title: Neutrophils are cytotoxic and growth-inhibiting for neuroblastoma cells with an anti-GD2 antibody but, without cytotoxicity, can be growth-stimulating.
abstract: Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells. To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside (GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence of target cells in 96-well plates after 6-18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive: SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by DIMSCAN, was correlated with neuroblastoma cell number in both assays (100-2000 cells/well). In the cytotoxicity test, both neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5-50:1) and mAb ch.14.18 dose (0.1-10 microg/ml). ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 microg/ml). Without antibody, neutrophils inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion, neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell growth if antibody is not present or if GD2 is not expressed.
authors.affiliation: Division of Hematology-Oncology, MS 57, Department of Pediatrics, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90054-0700, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Chen
authors.forename: C P
authors.initials: CP
authors.lastname: Reynolds
authors.forename: R C
authors.initials: RC
authors.lastname: Seeger
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D000920:Antibody-Dependent Cell Cytotoxicity; D000951:Antigens, Neoplasm / Q000276:immunology*; D002452:Cell Count; D002455:Cell Division; D017077:Culture Media, Conditioned; D004306:Dose-Response Relationship, Immunologic; D004801:Eosine Yellowish-(YS); D005452:Fluoresceins; D005456:Fluorescent Dyes; D005732:Gangliosides / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000276:immunology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000276:immunology; D009447:Neuroblastoma / Q000276:immunology / Q000473:pathology*; D009504:Neutrophils / Q000276:immunology / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D017077:Culture Media, Conditioned; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005732:Gangliosides; D011993:Recombinant Fusion Proteins; C085925:calcein AM; C019403:ganglioside, GD2; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004801:Eosine Yellowish-(YS); C018506:diacetylfluorescein