title: Molecular weight specific impact of soluble and immobilized hyaluronan on CD44 expressing melanoma cells in 3D collagen matrices.
abstract: Hyaluronan (HA) and its principal receptor CD44 are known to be involved in regulating tumor cell dissemination and metastasis. The direct correlation of CD44-HA interaction on proliferation and invasion of tumor cells in dependence on the molecular weight and the presentation form of HA is not fully understood because of lack of appropriate matrix models. To address this issue, we reconstituted 3D collagen (Coll I) matrices and functionalized them with HA of molecular weight of 30-50kDa (low molecular weight; LMW-HA) and 500-750kDa (high molecular weight; HMW-HA). A post-modification strategy was applied to covalently immobilize HA to reconstituted fibrillar Coll I matrices, resulting in a non-altered Coll I network microstructure and stable immobilization over days. Functionalized Coll I matrices were characterized regarding topological and mechanical characteristics as well as HA amount using confocal laser scanning microscopy, colloidal probe force spectroscopy and quantitative Alcian blue assay, respectively. To elucidate HA dependent tumor cell behavior, BRO melanoma cell lines with and without CD44 receptor expression were used for in vitro cell experiments. We demonstrated that only soluble LMW-HA promoted cell proliferation in a CD44 dependent manner, while HMW-HA and immobilized LMW-HA did not. Furthermore, an enhanced cell invasion was found only for immobilized LMW-HA. Both findings correlated with a very strong and specific adhesive interaction of LMW-HA and CD44+ cells quantified in single cell adhesion measurements using soft colloidal force spectroscopy. Overall, our results introduce an in vitro biomaterials model allowing to test presentation mode and molecular weight specificity of HA in a 3D fibrillar matrix thus mimicking important in vivo features of tumor microenvironments.
  
  
  
  Molecular weight and presentation form (bound vs. soluble) of hyaluronan (HA) are intensively discussed as key regulators in tumor progression and inflammation. We introduce 3D fibrillar collagen matrices with defined microstructure and stiffness allowing the presentation of specific molecular weight forms of HA in soluble and bound manner. Mimicking in that way important in vivo features of tumor microenvironments, we found that only low molecular weight HA (LMW-HA) in soluble form promoted proliferation of a melanoma cell line (BRO), while it enhanced cell invasion in bound form. The molecular weight specificity of LMW-HA was verified to be CD44 receptor dependent and was correlated to adhesive ligand-receptor interactions in quantitative colloidal force spectroscopy at single cell level.
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, Leipzig 04103, Germany.
authors.forename: Jiranuwat
authors.initials: J
authors.lastname: Sapudom
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, Leipzig 04103, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Ullm
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, Leipzig 04103, Germany.
authors.forename: Steve
authors.initials: S
authors.lastname: Martin
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, Leipzig 04103, Germany.
authors.forename: Liv
authors.initials: L
authors.lastname: Kalbitzer
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, Leipzig 04103, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Naab
authors.affiliation: INNOVENT e. V., Biomaterials Department, Prüssingstraße 27B, 07745 Jena, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Möller
authors.affiliation: INNOVENT e. V., Biomaterials Department, Prüssingstraße 27B, 07745 Jena, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schnabelrauch
authors.affiliation: Department of Dermatology, Venerology and Allergology, Universitätsklinikum Leipzig, Leipzig 04103, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Anderegg
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, Leipzig 04103, Germany; Heinrich-Heine-Universität, Institute for Organic and Macromolecular Chemistry, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Schmidt
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, Universität Leipzig, Leipzig 04103, Germany. Electronic address: tilo.pompe@uni-leipzig.de.
authors.forename: Tilo
authors.initials: T
authors.lastname: Pompe
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology*; D003102:Colloids; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000737:chemistry*; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology*; D008970:Molecular Weight; D009361:Neoplasm Invasiveness; D016062:Porosity; D051381:Rats; D012995:Solubility
chemical_list: C497483:CD44 protein, human; D003102:Colloids; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D003094:Collagen
keywords: CD44 receptor; Collagen; Extracellular matrix; Hyaluronan; Melanoma cells