title: Binding of trastuzumab to ErbB2 is inhibited by a high pericellular density of hyaluronan.
abstract: Although trastuzumab is an efficient drug, primary and acquired resistance is a challenging problem. The authors have previously shown in mouse xenograft experiments that masking ErbB2 by hyaluronan leads to diminished binding of the antibody and consequent resistance. In the current work, they correlated trastuzumab binding with the pericellular density of hyaluronan in ErbB2-overexpressing human breast cancer samples. A method for quantifying the relative binding of trastuzumab was developed involving constant and low-frequency background subtraction, segmenting the image to membrane and background pixels followed by evaluation of trastuzumab fluorescence, normalized with the expression level of ErbB2, only in the membrane. The normalized binding of trastuzumab showed a negative correlation with the pericellular density of hyaluronan (r = -0.52) with the effect being the most pronounced in the extreme cases (i.e., low and high hyaluronan densities predicted strong and weak binding of trastuzumab, respectively). Removal of hyaluronan by hyaluronidase digestion unmasked the trastuzumab binding epitope of ErbB2 demonstrated by a significantly increased normalized binding of the antibody. The results show that the accumulation of pericellular hyaluronan plays a crucial role in masking ErbB2.
authors.affiliation: Department of Biophysics and Cell Biology, Medical and Health Science Centre, University of Debrecen, Debrecen, Hungary.
authors.forename: Tímea
authors.initials: T
authors.lastname: Váradi
authors.forename: Tamás
authors.initials: T
authors.lastname: Mersich
authors.forename: Päivi
authors.initials: P
authors.lastname: Auvinen
authors.forename: Raija
authors.initials: R
authors.lastname: Tammi
authors.forename: Markku
authors.initials: M
authors.lastname: Tammi
authors.forename: Ferenc
authors.initials: F
authors.lastname: Salamon
authors.forename: István
authors.initials: I
authors.lastname: Besznyák
authors.forename: Ferenc
authors.initials: F
authors.lastname: Jakab
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Baranyai
authors.forename: János
authors.initials: J
authors.lastname: Szöllősi
authors.forename: Peter
authors.initials: P
authors.lastname: Nagy
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D061067:Antibodies, Monoclonal, Humanized / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism*; D000939:Epitopes; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D008875:Middle Aged; D011485:Protein Binding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D000939:Epitopes; D006820:Hyaluronic Acid; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C508053:ERBB2 protein, human