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.

3612 Background: NF1-related PN are locally invasive tumors characterized by increased activation of the RAS pathway causing significant morbidity including disfigurement, pain and functional limitations. Selumetinib has received breakthrough designation for NF1 PN based on phase I / II trials in children. We present results for the ongoing phase II study of selumetinib in adults with NF1 PN, which includes pharmacodynamic (PD) evaluation of serial tumor biopsies as well as functional/patient-reported outcomes (PROs). Methods: Open-label Simon 2-stage design. Eligibility: NF1 patients (pts) ≥18 years old with inoperable/symptomatic/progressive PN. First 2 pts received selumetinib 75 mg BID; subsequent pts received selumetinib 50 mg BID. Primary objective: response rate by volumetric MRI analysis (partial response [PR]; ≥20% volume decrease). Secondary objectives: PD studies on pre/on-treatment biopsies of PN and cutaneous neurofibromas, assessment of clinical benefit using PROs (Numeric Rating Scale-11, Pain Interference Index), and PN-specific functional assessments. Validated, fit-for-purpose, isozyme-specific measurements of pERK/ERK/pMEK/MEK performed using SOPs designed for labile phosphoproteins (PMID 27001313). Results: As of February 2020, 27 pts have enrolled. Outcomes are reported for 23 pts (74% male; median age 33 years, range 18-60). Most common PN-related morbidity: pain (19 pts). Sixteen pts achieved PR (69%), with 13/16 confirmed; no disease progression. Time to response: 11 months (range 5-25); median change in PN volume at best response: -22% (range -41% to +5.5%); median duration of treatment: 28 months (range 2-50). Selumetinib suppressed tumor pERK1,2/ERK1,2 but not pMEK1,2/MEK1,2 ratios from 1-10 hours following oral dosing (one t½). Pt-reported target tumor pain intensity and pain interference scores significantly improved (both p < 0.03). Pts 1 and 2 were dose-reduced due to grade 3 intolerable rash (n = 2) and pain (n = 1). Grade ≥3 drug-related toxicities on 50 mg (21 pts) include transaminitis (5 pts), rash (1 pt) and pancreatic enzyme elevation (1 pt). Two pts were dose reduced (rash = 1 pt, transaminitis = 1 pt). Two pts discontinued by choice, 2 pts withdrawn by PI (best interest of patient), and 1 pt each removed for transaminitis, surgical resection, serious concurrent medical illness, and non-compliance. Conclusions: Selumetinib shrinks the majority of adult PN and results in molecular target suppression and clinical benefit. Clinical trial information: NCT02407405 .

Smooth muscle cell (SMC) invasion into plaques and subsequent proliferation is a major factor in the progression of atherosclerosis. During disease progression, SMCs experience major changes in their microenvironment, such as what integrin-binding sites are exposed, the portfolio of soluble factors available, and the elasticity and modulus of the surrounding vessel wall. We have developed a hydrogel biomaterial platform to examine the combined effect of these changes on SMC phenotype. We were particularly interested in how the chemical microenvironment affected the ability of SMCs to sense and respond to modulus. To our surprise, we observed that integrin binding and soluble factors are major drivers of several critical SMC behaviors, such as motility, proliferation, invasion, and differentiation marker expression, and these factors modulated the effect of stiffness on proliferation and migration. Overall, modulus only modestly affected behaviors other than proliferation, relative to integrin binding and soluble factors. Surprisingly, pathological behaviors (proliferation, motility) are not inversely related to SMC marker expression, in direct conflict with previous studies on substrates coupled with single extracellular matrix (ECM) proteins. A high-throughput bead-based ELISA approach and inhibitor studies revealed that differentiation marker expression is mediated chiefly via focal adhesion kinase (FAK) signaling, and we propose that integrin binding and FAK drive the transition from a migratory to a proliferative phenotype. We emphasize the importance of increasing the complexity of in vitro testing platforms to capture these subtleties in cell phenotypes and signaling, in order to better recapitulate important features of in vivo disease and elucidate potential context-dependent therapeutic targets.

MET tyrosine kinase (TK) dysregulation is significantly implicated in many types of cancer. Despite over 20 years of drug development to target MET in cancers, a pure anti-MET therapeutic has not yet received market approval. The failure of two recently concluded phase III trials point to a major weakness in biomarker strategies to identify patients who will benefit most from MET therapies. The capability to interrogate oncogenic mutations in MET via circulating tumor DNA (ctDNA) provides an important advancement in identification and stratification of patients for MET therapy. However, a wide range in type and frequency of these mutations suggest there is a need to carefully link these mutations to MET dysregulation, at least in proof-of-concept studies. In this review, we elaborate how we can utilize recently developed and validated pharmacodynamic biomarkers of MET not only to show target engagement, but more importantly to quantitatively measure MET dysregulation in tumor tissues. The MET assay endpoints provide evidence of both canonical and non-canonical MET signaling, can be used as "effect markers" to define biologically effective doses (BEDs) for molecularly targeted drugs, confirm mechanism-of-action in testing combination of drugs, and establish whether a diagnostic test is reporting MET dysregulation. We have established standard operating procedures for tumor biopsy collections to control preanalytical variables that have produced valid results in proof-of-concept studies. The reagents and procedures are made available to the research community for potential implementation on multiple platforms such as ELISA, quantitative immunofluorescence assay (qIFA), and immuno-MRM assays.