Measuring mechanical cues for modeling the stromal matrix in 3D cell cultures.

A breast-cancer tumor develops within a stroma, a tissue where a complex extracellular matrix surrounds cells, mediating the cancer progression through biomechanical and -chemical cues. Current materials partially mimic the stromal matrix in 3D cell cultures but methods for measuring the mechanical properties of the matrix at cell-relevant-length scales and stromal-stiffness levels are lacking. Here, to address this gap, we developed a characterization approach that employs probe-based microrheometry and Bayesian modeling to quantify length-scale-dependent mechanics and mechanical heterogeneity as in the stromal matrix. We examined the interpenetrating network (IPN) composed of alginate scaffolds (for adjusting mechanics) and type-1 collagen (a stromal-matrix constituent). We analyzed viscoelasticity: absolute-shear moduli (stiffness/elasticity) and phase angles (viscous and elastic characteristics). We determined the relationship between microrheometry and rheometry information. Microrheometry reveals lower stiffness at cell-relevant scales, compared to macroscale rheometry, with dependency on the length scale (10 to 100 µm). These data show increasing IPN stiffness with crosslinking until saturation (≃15 mM of Ca2+). Furthermore, we report that IPN stiffness can be adjusted by modulating collagen concentration and interconnectivity (by polymerization temperature). The IPNs are heterogeneous structurally (in SEM) and mechanically. Interestingly, increased alginate crosslinking changes IPN heterogeneity in stiffness but not in phase angle, until the saturation. In contrast, such changes are undetectable in alginate scaffolds. Our nonlinear viscoelasticity analysis at tumor-cell-exerted strains shows that only the softer IPNs stiffen with strain, like the stromal-collagen constituent. In summary, our approach can quantify the stromal-matrix-related viscoelasticity and is likely applicable to other materials in 3D culture.

Quantifying the Influence of X-Ray Irradiation on Cell-Size-Scale Viscoelasticity of Collagen Type 1.

X-rays are widely used in mammography and radiotherapy of breast cancer. The research has focused on the effects of X-rays on cells in breast tissues, instead of the tissues' nonliving material, extracellular matrix. It is unclear what the influence of X-ray irradiation is on the matrix's mechanical cues, known to regulate malignant cancer-cell behaviors. Here, we developed a technique based on magnetic microrheology that can quantify the influence of X-ray irradiation on matrix viscoelasticity--or (solid-like) elastic and (liquid-like) viscous characteristics--at cell-size scales. To model breast-tissue extracellular matrix, we used the primary component of the tissue matrix, collagen type 1, as it is for control, and as irradiated by X-rays (tube voltage 50 kV). We used a magnetic microrheometer to measure collagen matrices using 10-µm-diameter magnetic probes. In each matrix, the probes were nanomanipulated using controlled magnetic forces by the microrheometer while the probes' displacements were detected to measure the viscoelasticity. The collagen-matrix data involve with a typical spatial variation in viscoelasticity. We find that higher irradiation doses (320 Gy) locally reduce stiffness (soften) collagen matrices and increase their loss tangent, indicating an elevated liquid-like nature. For lower, clinically relevant irradiation doses (54 Gy), we find insignificant matrix-viscoelasticity changes. We provide this irradiation-related technique for detection, and modification, of matrix viscoelastic cues at cell-size scales. The technique enables enhanced characterization of irradiated tissue constituents in a variety of breast-cancer radiotherapy types.

Fibrin Stiffness Regulates Phenotypic Plasticity of Metastatic Breast Cancer Cells.

The extracellular matrix (ECM)-regulated phenotypic plasticity is crucial for metastatic progression of triple negative breast cancer (TNBC). While ECM faithful cell-based models are available for in situ and invasive tumors, such as cell aggregate cultures in reconstituted basement membrane and in collagenous gels, there are no ECM faithful models for metastatic circulating tumor cells (CTCs). Such models are essential to represent the stage of metastasis where clinical relevance and therapeutic opportunities are significant. Here, CTC-like DU4475 TNBC cells are cultured in mechanically tunable 3D fibrin hydrogels. This is motivated, as in circulation fibrin aids CTC survival by forming a protective coating reducing shear stress and immune cell-mediated cytotoxicity and promotes several stages of late metastatic processes at the interface between circulation and tissue. This work shows that fibrin hydrogels support DU4475 cell growth, resulting in spheroid formation. Furthermore, increasing fibrin stiffness from 57 to 175 Pa leads to highly motile, actin and tubulin containing cellular protrusions, which are associated with specific cell morphology and gene expression patterns that markedly differ from basement membrane or suspension cultures. Thus, mechanically tunable fibrin gels reveal specific matrix-based regulation of TNBC cell phenotype and offer scaffolds for CTC-like cells with better mechano-biological properties than liquid.

