Abstract
BackgroundThe natural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to the NK cells from adult donors. This is due to the possibility 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 using IL-15, IGF-1 and SIS3 respectively.MethodsThe CD34 + cells were isolated from human UCB mononuclear cells through magnetic activation cell sorting (MACS) with purity of (≥ 90%) and were subjected to differentiate into NK cells. After 21 days of induction with SFTG36 (SCF, FLt-3L, TPO, GM-CSF, IL-3 and IL-6), IS721 (IGF-1, SIS3, IL-7 and IL-21) and IL-15/Hsp70 media, NK cells phenotypes were studied and their cytotoxicity against K562 human erythroleukemia cells and SKOV3 ovarian carcinoma cells was analyzed.ResultsThe NK cells induced in SFTG36/IS721 medium were selected for activation due to their higher expression of CD56 + 16 + CD3 − (93.23% ± 0.75) and mean fluorescence intensity (MFI) of NKG2D + (168.66 ± 20.00) and also a higher fold expansion potential (11.893 ± 1.712) compared to the other groups. These cells once activated with 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.ConclusionsThe 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.
Highlights
The natural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to the NK cells from adult donors
The aim of the present study was to differentiated to functional NK cells from UCB-CD34 + through manipulation of RAS/MAPK, PI3K/AKT, JAK/STAT and TGF-β as the critical activation and inhibition signaling pathways of NK cells starting from the early differentiation phase (Fig. 1)
Our results indicated that a higher number with larger colonies of CFU-BFU-E, CFU-GM and CFU-GEMM was formed in CD34 + cells treated with SFTG36 (P < 0.0001) (Fig. 2A, B)
Summary
The natural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to the NK cells from adult donors. Shokouhifar et al Cancer Cell Int (2021) 21:298 been made to apply the NK cells derived from peripheral blood to adoptive therapy of solid and hematopoietic cancers [1, 5] This approach has encountered several drawbacks including: insufficient cell number, limited donors, lower response to stimulants, decreased expansion potential and increased dysfunctionality. To address these limitations, other sources for NK cells such as bone marrow, embryonic cells, induced pluripotent and umbilical cord blood stem cells (UCB-HSC) have been tested [6,7,8,9,10]. The dysfunctional NK cell phenotypes are often determined as down regulation of specific surface activating receptors including NKG2D, CD16 and NCRs (NKp30, NKp44 and NKp46) [12,13,14] and up regulation of the inhibitory receptors including NKG2A, Tim and PD-1 [13, 15,16,17,18] on NK cells in tumors and chronic infections [13]
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