Overcoming the UCB HSCs –Derived NK Cells Dysfunctionality through Harnessing RAS/MAPK, IGF-1R and TGF-β Signaling Pathways

Abstract

BackgroundNatural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to NK cells from adult donors. This is due to opportunity to choose alloreactive donors and potentially more robust in vivo expansion. However, the cytotoxicity of UCB-HSC derived NK cells against cancer cells might be suboptimal. To overcome this obstacle, we attempted to generate NK cells with potent antitumor activity by targeting RAS/MAPK, IGF-1R and TGF-β signaling pathways.MethodsThe CD34+ cells isolated from human UCB mononuclear cells through MACS with purity of (≥90%) were used to be differentiated into NK cells. After 21 days of induction with SFTG36, IS721 and IL-15/Hsp70 media, NK cells phenotype was studied and their cytotoxicity against K562 human erythroleukemia cell and SKOV3 ovarian carcinoma cells was analyzed.ResultsThe induced NK cells treated with SFTG36/I721 and SFTG36/IS721 growth factor cocktail expressed a phenotype with CD56+16+CD3- and NKG2D+ with mean fluorescence intensity (MFI) of 92.7%±1.45-168.00±19.20 and 93.23%±0.75-168.66±20.00 respectively. These NK cells once activated by IL-15, demonstrated a higher cytotoxicity against K562 (≥90%) (P ≤ 0.001) and SKOV3 tumor cells (≥65%) (P ≤ 0.001) compared to IL-15/Hsp70 activated NK cells.Conclusion The differentiation of ex vivo-expanded CD34+ cells through manipulation of RAS/MAPK, IGF-1R and TGF-β signaling pathways is an efficient approach for generating functional NK cells that can be used for cancer immunotherapy.