A Hydrogel Platform to Understand Breast Cancer Metastasis to Bone Marrow
Lauren E. Jansen and Shelly R. Peyton
Department of Chemical Engineering, University of Massachusetts Amherst
Metastasis accounts for 90% of cancer-related deaths. Breast cancer most frequently spreads to bone marrow tissue, resulting in painful osteolytic tumors that disrupt normal bone remodeling and production of lymphatic cells. While research efforts have provided genetic factors likely involved in breast cancer spread to bone, these findings are exclusive to the cancer cell and neglect contributions from the bone marrow tissue. We hypothesize that metastasis depends on both cancer cells and local cell and extracellular matrix (ECM) properties of the secondary tissue site 1-3. I have designed a polymer hydrogel that mimics some aspects of human bone marrow tissue, which provides orthogonal control of scaffold stiffness, ligand presentation, and degradation. We have used this system to aid in isolating breast cancer cell phenotypes driven by local cells or matrix properties that may facilitate breast-to-bone metastasis.
Our system consists of a 4-arm star poly(ethylene glycol)-maleimide that encapsulates cells in a 3D microenvironment. Biochemically our system can be modified by crosslinking our scaffold with peptide sequences that degrade in the presence of cell-secreted enzymes, and by chemically incorporating peptides representing integrin-binding sites. Here, we have filtered for 12 peptide sequences that represent bone marrow tissue by capturing most of the unique integrin binding sites known to be present in marrow tissue4. We characterized the mechanics of porcine marrow using three different, but complementary techniques: rheology, indentation, and cavitation rheology5. Across techniques, the average Young’s modulus of marrow is 4.4±1.0 kPa at physiological temperature. This stiffness and elasticity were recreated in vitro using our PEG hydrogel (20wt%, 4-arm 20 kDa PEG cross-linked with 1.5 kDa linear PEG). Both bone marrow and the PEG hydrogel followed a Hertzian model upon compression, validating the use of a PEG-based hydrogel for bone marrow tissue mechanics
Using a competitive binding assay, we have validated that hMSCs adhere to a select combination of these bone marrow peptides (peptides collagen I (GFOGER), fibronectin (PHSRN-RGD), osteopontin (SVVYGLR), and tenascin C (AEIDGIEL)) better than RGD alone. Additionally, an RNA profile (collagen type I, osteopontin, and osteocalcin) of hMSCs encapsulated in our hydrogel, with these same peptides and a tuned stiffness, showed that these cells are more comparable to hMSCs in native bone marrow tissue than hMSCs on tissue culture plastic. Using these hydrogels, we have screened for phenotypes across 4 patient hMSCs and 5 breast cancer cell lines. We found patient-specific phenotypes, like hMSC proliferation in response to some breast cancer soluble factors, and consistent phenotypes, such as increased breast cancer displacement in the presence of stromal cells. A high throughput bead based ELISA (Magpix) screened for 15 proteins, across 5 breast cancer cell lines and 4 human marrow-derived stem cells (hMSC), known to regulate breast cancer metastasis to the bone marrow. The highly expressed cytokines and growth factors have been isolated in both cell types and, to this end, we are working to identify the soluble factors and/or the tissue features driving these responses.
We have fundamentally quantified the mechanical and proteomic properties of bone marrow tissue, and used this knowledge to design an in vitro bone marrow model of the extracellular matrix. We have used this model to isolate phenotypes between cancer cells, local hMSCs, and bone marrow ECM that could be implicated in successful breast cancer cell adhesion and proliferation in secondary tissues. In sum, we demonstrate the benefit of using tissue-specific hydrogels to improve our understanding of breast cancer metastasis.
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