Enhancing Immunogenicity of Embryonic Stem Cells by Expression of GM-CSF

Antigenic similarity between tumor cells and embryonic cells has been recognized for many years. However, attempts to utilize these shared antigens as targets for immunotherapy treatments in cancer patients have achieved little success, mostly due to the development of tumor-induced immune tolerance. An alternative to this strategy is prophylactic vaccination against tumor antigens, inducing immunity before cancer develops. Prior research has shown that a vaccine consisting of allogeneic murine embryonic stem cells (ES-D3) with STO fibroblasts expressing adjuvant granulocyte macrophage-colony stimulating factor (GM-CSF) to enhance immunogenicity significantly reduces the outgrowth of implanted and induced lung carcinomas in mice.

As the previous vaccine contained whole STO fibroblasts and ES-D3 stem cells, it cannot be tested in humans due to the risk of teratoma development and other side effects. The objective of this research was to eliminate these whole cells and work towards a final version that could eventually be injected into humans. To this purpose, we expressed GM-CSF in ES-D3 cells by two different approaches, eliminating the need for GM-CSF-expressing fibroblasts.

To improve the vaccine, we stably over­expressed GM-­CSF in ES-­D3 cells through two methods: transfection (selected by GFP) and lentiviral infection (selected by puromycin) to eliminate the need for STO fibroblasts. GM-CSF expression was confirmed by ELISA, which confirmed that appreciable concentrations of GM-CSF were obtained through these methods. Transfected cells showed GM-CSF concentrations only slightly lower than concentrations from the original STO fibroblasts, while lentivirally infected cells showed concentrations slightly higher than the STO fibroblasts. Additionally, we confirmed that ES-D3 cells maintained their pluripotency, and therefore their shared tumor antigens, through immunofluorescence and flow cytometry evaluating levels of SSEA-1, a murine stem cell marker for pluripotency, Oct-3/4, a murine transcription factor, and SSEA-4, a murine stem cell marker for differentiation. GM­-CSF expression did not appear to modulate pluripotency of ES­D3 cells, as shown by high-level expression of SSEA­1 and Oct­3/4 as well as low-level expression of SSEA­4 in GM­-CSF-­expressing ES­D3 cells.

Vaccine adjuvant GM-CSF was stably over-expressed in ES-D3 cells by two different approaches: transfection (selected by GFP) and lentiviral infection (selected by puromycin). Over-expression was very successful on two fronts, with GM-CSF-expressing ES-D3 cells showing appreciable concentrations of GM-CSF as well as maintained pluripotency. These results indicate that GM-CSF-expressing ES-D3 cells could be used to produce a new generation of anti-cancer vaccines.

Grant Support:

This work was supported by the James Graham Brown Cancer Center Summer Research Internship Program.