Background The field of cancer immunotherapy is growing at a fast pace, with new developments in this field leading to a change in cancer therapy. Dendritic cells (DCs) are one of the central tools in cellular anti-tumour immunotherapy and the production of clinical grade monocyte-derived DCs (Mo-DCs) is the most frequent approach for antitumor vaccines production. However, there is a large space for improvement of protocols and a clear need for the establishment of clinical standard operating procedures (CSOP). Cancer stem cells (CSCs) are a recently identified small cell population present in the tumour, resistant to radio/chemotherapy and known to be responsible for disease recurrence. Here, we aim to contribute to the standardization of CSOPs and to target and eradicate CSCs by developing a DC-based immunotherapy vaccine for pancreatic and non-small cells lung cancer (NSCLC), comparing DC loading with CSCs vs. classical tumour lysates.
Materials and Methods CSCs from PANC-1 (pancreatic cancer) and A549 (NSCLC) cell lines were isolated and characterized by RT-PCR and flow citometry. CSCs resistance to chemotherapy was also assessed. In vitro anti-tumour cytotoxicity assays were performed. We also defined and compared the effect of 4 culture media during human Mo-DCs production. Three Good Manufacturing Practice (GMP) serum-free culture media for clinical use were tested - DendriMACS, AIM-V and X-VIVO 15. RPMI was used as a comparative term given that it is largely used in pre-clinical research. We characterized DC viability, differentiation, maturation, internalization of tumour lysates, cytokines production and autologous T cell stimulatory capacity, as well as metabolomic profiles by Nuclear Magnetic Resonance (NMR) spectroscopy.
Results CSCs from PANC-1 and A549 cell lines were successfully isolated and overexpressed the stem-like markers NANOG, OCT4, SOX2 and CD133, with resistance to gemcitabine. In terms of differentiation, maturation, antigen uptake capacity and metabolic profiles, AIM-V and X-VIVO 15 present similar results. However, the use of X-VIVO 15 shows an enhanced DC production of IL-12. DCs cultured in X-VIVO 15 and AIM-V media are able to induce a superior stimulation of T cells (CTLs and Th1 responses) while DCs cultured in DendriMACS are more prone to induce Treg polarization. Our data show that X-VIVO 15 and AIM-V culture media are preferable to support the differentiation of DCs to be used in immunostimulatory approaches such as in cancer immunotherapy.
Conclusions Overall, our results demonstrate that blood monocytic precursors present considerable plasticity allowing a tailored differentiation of DCs just by changing the nutritive support. This highlights the need of critically defining the culture medium to be used in DC cancer immunotherapy, attaining the desired cell characteristics and consequent robust clinical responses. We are now assessing in vitro anti-tumour cytotoxicity to evaluate if DC loading with CSC antigens can be an efficient immunotherapy strategy to target and eliminate this specific and resistant cancer cell population.
Funding ImmunoDCs@CancerStemCells: Cellular Immunotherapy towards the elimination of cancer stem cells (Ref.: POCI-01-0247-FEDER-033532), co-funded by FEDER, COMPETE2020 and University of Coimbra.
Disclosure Information J. Calmeiro: None. M. Carrascal: A. Employment (full or part-time); Significant; Tecnimede Group. L. Mendes: None. I.F. Duarte: None. C. Gomes: None. J. Serra: A. Employment (full or part-time); Significant; Tecnimede Group. A. Falcão: None. M.T. Cruz: None. B.M. Neves: None.
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