title: Human colorectal cancer cells induce T-cell death through release of proapoptotic microvesicles: role in immune escape.
abstract: OBJECTIVE
  Normal and neoplastic cells release microvesicles, whose effects on the immune system still need to be elucidated. Because human colorectal cancer cells are hypothesized to escape immune recognition by expressing proapoptotic molecules, we investigated whether microvesicles bearing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand and inducing apoptosis of activated T cells are secreted by colorectal cancer cells both in vitro and in affected patients.
  
  
  METHODS
  Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand expression were analyzed in colorectal cancer cells and purified microvesicles by flow cytometry, Western blotting, and immunoelectron microscopy. Microvesicle tumor origin was assessed through simultaneous detection of lysosomal (CD63) and adenocarcinoma (carcinoembryonic antigen) markers. Proapoptotic activity of microvesicles was evaluated by annexin V/propidium iodide staining and caspase activation in T cells, including CD8+ T lymphocytes from colorectal cancer patients.
  
  
  RESULTS
  Colorectal cancer cells showed a granular pattern of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand expression, suggesting a secretory behavior. These proapoptotic molecules were detected on isolated microvesicles, together with class I HLA, CD63, and carcinoembryonic antigen. Microvesicles induced Fas ligand-mediated and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of activated CD8+ T cells generated from colorectal cancer patients. Microvesicles with comparable phenotypes and functions were found in plasma from patients with advanced disease, whereas vesicular structures expressing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand were also detected in colorectal cancer specimens.
  
  
  CONCLUSIONS
  These data show that colorectal cancer induces T-cell apoptosis through the release of Fas ligand-bearing and tumor necrosis factor-related apoptosis-inducing ligand-bearing microvesicles both in vitro and in vivo. This mechanism of immune escape has potential implications as a prognostic factor and could be targeted for the development of new antitumor therapies in colorectal cancer patients.
authors.affiliation: Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milan, Italy.
authors.forename: Veronica
authors.initials: V
authors.lastname: Huber
authors.forename: Stefano
authors.initials: S
authors.lastname: Fais
authors.forename: Manuela
authors.initials: M
authors.lastname: Iero
authors.forename: Luana
authors.initials: L
authors.lastname: Lugini
authors.forename: Paola
authors.initials: P
authors.lastname: Canese
authors.forename: Paola
authors.initials: P
authors.lastname: Squarcina
authors.forename: Annamaria
authors.initials: A
authors.lastname: Zaccheddu
authors.forename: Marisa
authors.initials: M
authors.lastname: Colone
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Arancia
authors.forename: Massimo
authors.initials: M
authors.lastname: Gentile
authors.forename: Ettore
authors.initials: E
authors.lastname: Seregni
authors.forename: Roberta
authors.initials: R
authors.lastname: Valenti
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Ballabio
authors.forename: Filiberto
authors.initials: F
authors.lastname: Belli
authors.forename: Ermanno
authors.initials: E
authors.lastname: Leo
authors.forename: Giorgio
authors.initials: G
authors.lastname: Parmiani
authors.forename: Licia
authors.initials: L
authors.lastname: Rivoltini
mesh_terms: D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D015179:Colorectal Neoplasms / Q000276:immunology* / Q000503:physiopathology*; D022162:Cytoplasmic Vesicles; D053222:Fas Ligand Protein; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*; D053221:TNF-Related Apoptosis-Inducing Ligand; D014407:Tumor Cells, Cultured; D019139:Tumor Escape / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha