Fig. 4

Neo-2/15 maintains mitochondrial fitness of CAR-NK cells within TME. a, b BBζ after being stimulated with IL-2 or Neo-2/15 and co-cultured with AsPC-1 cells or not at the E:T ratio of 1:1 for 4 hours is incubated with Mitotracker red (a) or JC-1 (b). For panel (a), data were analyzed using IncuCyte real-time imaging system to detect and quantify Mitotracker Red of mitochondrial image. Data are presented as mean ± SD (ns, not significant, *P < 0.05, n = 3). Scale bar = 200 μm. For panel (b), JC-1 aggregates are shown as red (indicating healthy mitochondria) whereas monomers are shown as green (indicating loss of mitochondrial membrane potential), and nuclei are stained with DAPI and are showing as blue. c Representative TEM images of BBζ treated with IL-2 or Neo-2/15 in the absence (top) or presence of AsPC-1 cells (middle), or with Mdivi-1 pretreatment and co-cultured with AsPC-1 cells (bottom). d IL-2 or Neo-2/15 stimulated BBζ pretreated with 10 nM Mdivi-1 and co-cultured with AsPC-1 cells for 4 hours at the E:T ratio of 1:1. The cytotoxicity is evaluated by the LDH assay. Data are presented as mean ± SD (*P < 0.05, n = 4). e Representative TEM images of BBζ treated with Neo-2/15 and co-cultured with AsPC-1 cells in the absence or presence of 10058-F4 pretreatment. Red arrows denote mitochondria. f IL-2 or Neo-2/15 stimulated BBζ pretreated with 60 μM 10058-F4 and co-cultured with AsPC-1 cells for 4 hours at the E:T ratio of 1:1. The cytotoxicity is evaluated by the LDH assay. Data are presented as mean ± SD (*P < 0.05, n = 4). g BBζ treated with IL-2 (red) or Neo-2/15 (blue) and co-cultured without (top) or with (bottom) AsPC-1 cells are stained with DCFH-DA and analyzed by flow cytometry