title: A simple and rapid protocol to non-enzymatically dissociate fresh human tissues for the analysis of infiltrating lymphocytes.
abstract: The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
authors.forename: Soizic
authors.initials: S
authors.lastname: Garaud
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Gu-Trantien
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
authors.forename: Jean-Nicolas
authors.initials: JN
authors.lastname: Lodewyckx
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
authors.forename: Anaïs
authors.initials: A
authors.lastname: Boisson
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
authors.forename: Pushpamali
authors.initials: P
authors.lastname: De Silva
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
authors.forename: Laurence
authors.initials: L
authors.lastname: Buisseret
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
authors.forename: Edoardo
authors.initials: E
authors.lastname: Migliori
authors.affiliation: Institut d'Immunologie Médicale, Université Libre de Bruxelles.
authors.forename: Myriam
authors.initials: M
authors.lastname: Libin
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles.
authors.forename: Céline
authors.initials: C
authors.lastname: Naveaux
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Flow Cytometry Core Facility, Université Libre de Bruxelles.
authors.forename: Hugues
authors.initials: H
authors.lastname: Duvillier
authors.affiliation: Molecular Immunology Unit, Université Libre de Bruxelles; Institut Jules Bordet, Université Libre de Bruxelles; Karen.Willard-Gallo@bordet.be.
authors.forename: Karen
authors.initials: K
authors.lastname: Willard-Gallo
mesh_terms: D001940:Breast / Q000166:cytology* / Q000276:immunology; D001943:Breast Neoplasms / Q000276:immunology / Q000473:pathology*; D003584:Cytological Techniques / Q000379:methods*; D005260:Female; D006801:Humans; D017493:Leukocyte Common Antigens / Q000737:chemistry; D016246:Lymphocytes, Tumor-Infiltrating / Q000166:cytology* / Q000276:immunology / Q000473:pathology
chemical_list: D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human