Devoloping Synthetic Environments to Study Cancer Progression and Metastasis

Cancer progression and metastasis is a complex, reciprocal communication between a tumor and the extracellular environment ^1-3.  While much of our basic understanding of fundamental cellular processes such as cell migration has been worked out in two dimensions ⁴, there remains a great need for improved 3-dimensional culture systems that capture the complexity of the natural tumor microenvironment ⁵.

The goal of this project is to study how the extracellular matrix (ECM) influences cancer cell migration and proliferation.   Our strategy is to synthetically develop a simplified but highly controlled ECM mimic that allows us to focus on specific aspects of cell/ECM interactions.   Currently, we are using a thiol-ene photopolymerization mechanism to copolymerize ene-functionalized poly(ethylene glycol) (PEG) precursors with thiol-containing peptides (see Figure 1) ⁶.  Based on this strategy, we form a hydrogel network using a broad range MMP-degradable crosslinker^{7, 8} and the common fibronectin-derived adhesion sequence RGDS ^{9, 10} to provide cells with a 3-dimensional environment permissive towards cell migration. 

Figure 1.  Schematic representation of thiol-ene polymerization.  20,000 M.W. 4-arm poly(ethylene glycol)-norbornene molecules are crosslinked with matrix metalloproteinase (MMP)-degradable peptides.  A pendant CRGDS peptide is included for cell adhesion.  Any thiol-containing molecule can be incorporated into the thiol-ene polymerization strategy, making it a highly versatile and controlled method for creating extracellular matrix mimics.

Using real-time microscopy, we have been able to quantitatively study cell migration and proliferation (Proliferation of HT-1080s in our thiol-ene hydrogel is demonstrated in Figure 2 and Movie 1).  Our results indicate that HT-1080 fibrosarcoma cells utilize a much more invasive migration mechanism than dermal fibroblasts.  Specifically, HT-1080 migration is characterized by a rounded morphology with a single leading edge protrusion whereas fibroblasts adopt a more spread morphology with multiple protrusions (see Figure 3 and Movies 2 and 3 below).  The invasiveness of HT-1080s relative to fibroblasts is quantified in Figure 3.  We are currently studying the details of the apparently unique migration mechanism utilized by HT-1080s and how the extracellular environment influences their behavior. 

Figure 2.  Dependence of proliferation on RGD concentration.  (a) Total cell count per field of view vs. RGD concentration after 1 day of incubation.  (b-e) Time lapse images of a dividing cell (See also Movie 1). 

Figure 3.  Comparison of migration for HT-1080s and dermal fibroblasts. (a,b) Observed morphologies for (a) HT-1080s and (b) dermal fibroblasts.  (a) While there were several morphologies observed for HT-1080s, most were rounded with a dynamic leading edge protrusion that defined the direction of migration (See also Movie 2) (b) Almost all fibroblasts adopted a typical spread, mesenchymal morphology (See also Movie 3).  (c) Migration parameters for fibroblasts normalized to HT-1080 values. 

Movie 1: Cell Division                           Movie 2: HT1080 Migration                   Movie 3: Fibroblast Migration

Other researchers on this project: Robert Rogers, Ben Fairbanks, and Lydia Everhart.

References

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2. Nelson, C.M. & Bissell, M.J. Of extracellular matrix, scaffolds, and signaling: Tissue architecture regulates development, homeostasis, and cancer. Annual Review of Cell and Developmental Biology 22, 287-309 (2006).

3. Erler, J.T. & Weaver, V.M. Three-dimensional context regulation of metastasis. Clin. Exp. Metastasis 26, 35-49 (2009).

4. Lauffenburger, D.A. & Horwitz, A.F. Cell migration: A physically integrated molecular process. Cell 84, 359-369 (1996).

5. Griffith, L.G. & Swartz, M.A. Capturing complex 3D tissue physiology in vitro. Nature Reviews Molecular Cell Biology 7, 211-224 (2006).

6. Fairbanks, B.D. et al. Versatile, bioresponsive hydrogels via thiol-ene photopolymerization. Submitted for Publication (2009).

7. Netzel-Arnett, S., Fields, G.B., Birkedal-Hansen, H., Van Wart, H.E. & Fields, G. Sequence specificities of human fibroblast and neutrophil collagenases [published erratum appears in J Biol Chem 1991 Nov 5;266(31):21326]. J. Biol. Chem. 266, 6747-6755 (1991).

8. Nagase, H. & Fields, G.B. Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptides. Biopolymers 40, 399-416 (1996).

9. Ruoslahti, E. & Pierschbacher, M.D. New Perspectives in Cell-Adhesion - Rgd and Integrins. Science 238, 491-497 (1987).

10. Pierschbacher, M.D. & Ruoslahti, E. Cell Attachment Activity of Fibronectin Can Be Duplicated by Small Synthetic Fragments of the Molecule. Nature 309, 30-33 (1984).