title: Real-time and label free determination of ligand binding-kinetics to primary cancer tissue specimens; a novel tool for the assessment of biomarker targeting.
abstract: In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative IHC analysis depends on both the specificity and affinity of the binding reagent, which are inherently difficult to quantifyin situ. Here we describe a label-free method that allows for the direct and real-time assessment of molecular binding kineticsin situon FFPE tissue specimens using quartz crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumour specimensin situ. rVAR2 interacted with FFPE human placenta and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody (anti-AR). As the KDvalue produced by this method is independent of the number of epitopes available, this readout offers a quantitative and unbiased readout forin situbinding-avidity and amount of binding epitopes. In summary, this method adds a new and important dimension to classical IHC-based molecular pathology by adding information about the binding characteristics in biologically relevant conditions. This can potentially be used to select optimal biologics for diagnostic and for therapeutic applications as well as guide the development of novel high affinity binding drugs.
authors.affiliation: Centre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada; Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Thomas Mandel
authors.initials: TM
authors.lastname: Clausen
authors.affiliation: Centre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Marina Ayres
authors.initials: MA
authors.lastname: Pereira
authors.affiliation: Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada; Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada; Molecular Pathology and Cell Imaging Laboratory, Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada.
authors.forename: Htoo Zarni
authors.initials: HZ
authors.lastname: Oo
authors.affiliation: Centre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Mafalda
authors.initials: M
authors.lastname: Resende
authors.affiliation: Centre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Tobias
authors.initials: T
authors.lastname: Gustavson
authors.affiliation: Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark; Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Mao
authors.affiliation: Institute for Molecular Science of Medicine, Aichi Medical University, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Sugiura
authors.affiliation: Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada; Molecular Pathology and Cell Imaging Laboratory, Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada.
authors.forename: Janet
authors.initials: J
authors.lastname: Liew
authors.affiliation: Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada; Molecular Pathology and Cell Imaging Laboratory, Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada.
authors.forename: Ladan
authors.initials: L
authors.lastname: Fazli
authors.affiliation: Centre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Thor G
authors.initials: TG
authors.lastname: Theander
authors.affiliation: Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada; Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada; Molecular Pathology and Cell Imaging Laboratory, Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada.
authors.forename: Mads
authors.initials: M
authors.lastname: Daugaard
authors.affiliation: Centre for Medical Parasitology at Department of Immunology and Microbiology, University of Copenhagen, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark.
authors.forename: Ali
authors.initials: A
authors.lastname: Salanti
keywords: Biomarker; Biosensor; Cancer; Malaria; Quartz crystal microscale; VAR2CSA