Flt3L and TK gene therapy eradicate multifocal glioma in a syngeneic glioblastoma model

Glioblastoma multiforme (GBM) is the most aggressive, rapidly progressing, and difficult to treat primary brain tumor. Classical GBMs do not have a single central tumor mass but disseminate throughout the brain.1–3 Even with the most aggressive treatments currently available, disease recurrence is nearly inevitable; only 29.1% of patients survive 1 year from diagnosis.4 During surgical resection of GBM, tumor cells cannot be removed completely due to the disseminated nature of this tumor, and this is believed to be the cause of tumor recurrence.5 Therefore, therapeutic approaches that are able to target and eliminate disseminated GBM foci would be anticipated to translate more efficaciously into the clinical setting. Gene therapy strategies utilizing oncolytic, replicating viral vectors have so far been the only treatment to show efficacy in models of multifocal GBM.6–10 Immunotherapy is another promising approach for treating disseminated GBM in human patients. Several immunotherapeutic approaches have used tumor vaccines to stimulate a highly specific and targeted immune response directed against tumor-specific antigens. These approaches require the recruitment and activation of antigen-presenting cells (APCs) within the tumor mass.11 Since dendritic cells (DCs) are the most efficient APCs,12,13 many preclinical and clinical trials employ DCs loaded with tumor lysates or specific tumor antigens14–18 to induce an immunotherapeutic antitumor effect. Flt3L (human soluble FMS-like tyrosine kinase 3 ligand) stimulates maturation and proliferation of DCs19–21 and natural killer (NK) cells.22 Treatment of solid tumors with recombinant Flt3L protein slows disease progression in models of breast cancer and leukemia and decreases the number of metastases in models of colon adenocarcinoma, sarcoma, and melanoma.23–25 Also, in sarcoma models, the administration of recombinant Flt3L protein induces significant tumor regression and immunological memory.26–28 However, Flt3L recombinant protein has not been efficacious in the treatment of sarcoma and melanoma tumors when implanted in the brain.23 This has been attributed to the dose, short half-life, or distribution of the cytokine, as well as to tumor antigen availability. We have recently demonstrated that injection of an adenoviral (Ad) vector expressing Flt3L (AdFlt3L) into the brain of rats induces a specific increase in the number of interferon alpha (IFNα)-secreting plasmacytoid dendritic cells (pDCs) within the parenchyma.19 In a syngeneic intracranial glioma model in rats, we found that AdFlt3L improves survival when tumors are treated within 3 days of tumor implantation.29 However, the efficacy of this treatment inversely correlates to tumor volume. Thus, we combined AdFlt3L with an Ad vector expressing herpes simplex virus type 1–thymidine kinase (AdTK), which prolonged survival in more than 70% of treated animals by inducing an antitumor immune response.30 In this experimental paradigm, TK in the presence of the prodrug ganciclovir (GCV) kills glioma cells,31,32 inducing the release of antigens that can be taken up by Flt3L-recruited DCs. Although preclinical models of GBM share many pathological features with human GBM,33 they do not fully mimic the disseminated nature of the human disease. In this study, we aimed to determine whether the antitumor response generated by combined treatment with AdFlt3L and AdTK would be effective in a multifocal intracranial GBM model in rat. Thus, we implanted CNS-1 rat glioma cells bilaterally in both brain hemispheres and administered AdFlt3L combined with AdTK unilaterally, leaving the second tumor untreated. The results of this study demonstrates that the antitumor response generated by AdFlt3L and AdTK is effective at eliminating both the primary treated and the secondary, distant untreated tumor. Our results suggest that this gene therapeutic approach would be effective in eliminating glioma cells that disseminate from the main tumor mass throughout the normal brain parenchyma, and support further development of TK in combination with Flt3L for translation into a phase I clinical trial.