title: On-demand dissolution of modular, synthetic extracellular matrix reveals local epithelial-stromal communication networks.
abstract: Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Valdez
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Christi D
authors.initials: CD
authors.lastname: Cook
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Caroline Chopko
authors.initials: CC
authors.lastname: Ahrens
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Wang
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Brown
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Manu
authors.initials: M
authors.lastname: Kumar
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Linda
authors.initials: L
authors.lastname: Stockdale
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rothenberg
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Kasper
authors.initials: K
authors.lastname: Renggli
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Gordon
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Douglas
authors.initials: D
authors.lastname: Lauffenburger
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Forest
authors.initials: F
authors.lastname: White
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: griff@mit.edu.
authors.forename: Linda
authors.initials: L
authors.lastname: Griffith
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002450:Cell Communication; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000494:pharmacology; D018836:Inflammation Mediators / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D012995:Solubility; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects
chemical_list: D001426:Bacterial Proteins; D018836:Inflammation Mediators; D036341:Intercellular Signaling Peptides and Proteins; D053583:Interleukin-1beta; D010455:Peptides; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Biomaterials; Matrix; Signaling; Sortase; Systems biology