Hadi Tavakoli Nia

Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy

Current measurements of the biomechanical properties of cells require physical contact with cells or lack subcellular resolution. Here we developed a label-free microscopy technique based on Brillouin light scattering that is capable of measuring an intracellular longitudinal modulus with optical resolution. The 3D Brillouin maps we obtained of cells in 2D and 3D microenvironments revealed mechanical changes due to cytoskeletal modulation and cell-volume regulation.

Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy

UNLABELLED It remains unclear how obesity worsens treatment outcomes in patients with pancreatic ductal adenocarcinoma (PDAC). In normal pancreas, obesity promotes inflammation and fibrosis. We found in mouse models of PDAC that obesity also promotes desmoplasia associated with accelerated tumor growth and impaired delivery/efficacy of chemotherapeutics through reduced perfusion. Genetic and pharmacologic inhibition of angiotensin-II type-1 receptor reverses obesity-augmented desmoplasia and tumor growth and improves response to chemotherapy. Augmented activation of pancreatic stellate cells (PSC) in obesity is induced by tumor-associated neutrophils (TAN) recruited by adipocyte-secreted IL1β. PSCs further secrete IL1β, and inactivation of PSCs reduces IL1β expression and TAN recruitment. Furthermore, depletion of TANs, IL1β inhibition, or inactivation of PSCs prevents obesity-accelerated tumor growth. In patients with pancreatic cancer, we confirmed that obesity is associated with increased desmoplasia and reduced response to chemotherapy. We conclude that cross-talk between adipocytes, TANs, and PSCs exacerbates desmoplasia and promotes tumor progression in obesity. SIGNIFICANCE Considering the current obesity pandemic, unraveling the mechanisms underlying obesity-induced cancer progression is an urgent need. We found that the aggravation of desmoplasia is a key mechanism of obesity-promoted PDAC progression. Importantly, we discovered that clinically available antifibrotic/inflammatory agents can improve the treatment response of PDAC in obese hosts. Cancer Discov; 6(8); 852-69. ©2016 AACR.See related commentary by Bronte and Tortora, p. 821This article is highlighted in the In This Issue feature, p. 803.

Poroelasticity of Cartilage at the Nanoscale

Atomic-force-microscopy-based oscillatory loading was used in conjunction with finite element modeling to quantify and predict the frequency-dependent mechanical properties of the superficial zone of young bovine articular cartilage at deformation amplitudes, δ, of ~15 nm; i.e., at macromolecular length scales. Using a spherical probe tip (R ~ 12.5 μm), the magnitude of the dynamic complex indentation modulus, |E*|, and phase angle, φ, between the force and tip displacement sinusoids, were measured in the frequency range f ~ 0.2-130 Hz at an offset indentation depth of δ(0) ~ 3 μm. The experimentally measured |E*| and φ corresponded well with that predicted by a fibril-reinforced poroelastic model over a three-decade frequency range. The peak frequency of phase angle, f(peak), was observed to scale linearly with the inverse square of the contact distance between probe tip and cartilage, 1/d(2), as predicted by linear poroelasticity theory. The dynamic mechanical properties were observed to be independent of the deformation amplitude in the range δ = 7-50 nm. Hence, these results suggest that poroelasticity was the dominant mechanism underlying the frequency-dependent mechanical behavior observed at these nanoscale deformations. These findings enable ongoing investigations of the nanoscale progression of matrix pathology in tissue-level disease.

Solid stress and elastic energy as measures of tumour mechanopathology

Solid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is challenging. Here, we show that two-dimensional spatial mappings of solid stress and the resulting elastic energy in excised or in situ tumours with arbitrary shapes and wide size ranges can be obtained via three distinct and quantitative techniques that rely on the measurement of tissue displacement after disruption of the confining structures. Application of these methods in models of primary tumours and metastasis revealed that: (i) solid stress depends on both cancer cells and their microenvironment; (ii) solid stress increases with tumour size; and (iii) mechanical confinement by the surrounding tissue significantly contributes to intratumoural solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to significant discoveries and new therapies.

