Sorafenib resistance and JNK signaling in carcinoma during extracellular matrix stiffening

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 β1integrin 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

PEG-Phosphorylcholine Hydrogels As Tunable and Versatile Platforms for Mechanobiology

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

Maximal Sharing in the Lambda Calculus with letrec

Increasing sharing in programs is desirable to compactify the code, and to avoid duplication of reduction work at run-time, thereby speeding up execution. We show how a maximal degree of sharing can be obtained for programs expressed as terms in the lambda calculus with letrec. We introduce a notion of `maximal compactness' for lambda-letrec-terms among all terms with the same infinite unfolding. Instead of defined purely syntactically, this notion is based on a graph semantics. lambda-letrec-terms are interpreted as first-order term graphs so that unfolding equivalence between terms is preserved and reflected through bisimilarity of the term graph interpretations. Compactness of the term graphs can then be compared via functional bisimulation. We describe practical and efficient methods for the following two problems: transforming a lambda-letrec-term into a maximally compact form; and deciding whether two lambda-letrec-terms are unfolding-equivalent. The transformation of a lambda-letrec-term $L$ into maximally compact form $L_0$ proceeds in three steps: (i) translate L into its term graph $G = [[ L ]]$; (ii) compute the maximally shared form of $G$ as its bisimulation collapse $G_0$; (iii) read back a lambda-letrec-term $L_0$ from the term graph $G_0$ with the property $[[ L_0 ]] = G_0$. This guarantees that $L_0$ and $L$ have the same unfolding, and that $L_0$ exhibits maximal sharing. The procedure for deciding whether two given lambda-letrec-terms $L_1$ and $L_2$ are unfolding-equivalent computes their term graph interpretations $[[ L_1 ]]$ and $[[ L_2 ]]$, and checks whether these term graphs are bisimilar. For illustration, we also provide a readily usable implementation.Comment: 18 pages, plus 19 pages appendi

Macropore effects on pesticides transport to groundwater

The objective of this investigation was to evaluate the leaching and degradation characteristics of atrazine and bromide in a field of alluvial soils under irrigated, no-till management. The experimental site was 0.1 ha in size. The soils were Sarpy (mixed, mesic Typic Udipsamments) with the surface texture varying from silt loam to loamy sand. Atrazine was applied at 2.2 kg/ha after sorghum (Sorghum bicolor) was planted. Bromide was applied at 115 kg/ha five days later. Soil cores were extracted to a depth of 150 cm which were segmented into 7.5 cm increments and were analyzed for each of the chemicals separately. The dates for sampling were one week, one month, two months, three months, and four months after application of the chemicals. As a result 1134 and 3542 soil samples were extracted for atrazine and bromide analysis, respectively. Atrazine was detected within the 15 to 22.5 cm depth increment one week after application. These data suggest that some of the atrazine can move to depth of 20 cm after one week which is probably due to the presence of macropores (1-5 mm diameter holes) open to the soil surface which were present in this field under no-till management. Atrazine was detected at very low concentrations at two and four months after application. Although extreme variability in atrazine concentrations occurred, the variations were not explained totally by differences in soil texture. The data in this study indicate some potential, although small, for atrazine contamination of groundwater.Project # G-1432-03 Agreement # 14-08-0001-G-1423-0

A Biomaterial Screening Approach Reveals Microenvironmental Mechanisms of Drug Resistance

Traditional drug screening methods lack features of the tumor microenvironment that contribute to resistance. Most studies examine cell response in a single biomaterial platform in depth, leaving a gap in understanding how extracellular signals such as stiffness, dimensionality, and cell–cell contacts act independently or are integrated within a cell to affect either drug sensitivity or resistance. This is critically important, as adaptive resistance is mediated, at least in part, by the extracellular matrix (ECM) of the tumor microenvironment. We developed an approach to screen drug responses in cells cultured on 2D and in 3D biomaterial environments to explore how key features of ECM mediate drug response. This approach uncovered that cells on 2D hydrogels and spheroids encapsulated in 3D hydrogels were less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. We found that transcriptomic differences between these in vitro models and tumor xenografts did not reveal mechanisms of ECM-mediated resistance to sorafenib. However, a systems biology analysis of phospho-kinome data uncovered that variation in MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by performing drug screening, phospho-kinome analysis, and systems biology across multiple biomaterial environments

Field evaluation and model calibration for agricultural pesticide transport to groundwater - phase II

