TRAIL on activated human NK cells contributes to cytotoxicity against human neuroblastoma cell lines that express TRAIL-R2

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Human neuroblastoma cells are susceptible to direct cytotoxicity of IL-2 activated natural killer (aNK) cells. The aim of this study was to examine the potential role of TRAIL expressed by aNK cells in aNK cytotoxicity against neuroblastoma cells. Examining a panel of 20 human neuroblastoma cell lines (multi-drug sensitive and resistant), we discovered that sensitivity to cytotoxicity mediated by purified (>96%) aNK cells (calcein-AM 6 hr assay) correlated with expression of TRAIL receptor-2 (TRAIL-R2) mRNA expression (Affymetrix U133A microarray analysis) and that mRNA correlated with surface TRAIL-R2 protein expression (p < 0.024). Flow cytometry demonstrated that 12 of the 20 cell lines expressed TRAIL-R2 at significant levels (Mean Fluorescence Intensity Index >4; MFI Index = MFI anti-TRAIL-R2 mAb/MFI isotype control mAb). No correlation was found between TRAIL-R2 expression and MYCN expression, p53 function, or drug sensitivity. Microarray analysis of primary tumors from patients with metastatic disease (stage 4) obtained at diagnosis (n=163), during induction chemotherapy (n=21), or at disease progression (n=18) demonstrated expression of TRAIL-R2 mRNA by neuroblastomas. Overall, 64% of these tumors expressed TRAIL-R2 mRNA at a level expected to result in protein expression. Using a neutralizing anti-TRAIL mAb, we showed significantly greater tumor cell survival for 6 of the 12 TRAIL-R2⁺ cell lines (median of 14% and range of 6% to 27% increased tumor cell survival in 31 experiments), demonstrating a role for TRAIL-R2 in aNK-mediated cytotoxicity. Of interest, 2 cell lines exhibiting an important role of TRAIL in aNK-mediated cytotoxicity were insensitive to soluble TRAIL. Six of eight cell lines exhibiting partial resistance to aNK cell-mediated killing expressed little or no TRAIL-R2 by flow cytometry. Even though TRAIL contributed to cytotoxicity of aNK cells, the perforin pathway dominated since concanamycin A, an inhibitor of the perforin pathway, almost completely inhibited killing in all 20 cell lines. Our data show TRAIL-R2 expression by both neuroblastoma cell lines and tumors from patients, and suggest that TRAIL-induced signaling can supplement perforin-dependent killing of approximately 50% of TRAIL-R2⁺ human neuroblastoma cell lines. Targeting the TRAIL pathway has become an important therapeutic strategy for many human malignancies, and it appears that aNK may be able to activate this death pathway for some neuroblastomas.