 mg/kg, 14 days). Four treatment cohorts were compared: Egr-Null/GCV + RT + Adeno-LacZ; Egr-Null/GCV + RT + Adeno-Flt3L; Egr-TK/GCV + RT + Adeno-LacZ; and Egr-TK/GCV + RT + Adeno-Flt3L. There was no primary
tumor regression in the Egr-Null
tumors after
radiation therapy alone. In contrast, Egr-TK
tumors had nearly complete
tumor regression for 3 weeks after
radiation therapy (P < 0.01), however, long-term follow-up demonstrated primary
tumor recurrence and death secondary to pulmonary
metastasis. Flt3L expression was confirmed by serum bioassay (mean = 88 ng/mL) in these animals and Western blotting of tissue culture medium in Adeno-Flt3L-infected BaF/huFlt3L cells.
Radiation therapy with Adeno-Flt3L gene therapy effectively retarded primary
tumor growth when compared to
radiation therapy alone. The trimodality
therapy (Egr-TK/GCV + RT + Adeno-Flt3L) was the most efficacious with 40% complete
tumor regression (>100 days) and <20% pulmonary
metastases, indicating the development of sustained antitumor immune response. These studies provide a rationale for triple modality
therapies with radiation-inducible HSV-TK gene therapy and Adeno-Flt3L when used in combination with primary
tumor radiation therapy for improved local and systemic control of HCC.