Magnetic microrheometry of tumor-relevant stiffness levels and probabilistic quantification of viscoelasticity differences inside 3D cell culture matrices.

The progression of breast cancer involves cancer-cell invasions of extracellular matrices. To investigate the progression, 3D cell cultures are widely used along with different types of matrices. Currently, the matrices are often characterized using parallel-plate rheometry for matrix viscoelasticity, or liquid-like viscous and stiffness-related elastic characteristics. The characterization reveals averaged information and sample-to-sample variation, yet, it neglects internal heterogeneity within matrices, experienced by cancer cells in 3D culture. Techniques using optical tweezers and magnetic microrheometry have measured heterogeneity in viscoelasticity in 3D culture. However, there is a lack of probabilistic heterogeneity quantification and cell-size-relevant, microscale-viscoelasticity measurements at breast-tumor tissue stiffness up to ≃10 kPa in Young's modulus. Here, we have advanced methods, for the purpose, which use a magnetic microrheometer that applies forces on magnetic spheres within matrices, and detects the spheres displacements. We present probabilistic heterogeneity quantification using microscale-viscoelasticity measurements in 3D culture matrices at breast-tumor-relevant stiffness levels. Bayesian multilevel modeling was employed to distinguish heterogeneity in viscoelasticity from the effects of experimental design and measurement errors. We report about the heterogeneity of breast-tumor-relevant agarose, GrowDex, GrowDex-collagen and fibrin matrices. The degree of heterogeneity differs for stiffness, and phase angle (i.e. ratio between viscous and elastic characteristics). Concerning stiffness, agarose and GrowDex show the lowest and highest heterogeneity, respectively. Concerning phase angle, fibrin and GrowDex-collagen present the lowest and the highest heterogeneity, respectively. While this heterogeneity information involves softer matrices, probed by ≃30 µm magnetic spheres, we employ larger ≃100 µm spheres to increase magnetic forces and acquire a sufficient displacement signal-to-noise ratio in stiffer matrices. Thus, we show pointwise microscale viscoelasticity measurements within agarose matrices up to Young's moduli of 10 kPa. These results establish methods that combine magnetic microrheometry and Bayesian multilevel modeling for enhanced heterogeneity analysis within 3D culture matrices.

Characterizing Aptamers with Reconfigurable Chiral Plasmonic Assemblies.

Aptamers have emerged as versatile affinity ligands and as promising alternatives to protein antibodies. However, the inconsistency in the reported affinities and specificities of aptamers has greatly hindered the development of aptamer-based applications. Herein, we present a strategy to characterize aptamers by using DNA origami-based chiral plasmonic assemblies as reporters and establishing a competitive hybridization reaction-based thermodynamic model. We demonstrate the characterization of several DNA aptamers, including aptamers for small molecules and macromolecules, as well as aptamers with high and low affinities. The presented characterization scheme can be readily adapted to a wide selection of aptamers. We anticipate that our approach will advance the development of aptamer-based applications by enabling reliable and reproducible characterization of aptamers.

Association between self-reported hypertension onset age and electrocardiographic left ventricular hypertrophy.

Objectively defined early-onset hypertension, based on repeated blood pressure measurements, is associated with greater odds of organ damage and cardiovascular mortality than late-onset hypertension. In this study we examined the association between two factors that are easily available in primary care, self-reported hypertension onset age and electrocardiographic left ventricular hypertrophy (ECG-LVH), in a nationwide population sample of 2864 Finns aged ≥50 years. We observed that, in contrast to prior findings, the odds of ECG-LVH were similar between self-reported hypertension onset age groups, and thus self-reported early-onset hypertension does not seem to associate with ECG-LVH more strongly than simple presence of hypertension.

Light-Responsive Dynamic DNA-Origami-Based Plasmonic Assemblies.

DNA nanotechnology offers a versatile toolbox for precise spatial and temporal manipulation of matter on the nanoscale. However, rendering DNA-based systems responsive to light has remained challenging. Herein, we describe the remote manipulation of native (non-photoresponsive) chiral plasmonic molecules (CPMs) using light. Our strategy is based on the use of a photoresponsive medium comprising a merocyanine-based photoacid. Upon exposure to visible light, the medium decreases its pH, inducing the formation of DNA triplex links, leading to a spatial reconfiguration of the CPMs. The process can be reversed simply by turning the light off and it can be repeated for multiple cycles. The degree of the overall chirality change in an ensemble of CPMs depends on the CPM fraction undergoing reconfiguration, which, remarkably, depends on and can be tuned by the intensity of incident light. Such a dynamic, remotely controlled system could aid in further advancing DNA-based devices and nanomaterials.

