(690b) Tunable Hydrogels to Understand the Role of the Microenvironment in Regulating Breast Cancer Dormancy and Recurrence

Breast cancer reoccurs in approximately 20 percent of all patients between 5 and 10 years after successful treatment of the primary tumor.¹  Many of these recurrences are believed to arise from tumor cells at distant metastatic sites that have reactivated after long periods of dormancy.  Remodeling of the extracellular matrix (ECM) at these sites over time is hypothesized to trigger the release of the tumor cells from dormancy.²  Two-dimensional (2D) and three-dimensional (3D) in vitro culture models have been developed to recreate some of the interactions between cancer cells and their microenvironment.^3,4  Lack of mechanical property control and differences between biochemical composition of these model systems and the native ECM make it challenging to identify key ECM signals that promote dormancy or activation.  Synthetic, hydrogel-based biomaterials are well suited for the creation of a new culture model to examine cancer recurrence, allowing user-directed control over the presentation of biomechanical and biochemical cues to direct cell behavior in vitro.^{5, 6}  Further, light-mediated hydrogel formation and modification permit spatiotemporal control over the presentation of biomechanical and biochemical cues to mimic the dynamic, nonhomogeneous in vivo microenvironment.⁷

We are designing a hydrogel-based 3D culture platform to mimic critical mechanical and biochemical properties of different metastatic site tissues where dormancy occurs, including the bone marrow, liver, and lung.  With this approach, we aim to identify cues that play a role in dormancy and reactivation of tumor cells toward the development of new therapeutics for recurrent breast cancer.  Specifically, poly(ethylene glycol)(PEG)-based hydrogels have been polymerized by a radically-initiated thiol−ene chemistry using PEG-4-thiol, prepared by modification of a four-arm PEG-OH monomer with thiols,⁸ and peptides modified with alloxycarbonyl-protected lysine residues to supply a biologically-orthogonal reactive functional group (−ene) for the formation of a step-growth hydrogel with ideal network structure.  Hydrogels formed within 5 minutes after the application of cytocompatible doses of UV light (10 mW/cm², 365 nm) in the presence of the lithium acylphosphinate photoinitiator.⁹  Mechanical properties of the hydrogels were measured by rheometry, and the elastic moduli of soft metastatic site tissues were achieved (~0.5-5 kPa).⁷  Further, biomimetic peptides have been spatially patterned within the matrix.

To establish matrix compositions that promote cancer dormancy or activation, we first compared the effects of soft and stiff substrates coated with relevant whole ECM proteins on tumor cell dormancy, including fibronectin (FN), laminin (LN), and collagen I and IV (Col I, Col IV) that are commonly found within metastatic tissue sites.  Cells were cultured on top of these soft and stiff substrates in the presence of various growth factors, including basic fibroblast growth factor (FGF2), and their continued activation or dormancy was evaluated by immunostaining for a marker of proliferation (Ki-67).  Finally, moving to 3D culture, human breast cancer cells (MCF7s) and human mesenchymal stem cells (hMSCs) were encapsulated within hydrogels decorated with integrin-binding peptides and remained viable post encapsulation as assessed by a membrane integrity assay (LIVE/DEAD) and metabolic activity assay (CellTiter96).  These results demonstrate the creation of a hydrogel-based synthetic ECM to investigate the role of the microenvironment in regulating cancer dormancy and recurrence.

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2          J Townson, et al. Cell Cycle. 2006, 5, 1744-1750.

3          J Barrios, et al. Cancer Microenviron. 2009, 2, 33-47.

4          D Barkan, et al. Cancer Res. 2008, 68, 6241-6250.

5          A Kloxin, et al. Adv Mater. 2010, 22, 3484-3494.

6          M Lutolf, et al. PNAS. 2003, 100, 5413-5418.

7          C DeForest, et al. Chem Mater. 2010, 22, 4783-4790.

8          B Fairbanks, et al. Macromolecules. 2011, 44, 2444-2450.

9          B Fairbanks, et al. Biomaterials. 2009, 30, 6702-6707.