title: Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.
abstract: BACKGROUND
  NY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC).
  
  
  RESULTS
  We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157-165) peptide specific chimeric antigen receptor (CAR) CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels.
  
  
  CONCLUSIONS
  These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.
authors.affiliation: Tissue Biology Research Unit, Department of Surgery, University Children's Hospital Zurich, Zurich, Switzerland; Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland.
authors.forename: Agnes S
authors.initials: AS
authors.lastname: Klar
authors.affiliation: Department of Oncology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Jakka
authors.initials: J
authors.lastname: Gopinadh
authors.affiliation: Department of Oncology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Sascha
authors.initials: S
authors.lastname: Kleber
authors.affiliation: Department of Oncology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Wadle
authors.affiliation: Department of Oncology, University Hospital Basel, Basel, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Renner
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D000077209:Decitabine; D005260:Female; D015789:HLA-A2 Antigen / Q000378:metabolism; D006801:Humans; D007167:Immunotherapy; D007371:Interferon-gamma / Q000378:metabolism; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009101:Multiple Myeloma / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D010455:Peptides / Q000276:immunology; D060888:Real-Time Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D008565:Membrane Proteins; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D000077209:Decitabine; D007371:Interferon-gamma; D001374:Azacitidine