In Vitro Generation of Murine Dendritic Cells for Cancer Immunotherapy: An Optimized Protocol.

BACKGROUND/AIM: Dendritic cell (DC) immunotherapy induces tumor-reactive T-cells. We optimized the maturation of murine DC against ovarian cancer.

MATERIALS AND

METHODS

Immature DC were generated from bone-marrow progenitor cells and loaded with hypericin-photodynamic-treated (Hyp-PDT) tumor cells (primary maturation). Lipopolysacharide (LPS 1 μg/ml) was used as a secondary maturation stimulus. After 24 h, maturation was assessed using flow cytometry. For in vivo experiments, C57BL/6 mice were vaccinated subcutaneously with matured, loaded mature DC. Immune response was evaluated through immunohistochemistry and cytometric bead array.

RESULTS

Based on the existing protocols for DC generation, therapeutic vaccination of tumor-bearing mice with mature ID8-fLuc Hyp-PDT DC induces an immunogenic response, but provides no survival benefit. As loading with Hyp-PDT lysate induces partial primary maturation, we reduced the dose of LPS to 0.125 μg/ml and the duration of maturation to 6 hours, improving viability of mature DC.

CONCLUSION

We optimized Hyp-PDT-based ID8-fLuc DC vaccine by reducing the amount of LPS and the duration of maturation.