Potential treatment approaches for malignant peritoneal mesothelioma: in vivo and in vitro experimental study of natural killer cell immunotherapy

Dynamic cellular and subcellular changes to MPM cells after co-culture with NK cells. (A) Dynamic interactions between NK and MPM cells were studied using live cell imaging techniques. Scale bar, 50 μm. Red circles represent MPM cells. (B) The process of NK cell-induced apoptosis in MPM cells. Scale bar, 50 μm. (C) Transmission electron microscopy was used to observe the internal structure of cells. Orange and red arrows represent NK and MPM cells, respectively. Yellow represents NK cell organelles. From left-to-right, top-to-bottom, the scale bars were 5 μm, 2 μm, 2 μm, 5 μm, 1 μm, 500 nm, 2 μm, and 2 μm, respectively. (D) The surface of cells was detected by scanning electron microscopy. Yellow and red represent NK and MPM cells, respectively. From left-to-right, top-to-bottom, the scale bars were 20 μm, 10 μm, 50 μm, 50 μm, 5 μm, 3 nm, 30 μm, and 5 μm, respectively. (E) Schematic diagram of the apoptosis process in MPM cells.

Effector molecules released by NK cells at the optimal NK cytotoxicity level. (A) Level of NK cell membrane CD107A expression for 4 h at a 10:1 E:T ratio. (B) Statistical results of CD107A expression. (C–F) Levels of effector molecule expression for 24 h at a 10:1 E:T ratio. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

NK cell cytotoxicity gradually decreased as tumor density increased. (A) Images of crystal violet staining after co-culturing NK and MPM cells (single tumor cell colony level) for 24 h. (B) CCK-8 experiment to detect the NK cell cytotoxicity against MPM cells at different densities for 24 h. (C) Images of Wright-Giemsa staining at the large tumor cell confluence level for 24 h. Scale bar, 50 μm. (D) Statistical results of the NK cell adhesion quantity at different tumor density levels for 24 h. (E) Flow cytometry analysis of NK cell activity after co-culturing with MPM cells (large tumor cell confluence level) for 24 h. (F) Statistical results of (E). (G) Immunofluorescence images of Ki-67 in MPM cells at the large tumor cell confluence level for 24 h. Yellow and red arrows represent NK and MPM cells, respectively. Scale bar, 20 μm. (H) Statistical results of Ki-67 expression in MPM cells for 24 h. (I–L) The level of different effector molecule expression (large tumor cell confluence level). (M) Potential mechanism diagram of infiltrating NK cell dysfunction.

NK cell adoptive transfer therapy reduced the tumor burden and prolonged survival of mice. (A) The workflow of the experimental design. (B, C) Gross anatomy of mice. C, colon; D, diaphragm; K, kidney; L, liver; P, pancreas; SP, spleen; ST, stomach; U, uterus; X, xiphoid; Tumor tissue is indicated by yellow arrows. (D) Results of small animal magnetic resonance. (E) Results of ePCI score. (F) Ascites in mice. (G) Survival time of mice. (H) Body weight changes in different groups. NK injection was indicated by orange arrows. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

NK cell releasing effector molecules inhibited tumor growth. (A) On day 30, IHC staining was performed to examine the infiltration of NK cells. NK cells infiltrated at the edge of the tumor tissue. Yellow and red arrows indicate NK and MPM cells, respectively (antibody: anti-CD56, species reaction: reacts with humans, but not with mice or rabbits). Scale bar, 50 μm. (B) On day 30, ELISA was used to detect the levels of effector molecule expression in tumor tissue. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.