Early Onset Hypertension Is Associated With Hypertensive End-Organ Damage Already by MidLife.

Early onset hypertension confers increased risk for cardiovascular mortality in the community. Whether early onset hypertension also promotes the development of target end-organ damage (TOD), even by midlife, has remained unknown. We studied 2680 middle-aged CARDIA study (Coronary Artery Risk Development in Young Adults) Study participants (mean age 50±4 years, 57% women) who underwent up to 8 serial blood pressure measurements between 1985 and 2011 (age range at baseline 18-30 years) in addition to assessments of echocardiographic left ventricular hypertrophy, coronary calcification, albuminuria, and diastolic dysfunction in 2010 to 2011. Age of hypertension onset was defined as the age at first of 2 consecutively attended examinations with blood pressure ≥140/90 mm Hg or use of antihypertensive medication. Participants were divided in groups by hypertension onset age (<35 years, 35-44 years, ≥45 years, or no hypertension). While adjusting for TOD risk factors, including systolic blood pressure, we used logistic regression to calculate odds ratios for cases (participants with TOD) versus controls (participants without TOD) to examine the relation of hypertension onset age and hypertensive TOD. Compared with normotensive individuals, hypertension onset at age <35 years was related to odds ratios of 2.29 (95% CI, 1.36-3.86), 2.94 (95% CI, 1.57-5.49), 1.12 (95% CI, 0.55-2.29), and 2.06 (95% CI, 1.04-4.05) for left ventricular hypertrophy, coronary calcification, albuminuria, and diastolic dysfunction, respectively. In contrast, hypertension onset at age ≥45 years was not related to increased odds of TOD. Our findings emphasize the importance of assessing age of hypertension onset in hypertensive patients to identify high-risk individuals for preventing hypertensive complications.

Electrocardiographic predictors of atrial fibrillation in nonhypertensive and hypertensive individuals.

OBJECTIVE: The aim of this study was to compare the predictive value of ECG abnormalities for atrial fibrillation in nonhypertensive versus hypertensive individuals. METHODS: We recorded ECG and measured conventional cardiovascular risk factors in a nationwide population-based sample of 5813 Finns. We divided the participants into nonhypertensive (n = 3148) and hypertensive (n = 2665) individuals and followed the participants for incident atrial fibrillation events. We evaluated the predictive ability of 12 ECG abnormalities for atrial fibrillation using multivariable-adjusted Fine-Gray models. RESULTS: During a follow-up of 11.9 ±â€Š2.9 years, 111 nonhypertensive and 301 hypertensive participants developed atrial fibrillation. Negative T wave in lateral leads predicted atrial fibrillation in both nonhypertensive [hazard ratio (HR), 4.59; 95% confidence interval (95% CI) 1.84-11.44] and hypertensive participants (HR, 1.81; 95% CI 1.16-2.84). In nonhypertensive participants, 1-SD increments in corrected QT interval (HR, 1.42; 95% CI, 1.18-1.71) and T-wave amplitude in lead augmented vector R (aVR) (HR, 1.40; 95% CI, 1.10-1.80) were related to atrial fibrillation. In hypertensive participants, prolonged PR interval (HR, 1.59; 95% CI 1.05-2.41), prolonged P-wave duration (HR, 1.43; 95% CI 1.07-1.91), left ventricular hypertrophy by Sokolow-Lyon criteria (HR, 1.55; 95% CI, 1.12-2.14) and poor R-wave progression (HR, 1.59; 95% CI, 1.02-2.48) predicted atrial fibrillation. Corrected QT interval and T-wave amplitude in lead aVR were stronger predictors of atrial fibrillation in nonhypertensive than in hypertensive participants. ECG abnormalities improved risk prediction only marginally (delta area under receiver-operating-characteristic curve = 0.000-0.005). CONCLUSION: Several ECG abnormalities associate with incident atrial fibrillation in hypertensive and nonhypertensive individuals but provide only marginal incremental predictive value. Corrected QT interval and T-wave amplitude in lead aVR may relate stronger to incident atrial fibrillation in nonhypertensive than in hypertensive individuals.

Thyroid-stimulating hormone and risk of sudden cardiac death, total mortality and cardiovascular morbidity.

