title: Infiltration of lymphocyte subpopulations into cancer microtissues as a tool for the exploration of immunomodulatory agents and biomarkers.
abstract: BACKGROUND
  The interaction between the immune system and malignant diseases is a proven key target for cancer therapy. We describe an innovative 3D cell culture system comprising both immune and cancer cells to evaluate their interaction and immune cell infiltration to provide an innovative in vitro screening of immunomodulatory agents and biomarkers.
  
  
  METHODS
  3D tumor microtissues were cultivated using a hanging drops system. Human non-small-cell lung cancer cell lines were incubated for 7 days to form microtissues. On day 5, peripheral blood mononuclear cells (PBMC) were added with or without interleukin-2 (IL-2) for 24 or 48h. Viability of cancer cells and the infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of tumor cells and PBMC and the infiltration of the PBMC into the tumor microtissues were analyzed by immunohistochemistry. Quantification of infiltration was measured by applying the TissueFAXS system.
  
  
  RESULTS
  Immunohistochemistry revealed PBMC infiltration in all cell lines which increased under IL-2 stimulation. Analysis of infiltrating populations showed both lymphocyte subpopulations and monocytes within the tumor microtissues. In all three co-cultures, CD3+CD8+ and CD3+CD8+CD45R0+CD28+ lymphocytes were increased with IL-2, whereas CD3+CD8+CD45R0-CD28+ PBMCs were decreased with and without IL-2 stimulation.
  
  
  CONCLUSIONS
  In summary, we present a novel cell culture system to study the interaction between cancer cells and immune cells in 3-dimensional microtissues. In addition, we report for the first time an in vitro infiltration assay based on 3D microtissues. This model has the potential to provide a tool for ex-vivo drug testing and biomarker screening of immunomodulatory agents.
authors.affiliation: Department of Internal Medicine V, Medical University of Innsbruck, Innsbruck, Tyrol, Austria; Tyrolean Cancer Research Institute, Innsbruck, Tyrol, Austria. Electronic address: Stefan.Koeck@i-med.ac.at.
authors.forename: Stefan
authors.initials: S
authors.lastname: Koeck
authors.affiliation: Department of Anatomy, Histology and Embryology, Medical University of Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Marit
authors.initials: M
authors.lastname: Zwierzina
authors.affiliation: Department of Internal Medicine V, Medical University of Innsbruck, Innsbruck, Tyrol, Austria; Tyrolean Cancer Research Institute, Innsbruck, Tyrol, Austria.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Huber
authors.affiliation: Department of Anatomy, Histology and Embryology, Medical University of Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Mario
authors.initials: M
authors.lastname: Bitsche
authors.affiliation: Department of Internal Medicine V, Medical University of Innsbruck, Innsbruck, Tyrol, Austria; Tyrolean Cancer Research Institute, Innsbruck, Tyrol, Austria.
authors.forename: Edith
authors.initials: E
authors.lastname: Lorenz
authors.affiliation: Department of Internal Medicine V, Medical University of Innsbruck, Innsbruck, Tyrol, Austria; Tyrolean Cancer Research Institute, Innsbruck, Tyrol, Austria.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gamerith
authors.affiliation: Department of Otorhinolaryngology, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Jozsef
authors.initials: J
authors.lastname: Dudas
authors.affiliation: InSphero AG, Schlieren, Canton of Zürich, Switzerland.
authors.forename: Jens M
authors.initials: JM
authors.lastname: Kelm
authors.affiliation: Department of Internal Medicine V, Medical University of Innsbruck, Innsbruck, Tyrol, Austria; Tyrolean Cancer Research Institute, Innsbruck, Tyrol, Austria.
authors.forename: Heinz
authors.initials: H
authors.lastname: Zwierzina
authors.affiliation: Department of Internal Medicine V, Medical University of Innsbruck, Innsbruck, Tyrol, Austria; Tyrolean Cancer Research Institute, Innsbruck, Tyrol, Austria.
authors.forename: Arno
authors.initials: A
authors.lastname: Amann
mesh_terms: D000954:Antigens, Surface / Q000378:metabolism; D015415:Biomarkers; D045744:Cell Line, Tumor; D002470:Cell Survival; D018920:Coculture Techniques; D003602:Cytotoxicity, Immunologic; D006801:Humans; D056747:Immunomodulation; D016130:Immunophenotyping; D016131:Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D009000:Monocytes / Q000276:immunology / Q000378:metabolism; D009369:Neoplasms / Q000276:immunology* / Q000473:pathology; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured
chemical_list: D000954:Antigens, Surface; D015415:Biomarkers
keywords: 3D; Biomarker; Cancer; Co-culture; Immune cells; Infiltration; Subpopulation