Cellular immunotherapy for murine neuroblastoma
Neuroblastoma (NB) is the most common extracranial paediatric tumour, and patients with disseminated disease have a poor long-term prognosis. Due to the significant mortality rate, alternative treatments to conventional therapies are continually sought. Using the A/J mouse model it has previously been demonstrated that a cellular vaccine of the syngeneic Neuro-2a NB cell line modified to express IL-2 and IL-12 abrogated the tumourigenicity of Neuro-2a cells, and mediated regression of established tumours. However, establishing cultures of primary NB cells can be problematic, making this approach difficult to implement clinically. This thesis describes the development of an alternative cellular vaccine to treat murine NB using a synthetic vector. Firstly, transfection of primary dendritic cells was optimised. Dendritic cells are potent antigen presenting cells and studies have shown them to generate anti-cancer responses. Optimal transfection levels of 5% were obtained but antigen presentation by these cells was limited. Therefore, an alternative approach was developed using fibroblasts engineered to express IL-2 and IL-12. Cytokine-expressing fibroblasts could be used in place of transfected tumour cells to provide sustained, high-level cytokine expression in the tumour locale. Transfection of syngeneic and allogeneic murine fibroblasts was optimised in vitro to produce therapeutic levels of IL-2 and IL-12. Cytokine-transfected fibroblasts were compared with cytokine-transfected Neuro-2a cells to prevent engraftment of wild-type Neuro-2a cells in vivo. The allogeneic cells prevented tumour engraftment in a nonspecific, cytokine-independent manner. Syngeneic fibroblasts expressing IL-2 and IL-12 inhibited tumour engraftment as effectively as cytokine expressing Neuro-2a cells, and this rejection was cytokine-dependent. The cytokine-transfected syngeneic fibroblasts induced protective immunity against rechallenge with wild-type Neuro-2a cells as effectively as cytokine-transfected-Neuro-2a cells. Intratumoural vaccination of cytokine-transfected syngeneic fibroblasts also demonstrated therapeutic efficacy against Neuro-2a-derived established tumours. Splenocytes from vaccinated mice demonstrated increased IL-2 and IFN-y expression and cytotoxicity compared with controls when co-cultured with wild-type Neuro-2a cells in vitro. Vaccinated tumours showed decreased vascularity and increased infiltration of CD45+ cells compared with controls. Therefore, cytokine-transfected syngeneic fibroblasts are a viable potential alternative vaccine for the treatment of minimal residual NB.