Anti-VEGF therapy induces ECM remodeling and mechanical barriers to therapy in colorectal cancer liver metastases

Anti-VEGF therapy enhances mechanical barriers to therapy in colorectal cancer liver metastases by increasing hyaluronan deposition. Stiff resistance to cancer therapy Antiangiogenic therapy with drugs that block vascular endothelial growth factor (VEGF) signaling to inhibit formation of new blood vessels in tumors is commonly used in colorectal cancer. Unfortunately, the effects of this therapy usually do not last for long, and a study by Rahbari et al. shows why this might be the case. The authors found that VEGF inhibition increased the stiffness of colorectal cancer liver metastases, making them more difficult to treat with chemotherapy. In a mouse model, the researchers were able to overcome this difficulty by using an enzyme to degrade a component of the extracellular matrix in liver metastases, suggesting that the matrix may be a target for future cancer therapies. The survival benefit of anti–vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy.

Aggrecan Nanoscale Solid–Fluid Interactions Are a Primary Determinant of Cartilage Dynamic Mechanical Properties

Poroelastic interactions between interstitial fluid and the extracellular matrix of connective tissues are critical to biological and pathophysiological functions involving solute transport, energy dissipation, self-stiffening and lubrication. However, the molecular origins of poroelasticity at the nanoscale are largely unknown. Here, the broad-spectrum dynamic nanomechanical behavior of cartilage aggrecan monolayer is revealed for the first time, including the equilibrium and instantaneous moduli and the peak in the phase angle of the complex modulus. By performing a length scale study and comparing the experimental results to theoretical predictions, we confirm that the mechanism underlying the observed dynamic nanomechanics is due to solid-fluid interactions (poroelasticity) at the molecular scale. Utilizing finite element modeling, the molecular-scale hydraulic permeability of the aggrecan assembly was quantified (kaggrecan = (4.8 ± 2.8) × 10(-15) m(4)/N·s) and found to be similar to the nanoscale hydraulic permeability of intact normal cartilage tissue but much lower than that of early diseased tissue. The mechanisms underlying aggrecan poroelasticity were further investigated by altering electrostatic interactions between the molecules constituent glycosaminoglycan chains: electrostatic interactions dominated steric interactions in governing molecular behavior. While the hydraulic permeability of aggrecan layers does not change across species and age, aggrecan from adult human cartilage is stiffer than the aggrecan from newborn human tissue.

High-bandwidth AFM-based rheology is a sensitive indicator of early cartilage aggrecan degradation relevant to mouse models of osteoarthritis

Murine models of osteoarthritis (OA) and post-traumatic OA have been widely used to study the development and progression of these diseases using genetically engineered mouse strains along with surgical or biochemical interventions. However, due to the small size and thickness of murine cartilage, the relationship between mechanical properties, molecular structure and cartilage composition has not been well studied. We adapted a recently developed AFM-based nano-rheology system to probe the dynamic nanomechanical properties of murine cartilage over a wide frequency range of 1 Hz to 10 kHz, and studied the role of glycosaminoglycan (GAG) on the dynamic modulus and poroelastic properties of murine femoral cartilage. We showed that poroelastic properties, highlighting fluid-solid interactions, are more sensitive indicators of loss of mechanical function compared to equilibrium properties in which fluid flow is negligible. These fluid-flow-dependent properties include the hydraulic permeability (an indicator of the resistance of matrix to fluid flow) and the high frequency modulus, obtained at high rates of loading relevant to jumping and impact injury in vivo. Utilizing a fibril-reinforced finite element model, we estimated the poroelastic properties of mouse cartilage over a wide range of loading rates for the first time, and show that the hydraulic permeability increased by a factor ~16 from knormal=7.80×10(-16)±1.3×10(-16) m(4)/N s to kGAG-depleted=1.26×10(-14)±6.73×10(-15) m(4)/N s after GAG depletion. The high-frequency modulus, which is related to fluid pressurization and the fibrillar network, decreased significantly after GAG depletion. In contrast, the equilibrium modulus, which is fluid-flow independent, did not show a statistically significant alteration following GAG depletion.

