Abstract: BACKGROUND: Immunotherapy enhances the anti-cancer immune response in many ways, so combined immunotherapy is a better choice. Ultrasound-targeted microbubble destruction technique delivers drugs, genes, antibodies and cytokines directly to the cytoplasm of immune cells and enhances the immune response. However, the application of ultrasound-targeted microbubble destruction technique in the treatment of ovarian cancer with both CXC chemokine receptor 4 antibody and programmed death-ligand 1 antibody has not been reported.
OBJECTIVE: To investigate the effect of ultrasound irradiation on the proliferation and migration of ovarian cancer cells with CXC chemokine receptor 4 antibody and programmed death-ligand 1 antibody double targeted nanobubbles.
METHODS: IOSE-80 normal ovarian epithelial cells, SKOV3 and CAOV3 ovarian cancer cells were cultured and expanded. Double labeling fluorescence immunoassay was used to co-locate CXC chemokine receptor 4 and programmed death-ligand 1 protein. Western blot assay was used to detect the relative expression of CXC chemokine receptor 4 and programmed death-ligand 1 protein in three kinds of cells and screen out the experimental cells, i.e., pure nanobubbles, nanobubbles carrying CXC chemokine receptor 4 antibody, nanobubbles carrying CXC chemokine receptor 4 and programmed death-ligand 1 antibody. SKOV3 ovarian cancer cells in the logarithmic growth phase were taken and divided into six groups for treatment. Group A was added with McCoy's 5A medium. Group B was added with McCoy's 5A medium containing stromal cell-derived factor-1. Group C was added with pure nanobubble solution and McCoy's 5A medium containing stromal cell-derived factor-1. Group D was added with nanobubble solution containing CXC chemokine receptor 4 antibody and McCoy's 5A medium containing stromal cell-derived factor-1. Group E was added with nanobubble solution containing CXC chemokine receptor 4 and programmed death-ligand 1 antibody and McCoy's 5A medium containing stromal cell-derived factor-1. Pure nanobubble solution was added in group F. After ultrasonic irradiation for 120 seconds and incubation for 48 hours, the survival rate of cells was measured by CCK-8 assay, and the healing and migration ability of cells in groups B-E were measured by wound healing test.
RESULTS AND CONCLUSION: (1) Immunofluorescence staining showed that CXC chemokine receptor 4 and programmed death-ligand 1 protein could be expressed in all three kinds of cells. Western blot assay showed that the expression levels of CXC chemokine receptor 4 and programmed death-ligand 1 in SKOV3 and CAOV3 ovarian cancer cells were significantly higher than those in IOSE-80 normal ovarian epithelial cells (P < 0.05). (2) CCK-8 assay results exhibited that the cell survival rate of group B was higher than that of group A (P < 0.05). The cell survival rate of group F was lower than that of group A (P < 0.05). The cell survival rate of groups B-E decreased gradually, and there were significant differences between the two groups (P < 0.05). (3) Wound healing test demonstrated that the cell healing rate of groups B-E decreased gradually, and there were significant differences between the two groups (P < 0.05). (4) The results show that the use of CXC chemokine receptor 4 antibody and programmed death-ligand 1 antibody double targeted nanobubbles under ultrasound-targeted microbubble destruction can significantly inhibit the proliferation and migration of ovarian cancer cells. 

Key words: ovarian tumor, proliferation, targeted nanobubble, ultrasound-targeted microbubble destruction, CXC chemokine receptor 4, programmed death-ligand 1