BACKGROUND: Previous data on the association of thyroid function with total mortality, cardiovascular disease (CVD) outcomes and sudden cardiac death (SCD) are conflicting or limited. We investigated associations of thyroid-stimulating hormone (TSH) with these outcomes in a nationwide population-based prospective cohort study. METHODS: We examined 5211 participants representative of the Finnish population aged ≥30 years in 2000-2001 and followed them for a median of 13.2 years. Using Cox proportional hazards regression models adjusted for baseline age, gender, smoking, diabetes, systolic blood pressure and total and high-density lipoprotein cholesterol, we assessed the associations of continuous baseline TSH and TSH categories (low [<0.4 mU/L], reference range [0.4-3.4 mU/L] and high [>3.4 mU/L]) with incident total mortality, SCD, coronary heart disease events, stroke, CVD, major adverse cardiac events and atrial fibrillation. RESULTS: High TSH at baseline was related to a greater risk of total mortality (HR 1.34, 95% CI 1.02-1.76) and SCD (HR 2.28, 95% CI 1.13-4.60) compared with TSH within the reference range. High TSH was not associated with the other outcomes (P ≥ .51), whereas low TSH was not associated with any of the outcomes (P ≥ .09). TSH at baseline over the full range did not have a linear relation with any of the outcomes (P ≥ .17). TSH showed a U-shaped association with total mortality after a restricted cubic spline transformation (P = .01). CONCLUSIONS: Thyroid function abnormalities could be linked with higher risks of total mortality and SCD. Large-scale randomized studies are needed for evidence-based recommendations regarding treatment of mild thyroid failure.

Incidence rates, correlates, and prognosis of electrocardiographic P-wave abnormalities - a nationwide population-based study.

BACKGROUND: Scant data exist on incidence rates, correlates, and prognosis of electrocardiographic P-wave abnormalities in the general population. METHODS: We recorded ECG and measured conventional cardiovascular risk factors in 5667 Finns who were followed up for incident atrial fibrillation (AF). We obtained repeat ECGs from 3089 individuals 11years later. RESULTS: The incidence rates of prolonged P-wave duration, abnormal P terminal force (PTF), left P-wave axis deviation, and right P-wave axis deviation were 16.0%, 7.4%, 3.4%, and 2.2%, respectively. Older age and higher BMI were associated with incident prolonged P-wave duration and abnormal PTF (P≤0.01). Higher blood pressure was associated with incident prolonged P-wave duration and right P-wave axis deviation (P≤0.01). During follow-up, only prolonged P-wave duration predicted AF (multivariable-adjusted hazard ratio, 1.38; P=0.001). CONCLUSIONS: Modifiable risk factors associate with P-wave abnormalities that are common and may represent intermediate steps of atrial cardiomyopathy on a pathway leading to AF.

Prevalence and prognosis of ECG abnormalities in normotensive and hypertensive individuals.

OBJECTIVE: To define the prevalence and prognosis of ECG abnormalities in hypertensive individuals. METHODS: ECG, blood pressure and other cardiovascular risk factors were recorded in a nationwide population sample of 5800 Finns. The presence of 15 ECG abnormalities was evaluated. Participants were divided into categories by blood pressure and followed for coronary heart (CHD) and cardiovascular disease (CVD) events. RESULTS: Mean follow-up was 10.4 ±â€Š2.2 years. The age- and sex-adjusted prevalence rates of ECG abnormalities were generally higher in the hypertensive participants than in normotensive individuals. In multivariable-adjusted Cox models, the following ECG abnormalities predicted CHD in hypertensive participants: left ventricular hypertrophy (LVH) by Sokolow-Lyon criteria [hazard ratio, 1.47; 95% confidence interval (CI), 1.07-2.01; P = 0.02], LVH with ST-depression and negative T wave (ST/T changes) (hazard ratio, 2.31; 95% CI, 1.20-4.43, P = 0.01), ST/T changes (hazard ratio, 2.12; 95% CI, 1.34-3.36; P = 0.001), positive T wave in lead aVR (AVRT+) (hazard ratio, 1.74; 95% CI, 1.15-2.64; P = 0.009) and poor R-wave progression (hazard ratio, 2.02; 95% CI, 1.27-3.22; P = 0.003). These ECG abnormalities were also significant predictors of CVD in hypertensive participants (P ≤ 0.03 for all). Nonspecific intraventricular conduction delay predicted CVD in the whole population (hazard ratio, 1.50; 95% CI, 1.06-2.13; P = 0.02). Prolonged QT interval, abnormal P-wave indices, left axis deviation and early repolarization pattern were not associated with CHD or CVD. CONCLUSION: ECG abnormalities are highly prevalent in hypertensive individuals. LVH is still the cornerstone of cardiovascular risk assessment in hypertensive patients. The additional assessment of ST/T changes, AVRT+ and poor R-wave progression in ECGs could improve risk prediction in hypertensive patients.