Nanomechanical phenotype of chondroadherin-null murine articular cartilage

Chondroadherin (CHAD), a class IV small leucine rich proteoglycan/protein (SLRP), was hypothesized to play important roles in regulating chondrocyte signaling and cartilage homeostasis. However, its roles in cartilage development and function are not well understood, and no major osteoarthritis-like phenotype was found in the murine model with CHAD genetically deleted (CHAD−/−). In this study, we used atomic force microscopy (AFM)-based nanoindentation to quantify the effects of CHAD deletion on changes in the biomechanical function of murine cartilage. In comparison to wild-type (WT) mice, CHAD-deletion resulted in a significant≈70–80% reduction in the indentation modulus, Eind, of the superficial zone knee cartilage of 11 weeks, 4 months and 1 year old animals. This mechanical phenotype correlates well with observed increases in the heterogeneity collagen fibril diameters in the surface zone. The results suggest that CHAD mainly plays a major role in regulating the formation of the collagen fibrillar network during the early skeletal development. In contrast, CHAD-deletion had no appreciable effects on the indentation mechanics of middle/deep zone cartilage, likely due to the dominating role of aggrecan in the middle/deep zone. The presence of significant rate dependence of the indentation stiffness in both WT and CHAD−/− knee cartilage suggested the importance of both fluid flow induced poroelasticity and intrinsic viscoelasticity in murine cartilage biomechanical properties. Furthermore, the marked differences in the nanomechanical behavior of WT versus CHAD−/− cartilage contrasted sharply with the relative absence of overt differences in histological appearance. These observations highlight the sensitivity of nanomechanical tools in evaluating structural and mechanical phenotypes in transgenic mice.

Mechanical Reinforcement of Polymeric Fibers through Peptide Nanotube Incorporation

High aspect ratio nanotubular assemblies can be effective fillers in mechanically reinforced composite materials. However, most existing nanotubes used for structural purposes are limited in their range of mechanical, chemical, and biological properties. We demonstrate an alternative approach to mechanical reinforcement of polymeric systems by incorporating synthetic D,L-cyclic peptide nanotube bundles as a structural filler in electrospun poly D-, L-lactic acid fibers. The nanotube bundles self-assemble through dynamic hydrogen bonding from synthetic cyclic peptides to yield structures whose dimensions can be altered based on processing conditions, and can be up to hundreds of micrometers long and several hundred nanometers wide. With 8 wt % peptide loading, the composite fibers are >5-fold stiffer than fibers composed of the polymer alone, according to atomic force microscopy-based indentation experiments. This represents a new use for self-assembling cyclic peptides as a load-bearing component in biodegradable composite materials.

Laser Speckle Rheology for evaluating the viscoelastic properties of hydrogel scaffolds

Natural and synthetic hydrogel scaffolds exhibit distinct viscoelastic properties at various length scales and deformation rates. Laser Speckle Rheology (LSR) offers a novel, non-contact optical approach for evaluating the frequency-dependent viscoelastic properties of hydrogels. In LSR, a coherent laser beam illuminates the specimen and a high-speed camera acquires the time-varying speckle images. Cross-correlation analysis of frames returns the speckle intensity autocorrelation function, g2(t), from which the frequency-dependent viscoelastic modulus, G*(ω), is deduced. Here, we establish the capability of LSR for evaluating the viscoelastic properties of hydrogels over a large range of moduli, using conventional mechanical rheometry and atomic force microscopy (AFM)-based indentation as reference-standards. Results demonstrate a strong correlation between |G*(ω)| values measured by LSR and mechanical rheometry (r = 0.95, p < 10−9), and z-test analysis reports that moduli values measured by the two methods are identical (p > 0.08) over a large range (47 Pa – 36 kPa). In addition, |G*(ω)| values measured by LSR correlate well with indentation moduli, E, reported by AFM (r = 0.92, p < 10−7). Further, spatially-resolved moduli measurements in micro-patterned substrates demonstrate that LSR combines the strengths of conventional rheology and micro-indentation in assessing hydrogel viscoelastic properties at multiple frequencies and small length-scales.