Ethical statement
Umbilical cord blood specimens were collected for research purposes following protocols approved by the Ethics Committee of the Medical Genetics Institute, number 02/2024/CT-VDTYH. Informed consent was obtained from pregnant women prior to labor and birth. All methods adhered to approved guidelines and regulations set forth by this committee. The samples were collected at the Department of Gynecology and Obstetrics of the University Hospital of the Ho Chi Minh City University of Medicine and Pharmacy.
Cell lines
The K562 cell line was procured from the American Type Culture Collection (ATCC). Engineered K562 cell lines expressing CD80, 4-1BBL, or membrane-bound IL-21, either individually or in combination, were generated by transducing K562 cells with CD80-, 4-1BBL-, or mbIL-21-lentivirus and subsequently sorted using the WOLFG2 Cell Sorter. These cells and their derivatives were cultured in RPMI 1640 (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C under 5% CO2. They were validated by flow cytometry with the antibodies against CD80 (#375409), 4-1BBL (#311505), and IL21 (#513003) (Biolegend). The Lenti-X293T cell line was purchased from Takara Bio (USA) and maintained in DMEM (Gibco, USA) supplemented with 10% FBS and 1% penicillin-streptomycin. MKN-45 cells were generously provided by Dr. Phu-Hung Nguyen (Thai Nguyen University of Science, Thai Nguyen, Viet Nam) and cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Additionally, MCF-7 cells were kindly provided by Dr. Thuy-Vy Nguyen (Ho Chi Minh City University of Science, Ho Chi Minh City, Viet Nam) and maintained in DMEM (Gibco, USA) medium supplemented with 10% FBS and 1% penicillin-streptomycin. Before experimentation, all cell lines were rigorously tested and confirmed negative for mycoplasma contamination.
Peripheral blood mononuclear cell and NK cell isolation
Umbilical cord blood, collected using CPDA-1 anticoagulant, was processed immediately upon retrieval. Blood samples were diluted at a 1:2 ratio with phosphate-buffered saline (PBS) and carefully layered onto Lymphoprep (Stemcell Technologies, Canada) while maintaining a 2:1 ratio. Density-gradient centrifugation was performed for 30 minutes at 1000 x g without applying the brake. The mononuclear cells were isolated from the whitish ring formed at the interface between the Lymphoprep and plasma, followed by a washing step using PBS.
NK cell isolation and expansion
Each cord-blood sample was processed using 108 mononuclear cells to isolate NK cells employing the MojoSort Human NK Cell Isolation Kit (Biolegend, USA). The isolated NK cells were cultured in AIM-V medium (Gibco, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) from Cytiva (Sweden), 500 IU/ml IL-2 (PeproTech, USA), and 20 ng/ml IL-15 (PeproTech, USA), with or without feeder cells. The culture medium was refreshed every 3 days. For feeder-cell-dependent expansion, freshly isolated NK cells were stimulated at a 1:2 ratio with either K562 cells or engineered K562 cells, irradiated with 100 Gy. Feeder cells were replenished every 7 days. Cell numbers were determined by staining with trypan blue and counting via trypan blue exclusion using a hemocytometer. To conduct phenotype analysis, cells were harvested and stained for CD3 (#300308), CD56 (#362526), NKG2D (#320807), NKp46 (#331917), NKp44 (#325107), NKp30 (#325209), CD16 (#302007), NKG2A(#375107), and TIGIT (#372703) (Biolegend) markers, followed by analysis using a flow cytometer.
Lentivirus production and NK cell transduction
The Lenti-X293T cell line obtained from Takara Bio (US) served as the packaging cell line for lentivirus production. Lentiviruses were generated via transient transfection of pMDLg/pRRE, pMD2.G, and pRSV-Rev plasmids (NovoPro Bioscience, China) alongside pCDH-EF1a-DNAM-1 CAR-IRES-EGFP constructs (Epoch Life Science, USA), forming a chimeric antigen receptor. For BaEV-pseudotype lentiviral vectors, transfection involved BaEVRLess encoding plasmid along with pMDLg/pRRE, pRSV-Rev, pCDH-EF1a-DNAM-1 CAR-IRES-EGFP, with or without pMD2.G. The BaEVRLess plasmid was generously provided by Prof. Verhoeyen Els (CIRI, Ecole Normal Supérieure, Lyon, France).
NK cell expansion was conducted using K562-CD80-mbIL-21 cells at a 1:2 ratio for 5 days in AIM-V medium supplemented with 10% FBS, 500 IU/ml IL-2, and 20 ng/ml IL-15. Subsequently, restimulation was carried out with K562-CD80-mbIL-21 cells at a 1:1 ratio for 2 days before the transduction process with CAR-lentivirus. The transduction of NK cells was performed in 24-well plates precoated overnight at 4°C with Retronectin (Takara Bio, USA) at 5 μg/cm2. On the transduction day, lentiviral supernatant was introduced into the wells, followed by a 6-hour incubation at 37°C before adding NK cells. Cells underwent centrifugation at 1000 x g for 1.5 hours at 32°C. Post-transduction, the medium was refreshed the following day and subsequently every 3 days, with CAR expression assessment performed 5 days after transduction.
Cytotoxicity assay
For cytotoxicity assessment, CAR-NK and CAR-T cells were co-cultured with MKN-45 or MCF-7 cells labeled with CellTrace Far Red (Invitrogen, USA) at multiple E:T ratios. Following a 4-hour incubation, cells were harvested and stained with Zombie Red (Biolegend, USA) for viability analysis using flow cytometry. The cytotoxicity index was determined by calculating the ratio of target cell death percentage in the co-culture to the rate of cell death in the target cell monoculture.
Spheroid formation
Spheroid formation was conducted as described previously33. Briefly, MKN-45 cells were cultured in 10-cm culture dishes using RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Upon reaching confluency, cells were trypsinized and suspended in serum-free RPMI 1640 medium supplemented with MaxGel (Sigma-Aldrich, USA) at a 1:100 ratio, along with 100 ng/ml EGF and 20 ng/ml FGF. The parental cells were then seeded at a concentration of 1000 cells in 100 µl medium per well, in an ultra-low attachment plate sourced from Corning (USA). Labeled effector cells (NK/T cells) were incubated with the spheroids for 48 hours. After that, the spheroids were washed and trypsinized. Then, suspended cells were analyzed by flow cytometry. The percentage of the effector cells detected in the spheroids was a measure for spheroid infiltration.
Spheroid infiltration and cell death induced by immune cells
T cells and NK cells were washed with PBS then resuspended in AIM-V 10% FBS. Each spheroid was co-cultured with 104 immune cells for 48 hours, then the cells were collected, trypsinized, and stained with either anti-CD3 or anti-CD56 antibodies to quantify T cell and NK cell infiltration. Cell death was assessed by staining with Zombie Red fixable viability dye (Biolegend, USA). T cell and NK cell infiltration was measured by the percentage of CD3+ Zombie- or CD56+ Zombie- cells in total cells. Cell death was quantified by the percentage of CD3- Zombie+ or CD56- Zombie+ cells in total cells.
Statistical analysis
Statistical analyses were conducted using Prism-GraphPad software. Specifically, Mann-Whitney tests were employed for comparing transduction rates, while Dunn’s multiple comparison tests were utilized to assess cytotoxicity indices in co-culture with MKN-45 or MCF-7 cells.