Students supported: 1 MS StudentCertain soil physical and chemical property data are needed to predict transport of agricultural chemicals (pesticides, herbicides, etc.) to groundwater. The objective of this investigation was to evaluate the soil variability of selected soil physical and chemical properties in a field used to study atrazine and bromide leaching. The site was divided into three areas due to differences in surface texture. Area I had a surface texture of sand, Area II sandy loam, and Area III loam. Soil physical properties were measured on 455 undisturbed soil samples taken systematically at 91 locations at five selected depths (15 to 20 cm depth increments to a depth of 85 cm) throughout the field. Additional samples were taken for measurement of organic matter content and pH. Organic matter content values of the three soil areas were similar throughout the soil profile. However, there was an additional peak (besides that at the soil surface) of organic matter content at the 100 cm depth in all areas. This was probably due to buried plant materials. Salt pH of Area I was higher down to the 65 cm depth compared to Areas II and III. Soil bulk density values throughout the soil profile were similar for the three soil areas. Interestingly, bulk density decreased with increasing soil depth which was attributed to the coarser texture of soil particles with increasing depth. Below the third depth, over 80 percent of the samples had 90 percent or more sand of which at least 85 percent was very coarse (1.0 to 2.0 mm). Saturated hydraulic conductivity values 7 of the three areas were similar for the shallow depths. At the 55 cm depth however, Area I had higher saturated hydraulic conductivity values than Areas II and III. The soil water characteristics of the three areas were similar for the five measured depths.Project # G-1572-02 Agreement # 14-08-0001-G-1572-0

Interfacial-Redox-Induced Tuning of Superconductivity in YBa2Cu3O7-δ.

Solid-state ionic approaches for modifying ion distributions in getter/oxide heterostructures offer exciting potentials to control material properties. Here, we report a simple, scalable approach allowing for manipulation of the superconducting transition in optimally doped YBa2Cu3O7-δ (YBCO) films via a chemically driven ionic migration mechanism. Using a thin Gd capping layer of up to 20 nm deposited onto 100 nm thick epitaxial YBCO films, oxygen is found to leach from deep within the YBCO. Progressive reduction of the superconducting transition is observed, with complete suppression possible for a sufficiently thick Gd layer. These effects arise from the combined impact of redox-driven electron doping and modification of the YBCO microstructure due to oxygen migration and depletion. This work demonstrates an effective step toward total ionic tuning of superconductivity in oxides, an interface-induced effect that goes well into the quasi-bulk regime, opening-up possibilities for electric field manipulation

Smooth Muscle Stiffness Sensitivity is Driven by Soluble and Insoluble ECM Chemistry

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 expres- sion, 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, patho- logical behaviors (proliferation, motility) are not inversely related to SMC marker expression, in direct conflict with previous studies on substrates coupled with single extracel- lular matrix (ECM) proteins. A high-throughput bead-based ELISA approach and inhibitor studies revealed that differ- entiation 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

Ionic Tuning of Cobaltites at the Nanoscale

Control of materials through custom design of ionic distributions represents a powerful new approach to develop future technologies ranging from spintronic logic and memory devices to energy storage. Perovskites have shown particular promise for ionic devices due to their high ion mobility and sensitivity to chemical stoichiometry. In this work, we demonstrate a solid-state approach to control of ionic distributions in (La,Sr)CoO$_{3}$ thin films. Depositing a Gd capping layer on the perovskite film, oxygen is controllably extracted from the structure, up-to 0.5 O/u.c. throughout the entire 36 nm thickness. Commensurate with the oxygen extraction, the Co valence state and saturation magnetization show a smooth continuous variation. In contrast, magnetoresistance measurements show no-change in the magnetic anisotropy and a rapid increase in the resistivity over the same range of oxygen stoichiometry. These results suggest significant phase separation, with metallic ferromagnetic regions and oxygen-deficient, insulating, non-ferromagnetic regions, forming percolated networks. Indeed, X-ray diffraction identifies oxygen-vacancy ordering, including transformation to a brownmillerite crystal structure. The unexpected transformation to the brownmillerite phase at ambient temperature is further confirmed by high-resolution scanning transmission electron microscopy which shows significant structural - and correspondingly chemical - phase separation. This work demonstrates room-temperature ionic control of magnetism, electrical resistivity, and crystalline structure in a 36 nm thick film, presenting new opportunities for ionic devices that leverage multiple material functionalities