TRAIL is a cell surface death ligand that can kill most tumor cell types expressing TRAIL-receptor 1 (TRAIL-R1) or TRAIL-R2. Observing that TRAIL was expressed by natural killer (NK) cells which were activated (aNK) with IL2 plus IL15, we characterized the role of TRAIL in aNK cytotoxicity against neuroblastoma cells, a tumor type reported to be resistant to soluble, recombinant TRAIL. We examined a panel of 17 human neuroblastoma cell lines (multi-drug sensitive and resistant) and found that sensitivity to cytotoxicity mediated by purified (>98%) aNK cells (calcein-AM 8 hr assay) correlated with expression of TRAIL receptor-2 (TRAIL-R2) mRNA expression (Affymetrix U133A oligonucleotide microarray analysis) and with surface protein expression (flow cytometry). mRNA correlated with surface TRAIL-R2 protein expression (p < 0.024). Little or no TRAIL-R2 was expressed by 4 of the 6 cell lines exhibiting partial resistance to aNK killing. TRAIL-R1 was not expressed by any of the cell lines, and no correlation was observed with TRAIL-R3 or TRAIL-R4. A neutralizing mAb (75411.11) that blocks TRAIL on aNK cells significantly increased the survival of 9 of the 13 TRAIL-R2+ tumor cell lines (median 14%, range 6%-27%), demonstrating a measurable role for TRAIL-R2 in aNK cytotoxicity in vitro. Even though TRAIL contributed to the cytotoxicity of aNK cells, perforin was the dominant pathway since concanamycin A, an inhibitor of the perforin pathway, almost completely inhibited killing in all 17 cell lines. Microarray analysis of primary neuroblastomas from 153 patients with stage 4 (metastatic) disease revealed that almost all expressed higher TRAIL-R2 mRNA levels than were expressed by most normal neural or neural crest derived tissues. Progression-free survival (PFS) for patients diagnosed after 12 months of age correlated inversely with expression of TRAIL-R2 independent of MYCN status; PFS associated with high and low expression (above and below the median value) was 35% and 75%, respectively (p<0.0012). This, as well as our in vitro data wherein 4 of 13 TRAIL-R2+ cell lines were insensitive to aNK expression of TRAIL, suggests that neuroblastoma cells with high expression can survive by disabling the TRAIL-R2 mediated death pathway. Definition of mechanisms of resistance may lead to therapeutic strategies that restore sensitivity to TRAIL.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]