Immune activation of the monocyte-derived dendritic cells using patients own circulating tumor cells

摘要

Background Dendritic cell (DC) therapy counts to the promising strategies how to weaken and eradicate cancer disease. We aimed to develop a good manufacturing practice (GMP) protocol for monocyte-derived DC (Mo-DC) maturation using circulating tumor cells lysates with subsequent experimental T-cell priming in vitro. Methods DC differentiation was induced from a population of immunomagnetically enriched CD14 + monocytes out of the leukapheresis samples (n= 6). The separation was provided automatically, in a closed bag system, using CliniMACS Prodigy (R) separation protocols (Miltenyi Biotec). For differentiation and maturation of CD14+cells, DendriMACs (R) growing medium with supplements (GM-CSF, IL-4, IL-6, IL-1B, TNFa, PGE) was used. Immature Mo-DCs were loaded with autologous circulating tumor cell (CTCs) lysates. Autologous CTCs were sorted out by size-based filtration (MetaCell (R)) of the leukapheresis CD14-negative fraction. A mixture of mature Mo-DCs and autologous non-target blood cells (NTBCs) was co-cultured and the activation effect of mature Mo-DCs on T-cell activation was monitored by means of multimarker gene expression profiling. Results New protocols for mMo-DC production using automatization and CTC lysates were introduced including a feasible in vitro assay for mMo-DC efficacy evaluation. Gene expression analysis revealed elevation for following genes in NTBC (T cells) subset primed by mMo-DCs: CD8A, CD4, MKI67, MIF, TNFA, CD86, and CD80 (p <= 0.01). Conclusion Summarizing the presented data, we might conclude mMo-DCs were generated using CliniMACS Prodigy (R) machine and CTC lysates in a homogenous manner showing a potential to generate NTBC activation in co-cultures. Identification of the activation signals in T-cell population by simple multimarker-qPCRs could fasten the process of effective mMo-DC production.