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

Abstract

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.

Keywords: Dendritic cell; LPS; immunotherapy; maturation; mouse.