3D Hyaluronan Dual-Click Chemistry Hydrogels as Matrices for Breast Spheroid Formation

Alexander E. G. Baker (University of Toronto)
Roger Y. Tam (University of Toronto)
Senthil Muthuswamy (University of Toronto)
Molly S. Shoichet (University of Toronto)


Terminal ductal lobular structures are the main functioning unit of mammary glands and the most common site for breast carcinogenesis (the cited reference is missing author and title details).1 Mammary ducts are lined with luminal epithelial cells surrounded by myoepithelial cells encircled by basement membrane. These luminal cells are highly proliferative epithelial cells and are the most susceptible to malignant transformation.2 In conventional 2D cultures of breast epithelial cells, they instead grow as flat monolayers. A substitute for recapitulating human breast tumors are xenografts using immunocompromised mice which fail to fully represent human breast cancer and requires time consuming analyses.3 This supports the need for a representative in vitro tool to study breast cancer and drug development. Matrigel® is a material derived from mouse sarcoma, and supports the in vitro culture of breast epithelial cells; however, it suffers from batch heterogeneity, reducing reproducibility. Therefore, a well-defined and tunable 3D scaffold which mimics tumour microenvironment is needed.4 We have synthesized a bifunctional hyaluronan (HA) hydrogel cross-linked with polyethylene glycol and ECM peptide mimics with tunable chemical and physical properties permitting the formation of 3D spheroid structures from human breast tumour epithelial cells MCF-7 and T-47D.

Materials and Methods

Hyaluronan based scaffolds with independent click chemical crosslinking and ligand immobilization were prepared through sequential synthesis. HA hydrogels were crosslinked with poly(ethylene glycol). Maleimide functionalized peptides prepared using solid phase synthesis, were immobilized using Diels Alder chemistry with furan modified HA. Human ductal breast epithelial tumor cells T47D (human epidermal growth factor receptor 2 negative, HER2-) and breast adenocarcinoma cells MCF-7 (HER2-) were seeded on the gels and the cell morphology was assessed by confocal microscopy. Cell spheroid size was assessed of cells grown on 3D HA hydrogels vs. those grown on Matrigel® and 2D tissue culture polystyrene.


The crosslinked HA was assessed for swelling and mechanical strength. The use of ECM peptide mimics, including fibronectin- and laminin-derived ligands, were synthesized by solid phase peptide synthesis and used to promote cell adhesion. Using these HA-based hydrogels, we were able to generate 3D multicellular structures comparable to Matrigel (see Figure 1). These hydrogels showed long-term stability over four weeks of cell culture. Cell viability and morphology of the 2D monolayer and 3D spheroids were assessed using growth curves and spheroid size was compared to Matrigel.

Discussion and Conclusion

These hyaluronan scaffolds offer extended hydrogel stability and accommodate the formation of luminal A breast epithelial spheroids. This enables their further exploration in cancer research.

Figure 1: Bright-field confocal images comparing T-47D breast epithelial spheroids cultured on (a) growth factor reduced Matrigel compared to (b) crosslinked hyaluronan modified with cell-adhesive peptides after 8 days.


We are grateful to the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support.


1. Anderson, E.; Clarke, R. B., Steroid receptors and cell cycle in normal mammary epithelium. Journal of mammary gland biology and neoplasia 2004, 9 (1), 3-13. 2. Clarke, R. B.; Anderson, E.; Howell, A., Steroid receptors in human breast cancer. Trends in endocrinology and metabolism: TEM 2004, 15 (7), 316-23. 3. Perou, C. M.; et al., Molecular portraits of human breast tumours. Nature 2000, 406 (6797), 747-52. 4. Owen, S. C.; Fisher, S. A.; Tam, R. Y.; Nimmo, C. M.; Shoichet, M. S., Hyaluronic Acid Click Hydrogels Emulate the Extracellular Matrix. Langmuir 2013, 29 (24), 7393-7400.

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