On-demand dissolution of modular, synthetic extracellular matrix reveals local epithelial-stromal communication networks

Biomaterials. 2017 Jun:130:90-103. doi: 10.1016/j.biomaterials.2017.03.030. Epub 2017 Mar 23.

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.

Keywords: Biomaterials; Matrix; Signaling; Sortase; Systems biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aminoacyltransferases / metabolism
  • Bacterial Proteins / metabolism
  • Cell Communication
  • Cell Line, Tumor
  • Coculture Techniques
  • Cysteine Endopeptidases / metabolism
  • Epithelial Cells / cytology*
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism
  • Extracellular Matrix / drug effects
  • Extracellular Matrix / metabolism*
  • Humans
  • Hydrogel, Polyethylene Glycol Dimethacrylate / pharmacology
  • Inflammation Mediators / metabolism
  • Intercellular Signaling Peptides and Proteins / metabolism
  • Interleukin-1beta / metabolism
  • Kinetics
  • Peptides / chemistry
  • Solubility
  • Stromal Cells / cytology
  • Stromal Cells / drug effects

Substances

  • Bacterial Proteins
  • Inflammation Mediators
  • Intercellular Signaling Peptides and Proteins
  • Interleukin-1beta
  • Peptides
  • Hydrogel, Polyethylene Glycol Dimethacrylate
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases