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.

We report here the synthesis of a new class of hydrogels with an extremely wide range of mechanical properties suitable for cell studies. Mechanobiology has emerged as an important field in bioengineering, in part due to the development of synthetic polymer gels and fibrous protein biomaterials to control and quantify how cells sense and respond to mechanical forces in their microenvironment. To address the problem of limited availability of biomaterials, in terms of both mechanical range and optical clarity, we have prepared hydrogels that combine poly(ethylene glycol) (PEG) and phosphorylcholine (PC) zwitterions. Our goal was to create a hydrogel platform that exceeds the range of Young's moduli reported for similar hydrogels, while being simple to synthesize and manipulate. The Young's modulus of these "PEG-PC" hydrogels can be tuned over 4 orders of magnitude, much greater than commonly used hydrogels such as PEG-diacrylate, PEG-dimethacrylate, and polyacrylamide, with smaller average mesh sizes and optical clarity. We prepared PEG-PC hydrogels to study how substrate mechanical properties influence cell morphology, focal adhesion structure, and proliferation across multiple mammalian cell lines, as a proof of concept. These novel PEG-PC biomaterials represent a new and useful class of mechanically tunable hydrogels for mechanobiology.

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