(Ad) that express the immunostimulatory cytokine human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L, Ad-hsFlt3L), and the conditionally cytotoxic Herpes Simplex Type 1- thymide kinase (TK, Ad-TK) induces tumor regression, long-term survival, and immunological memory in rats and mice bearing large intracranial syngeneic glioblastomas or intracranial melanomas [7–10]. Intratumoral expression of Ad-hsFlt3L recruits dendritic cells into the brain parenchyma [11], improving tumor antigen presenta- tion, while Ad-TK exerts a cytotoxic effect exclusively in proliferating GBM cells in the presence of ganciclovir (GCV), leading to the release of tumor antigens and inflammatory molecules from dying tumor cells [7,8,10]. Spontaneous canine GBM constitutes an attractive animal model for testing novel therapies for GBM. GBM is the most common primary brain tumor in dogs, specially in certain breeds, such as Boston terriers and Boxers [11–16]. Spontaneous GBM in dogs exhibit the same histopathological features, clinical signs and standard treatment as the human disease [17–20]. A clinical trial in canine GBM patients would provide very useful data: i) due to the infiltrative nature of spontaneous GBM in dogs, they will allow testing the efficacy of novel therapies, and their toxicity to the normal brain, ii) due to the large size of the dog brain, they would be useful for assessing the doses and volumes needed in order to optimize treatment protocols, iii) the fact that dogs undergo similar standard care for GBM allows performing clinical trials that mimic more closely the clinical scenario, in which new therapies are implemented in conjunction with standard of care, iv) the individual variability of outbreed dogs better mimics the clinical scenario. Thus we aim to test the efficacy of Ad-hsFlt3L and Ad-TK in dog patients bearing spontaneous GBM. To this end we and others have previously demonstrated the feasibility of delivering therapeutic transgenes to dog GBM cells in vitro and dog brain cells in vivo upon intracranial injection of gene therapy vectors, such as type 5 adenoviral vectors [21–23] and adeno-associated viral vectors [24]. Also, we have shown that TK expression exerts a powerful cytotoxic effect on dog GBM cells [21,22]. However, the functionality of human soluble Flt3L on dog dendritic cells (DCs) has been controversial [25–27]. Considering that the efficacy of this treatment depends on an anti-tumor immune response triggered by dendritic cells upon Flt3L stimulation [10], and as a prelude for a clinical trial in canine GBM patients, we aimed to evaluate whether hsFlt3L expressed by the therapeutic Ad-hsFlt3L would exert a trophic effect on dog dendritic cells. It has been previously demonstrated that DCs can be successfully obtained from dog peripheral blood mononuclear cells (PBMC) using canine IL-4 and GM-CSF, in vitro [28]. In order to evaluate the ability of hsFlt3L to generate DCs from dog PBMC we compared it with canine IL-4 and GM-CSF. Our results demonstrate that hsFLT3L is capable of generating DCs from PBMC cultures with very similar characteristics to canine IL- 4 and GM-CSF-cultured DCs. Thus, indicating that hsFlt3L is able to modulate the function of dog dendritic cells. We found that dendritic cells generated using hsFlt3L express monocytic and dendritic cell markers, they are capable of phagocytosis and overexpression of activation markers and pro-inflammatory cytokines upon maturation. Also, they are effective at antigen presentation to T cells. Thus, this report supports the use hsFlt3L encoded within an Ad in combination with Ad-TK (plus GCV) in dog GBM patients. Our data will be critical for translating the use of hsFlt3L in both dendritic cells’ vaccination approaches and also in gene therapeutic strategies from rodents to canine patients and ultimately to human patients. Human Flt3L Expands Dog DCs Results Morphological features of DCs cultured with hsFlt3L or canine IL-4 and GM-CSF Adherent dog peripheral blood leucocytes were cultured with hsFlt3L derived from Ad-hsFlt3L conditioned medium or with canine IL-4 and GM-CSF. Although the gross appearance of both cultures was similar and adherent cellular aggregates were readily observed after 4–6 days of culture in both conditions, the amount of cells outside the aggregates was higher in the presence of canine- IL-4 and GM-CSF (Fig. 1). After 1 week, cultured cells that differentiated into non-adherent cells exhibiting thin cytoplasmic processes were observed in hsFlt3L as well as in IL-4+GM-CSF cultures (Inset Fig. 1). At day 7–9 floating cells were collected and cellular aggregates were dislodged for flow cytometric analysis of immune cell markers or to activate the immature DCs. The average yield of cells collected from these cultures (n = 27 dogs) was 456104 (67.56104) and 1016104 (6146104) for hsFlt3L and canine-IL-4/GM-CSF cultures, respectively (Fig. 1C). Phenotypic characterization of canine DCs cultured in the presence hsFlt3L or canine IL-4 and GM-CSF In order to determine the ability of hdFlt3L to generate DCs from of peripheral blood precursors, we assessed the expression of DC markers. We studied the phenotype of peripheral blood DC cultures 1 week after incubation in the presence of hsFlt3L or canine IL-4 and GM-CSF. In peripheral blood cultures incubated with hsFlt3L, we found that around half of the non-adherent cells expressed the monocytic marker CD14 (Fig. 2). Expression of DC marker CD11c and macrophage CD18 was observed in 20% and 10% of cells, respectively. These results were comparable with peripheral blood cultures incubated in the presence of IL-4 and GM-CSF (Fig. 2). Activation status of canine DCs cultured in the presence of hsFlt3L or canine IL-4 and GM-CSF Optimal antigen presentation requires maturation of DCs; this is crucial to mount an anti-tumor immune response. Thus, we evaluated the activation status of peripheral blood DC cultures incubated with hsFlt3L or canine IL-4/GM-CSF. After 7 days in culture 40–50% of cells in both peripheral blood cultures expressed intermediate levels of MHCII, while ,10% expressed high levels of MHCII (Fig. 3 A). In addition ,65% cells in both cultures expressed the co-activation marker CD80 (Fig. 3 B). In order to induce maturation of DCs, cells were collected 7 days after culture, cytokines were removed and cells were incubated with ODN CpG or LPS for 24h. Activation led to over expression of MHCII in hsFlt3L- and IL-4/GM-CSF-derived DCs under the three conditions tested (cytokine withdrawal +/2 CpG or LPS). MHCII expression was observed in over 70% of the cells, half of which were MHCIIhigh (Fig. 3 A) and CD80 expression was detected in ,80– 90% of the cells, regardless the activation stimulus (Fig. 3 B). We also measured the release of cytokines into the cell culture supernatant during 24 h activation (Fig. 4). We found that LPS strongly stimulated the release of IL-6 and TNF-a from hsFlt3L- and IL-4/GM-CSF-derived canine DCs, while CpG increased only slightly the levels of IL-6 and TNF-a (Fig. 4 A and B). On the other hand, both CpG and LPS increased the release of IFN-c from hs- Flt3L and IL-4/GM-CSF-cultured canine DCs (Fig. 4 C). Taken together these results suggest that the maturation of DCs generated from peripheral blood with hsFlt3L is comparable to IL-4/GM- CSF. Also, there were no differences in the sensitivity of both types of cultures to TLR agonists. PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11074