O2166: Oncolytic Virus Improves Attraction of Natural Killer Cells to Neuroblastoma Cells

Background: Neuroblastoma is the most common extracranial solid tumor in children and remains responsible for about 1 in 8 pediatric cancer associated deaths. Patients with high-risk disease have particularly poor outcomes with a 50% chance of disease recurrence and are subjected to a therapeutic regimen that involves considerable toxicities. Immunotherapies are a novel approach to targeting neuroblastoma and they may result in less harmful side effects. Immunotherapy has realized minimal success in pediatric solid tumors due to a host of factors. For example, in neuroblastoma, MYCN supports an immunosuppressive tumor microenvironment. We have previously demonstrated that an oncolytic Herpes Simplex Virus (oHSV) effectively targets, replicates within, and kills neuroblastoma cell lines and patient derived xenograft tumors. Natural killer (NK) cells constitute a growing area of investigation for use as an immunotherapy. NK cells innately kill tumor cells, as well as virus infected cells. We hypothesized that infecting neuroblastoma cells with oHSV would increase attraction of NK cells toward the tumor cells.

Methods: Both SK-N-AS (MYCN non-amplified) and SK-N-BE(2) (MYCN amplified) neuroblastoma cell lines were used. A cultured NK cell line, NK92 MI, previously proven safe for clinical applications, were utilized for these studies. SK-N-AS (5x104) and SK-N-BE(2) (2.5x104) cells were either not infected or infected with 1 plaque forming unit (PFU) of oHSV for 24 hours. Following infection, CMFDA+ stained NK cells (5x105) were added to the top of a Boyden chamber and allowed to migrate for 24 hours to the bottom well where there were either no, non-infected, or oHSV infected neuroblastoma cells. NK cells were collected from the bottom well and counted via flow cytometry (Figure 1A). Similar studies were performed with NK cells that had been previously primed by exposing them to oHSV infected SK-N-BE(2) cells.

Results: At baseline, there was no significant difference between the number of NK cells migrating to SK-N-AS compared to SK-N-BE(2) cells (612 ± 296 vs. 504 ± 224, p = 0.78, Figure 1B). Oncolytic HSV infection of the neuroblastoma cells resulted in an increase in NK cell migration by nearly 2 times in SK-N-AS and nearly 3 times in SK-N-BE(2) cells compared to cells not infected with oHSV (Figure 1C). When NK cells were primed with previous exposure to SK-N-BE(2) cells infected with oHSV, these primed NK cells migrated to non-infected SK-N-BE(2) cells significantly better than non-primed NK cells (440 ± 55 vs 176 ± 69, p < 0.05, Figure 1D).

Conclusion: We found that oHSV infection increased NK cell attraction to neuroblastoma tumor cells. These results suggest that oHSV may expand the feasibility of using NK cells as an immunotherapeutic adjunct against solid tumors that have been historically immunologically cold.