454 Oncolytic parainfluenza virus 5 vector enhances natural killer cell killing of lung tumor cells in 2D and 3D spheroid cultures

Background Natural killer (NK) cells are innate immune cells with natural cytotoxicity towards both tumor cells and virus infected cells. We have developed a particle-based method for in vitro specific expansion of NK cells that yields highly cytotoxic NK cells (PM21-NK cells). There is intense interest in the use of novel oncolytic viruses with the potential to synergize with immune cells to kill tumor cells. Here we have tested the hypothesis that infection with a tumor-selective cytopathic Parainfluenza virus 5 (PIV5-P/V) vector will enhance PM21-NK cell-mediated killing of lung cancer cells in both 2-dimensional (2D) and 3-dimensional (3D) cultures. Methods In 2D cultures, live cell time-lapse imaging, flow cytometry and luminescence-based methods were used to assess the killing efficiency of PM21-NK cells against A549 lung tumor cells infected with PIV5-P/V. Blocking antibodies were used to evaluate different NK cell activating receptors involved in recognition of infected tumor cells. IncuCyte live cell imaging system was used to assess real time killing of 3D lung spheroids by a combination of NK cells and PIV5-P/V virus. Z-stack spheroid images were captured using Keyence microscope. Results In 2D cultures, PM21 NK cells efficiently kill A549 cells that have been infected with P/V CPI- virus and enhance the overall rate of killing compared to uninfected cell targets. Antibody blocking showed that the viral Hemagglutinin-Neuraminidase (HN) glycoprotein and NK cell receptors NKp30, NKp46 and NKG2D were involved in PM21-NK cell recognition of PIV5-P/V infected A549 cells. In 3D cultures of A549 tumor spheroids, PIV5-P/V infection was limited to the outer layer of the spheroid, with restricted spread of the infection to inner compartments. However, addition of PM21-NK cells to PIV5-P/V-infected spheroids resulted in killing of not only the infected surface of the spheroid but continued to the uninfected cells located at the center of the spheroid. Conclusions Our data support the potential of combining oncolytic virotherapy along with PM21-NK cell adoptive therapy against lung cancer.