Immunotherapy against melanoma: Novel protocol for immunogenic maturation of dendritic cells

FÁTIMA MARIA MENTUCCI; MACCIONI, MARIANA; N. BELÉN RUMIE VITTAR; LAMBERTI, MARÍA JULIA

Mar del Plata

Congreso; Annual meeting of bioscience societies- SAIC, SAFE, SAP, SAB, AACYTAL, NANOMED-ar, HCS.; 2019

Abstract/Resumen: Dendritic cells (DCs) have been used as immunotherapeutic in multiple clinical trials with varying success, but strategies for activating are not yet fully elucidated. Recently, have been demonstrated that immunogenic cell death (ICD) enhances DC maturation and DC ability to stimulate immune effector cells, thus ICD could represent a novel tool for improving DCs-based immunotherapy. Photodynamic Therapy (PDT), an antitumor therapeutic that combines photosensitizing agents, O2 and light to create a harmful photochemical reaction, is a well described inducer of ICD. Based on these findings, we propose to optimize DCs maturation protocol by loading them with PDT-treated B16-OVA melanoma cells (Mel-PDT). Firstly, we induced bone marrow-derived DCs differentiation by using 10 or 30 % J558-conditioned medium (CM) containing GM-CSF. Floating and attached cells were separately examined for their CD11c differentiation marker expression by flow cytometry. Attached cells showed to be the optimal condition DC for differentiation process (75.2 % CD11c+). Next, the ability of tumor photosensitization to promote DCs maturation was compared to activation triggered by known DC activating LPS (0.5 μg/ml). In the former protocol, DCs were loaded with Mel-PDT in 1:1, 1:2 and 1:3 ratios (DCs:TCs ratio) and the maturation marker was assessed by flow cytometry. The best maturation regimen were DCs co-cultured with Mel-PDT in 1:1 ratio (53.3 % CD86+), even higher than LPS-induced maturation (45.6 % CD86+). To assess the PDT implication on melanoma DCs phagocytosis, bright-field photographs were taken after 24 h of cellular co-incubation. Interestingly, it was observed that DCs preferentially phagocytes Mel-PDT. Overall, our experiments focused on a novel method to obtain tumor lysates from cells putatively undergoing ICD to be used for DC pulsing and to test the maturation of the generated DCs for their future application on antitumor vaccine development.