Saturday, April 19, 2014

Extracellular Matrix Environment and Chemotherapeutics

Substrate Matters!

Neuromics' has been promoting a variety of 3-D Cell Based Assay Solutions for the past several years. These include: Nanofibers, Hydrogels and Extracellular Matrix (ECM) Proteins. We have found that the adoption rate for these as standard tools for drug discovery is slower that we anticipated.

We believe substrate matters so I am pleased to share a recent publication that references use of our Collagen IV and other ECM proteins. This confirms the importance of using a more in vivo like environment in testing chemotherapeutics: Thuy V. Nguyen,Marianne Sleiman,Timothy Moriarty,William G. Herrick,Shelly R. Peyton. Sorafenib resistance and JNK signaling in carcinoma during extracellular matrix stiffening. Publication: Biomaterials. Elsevier. 13 April 2014.

Abstract: Tumor progression is coincident with mechanochemical changes in the extracellular matrix (ECM). We hypothesized that tumor stroma stiffening, alongside a shift in the ECM composition from a basement membrane-like microenvironment toward a dense network of collagen-rich fibers during tumorigenesis, confers resistance to otherwise powerful chemotherapeutics. To test this hypothesis, we created a high-throughput drug screening platform based on our poly(ethylene glycol)-phosphorylcholine (PEG-PC) hydrogel system, and customized it to capture the stiffness and integrin-binding profile of in vivo tumors. We report that the efficacy of a Raf kinase inhibitor, sorafenib, is reduced on stiff, collagen-rich microenvironments, independent of ROCK activity. Instead, sustained activation of JNK mediated this resistance, and combining a JNK inhibitor with sorafenib eliminated stiffness-mediated resistance in triple negative breast cancer cells. Surprisingly, neither ERK nor p38 appears to mediate sorafenib resistance, and instead, either ERK or p38 inhibition rescued sorafenib resistance during JNK inhibition, suggesting negative crosstalk between these signaling pathways on stiff, collagen-rich environments. Overall, we discovered that β1 integrin and its downstream effector JNK mediate sorafenib resistance during tumor stiffening. These results also highlight the need for more advanced cell culture platforms, such as our high-throughput PEG-PC system, with which to screen chemotherapeutics.

Figure: High-throughput biomaterial platform for drug screening. (A) The high-throughput platform consists of a black-walled, glass bottom plate, with PEG-PC gels cast in each of the inner 6x10 wells. (B) Gels can be functionalized with any protein or peptide of interest, and they support the adhesion and growth of carcinoma cells. We used this platform to test carcinoma cell response to a kinase inhibitor (sorafenib) as a function of underlying gel stiffness and ECM adhesive protein cocktail. (C) A representative graph of SkBr3 proliferation (y-axis) in response to sorafenib (x-axis) across a range of gel stiffness (colors) demonstrates the IC-50 calculation.

This confirms the importance of considering your substrate environment when developing your in vitro assays for High Content and High Throughput Drug Discovery. We will continue to provide updates.

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