Different contractility modes control cell escape from multicellular spheroids and tumor explants.

Cells can adapt their active contractile properties to switch between dynamical migratory states and static homeostasis. Collective tissue surface tension, generated among others by the cortical contractility of single cells, can keep cell clusters compact, while a more bipolar, anisotropic contractility is predominantly used by mesenchymal cells to pull themselves into the extracellular matrix (ECM). Here, we investigate how these two contractility modes relate to cancer cell escape into the ECM. We compare multicellular spheroids from a panel of breast cancer cell lines with primary tumor explants from breast and cervical cancer patients by measuring matrix contraction and cellular spreading into ECM mimicking collagen matrices. Our results in spheroids suggest that tumor aggressiveness is associated with elevated contractile traction and reduced active tissue surface tension, allowing cancer cell escape. We show that it is not a binary switch but rather the interplay between these two contractility modes that is essential during this process. We provide further evidence in patient-derived tumor explants that these two contractility modes impact cancer cells' ability to leave cell clusters within a primary tumor. Our results indicate that cellular contractility is an essential factor during the formation of metastases and thus may be suitable as a prognostic criterion for the assessment of tumor aggressiveness.

Sensitivity of magnetic resonance elastography to extracellular matrix and cell motility in human prostate cancer cell line-derived xenograft models.

Prostate cancer (PCa) is a significant health problem in the male population of the Western world. Magnetic resonance elastography (MRE), an emerging medical imaging technique sensitive to mechanical properties of biological tissues, detects PCa based on abnormally high stiffness and viscosity values. Yet, the origin of these changes in tissue properties and how they correlate with histopathological markers and tumor aggressiveness are largely unknown, hindering the use of tumor biomechanical properties for establishing a noninvasive PCa staging system. To infer the contributions of extracellular matrix (ECM) components and cell motility, we investigated fresh tissue specimens from two PCa xenograft mouse models, PC3 and LNCaP, using magnetic resonance elastography (MRE), diffusion-weighted imaging (DWI), quantitative histology, and nuclear shape analysis. Increased tumor stiffness and impaired water diffusion were observed to be associated with collagen and elastin accumulation and decreased cell motility. Overall, LNCaP, while more representative of clinical PCa than PC3, accumulated fewer ECM components, induced less restriction of water diffusion, and exhibited increased cell motility, resulting in overall softer and less viscous properties. Taken together, our results suggest that prostate tumor stiffness increases with ECM accumulation and cell adhesion - characteristics that influence critical biological processes of cancer development. MRE paired with DWI provides a powerful set of imaging markers that can potentially predict prostate tumor development from benign masses to aggressive malignancies in patients. STATEMENT OF SIGNIFICANCE: Xenograft models of human prostate tumor cell lines, allowing correlation of microstructure-sensitive biophysical imaging parameters with quantitative histological methods, can be investigated to identify hallmarks of cancer.

Effect of non-linear strain stiffening in eDAH and unjamming.

In cell clusters, the prominent factors at play encompass contractility-based enhanced tissue surface tension and cell unjamming transition. The former effect pertains to the boundary effect, while the latter constitutes a bulk effect. Both effects share outcomes of inducing significant elongation in cells. This elongation is so substantial that it surpasses the limits of linear elasticity, thereby giving rise to additional effects. To investigate these effects, we employ atomic force microscopy (AFM) to analyze how the mechanical properties of individual cells change under such considerable elongation. Our selection of cell lines includes MCF-10A, chosen for its pronounced demonstration of the extended differential adhesion hypothesis (eDAH), and MDA-MB-436, selected due to its manifestation of cell unjamming behavior. In the AFM analyses, we observe a common trend in both cases: as elongation increases, both cell lines exhibit strain stiffening. Notably, this effect is more prominent in MCF-10A compared to MDA-MB-436. Subsequently, we employ AFM on a dynamic range of 1-200 Hz to probe the mechanical characteristics of cell spheroids, focusing on both surface and bulk mechanics. Our findings align with the results from single cell investigations. Specifically, MCF-10A cells, characterized by strong contractile tissue tension, exhibit the greatest stiffness on their surface. Conversely, MDA-MB-436 cells, which experience significant elongation, showcase their highest stiffness within the bulk region. Consequently, the concept of single cell strain stiffening emerges as a crucial element in understanding the mechanics of multicellular spheroids (MCSs), even in the case of MDA-MB-436 cells, which are comparatively softer in nature.

Anatomy of the fetal membranes: insights from spinning disk confocal microscopy.

PURPOSE: The fetal membranes are essential for the maintenance of pregnancy, and their integrity until parturition is critical for both fetal and maternal health. Preterm premature rupture of the membranes (pPROM) is known to be an indicator of preterm birth, but the underlying architectural and mechanical changes that lead to fetal membrane failure are not yet fully understood. The aim of this study was to gain new insights into the anatomy of the fetal membrane and to establish a tissue processing and staining protocol suitable for future prospective cohort studies. METHODS: In this proof of principle study, we collected fetal membranes from women undergoing vaginal delivery or cesarean section. Small membrane sections were then fixed, stained for nucleic acids, actin, and collagen using fluorescent probes, and subsequently imaged in three dimensions using a spinning disk confocal microscope. RESULTS: Four fetal membranes of different types were successfully processed and imaged after establishing a suitable protocol. Cellular and nuclear outlines are clearly visible in all cases, especially in the uppermost membrane layer. Focal membrane (micro) fractures could be identified in several samples. CONCLUSION: The presented method proves to be well suited to determine whether and how the occurrence of membrane (micro) fractures and cellular jamming correlate with the timing of membrane rupture and the mode of delivery. In future measurements, this method could be combined with mechanical probing techniques to compare optical and mechanical sample information.

Re-evaluation of publicly available gene-expression databases using machine-learning yields a maximum prognostic power in breast cancer.

Gene expression signatures refer to patterns of gene activities and are used to classify different types of cancer, determine prognosis, and guide treatment decisions. Advancements in high-throughput technology and machine learning have led to improvements to predict a patient's prognosis for different cancer phenotypes. However, computational methods for analyzing signatures have not been used to evaluate their prognostic power. Contention remains on the utility of gene expression signatures for prognosis. The prevalent approaches include random signatures, expert knowledge, and machine learning to construct an improved signature. We unify these approaches to evaluate their prognostic power. Re-evaluation of publicly available gene-expression data from 8 databases with 9 machine-learning models revealed previously unreported results. Gene-expression signatures are confirmed to be useful in predicting a patient's prognosis. Convergent evidence from [Formula: see text] 10,000 signatures implicates a maximum prognostic power. By calculating the concordance index, which measures how well patients with different prognoses can be discriminated, we show that a signature can correctly discriminate patients' prognoses no more than 80% of the time. Additionally, we show that more than 50% of the potentially available information is still missing at this value. We surmise that an accurate prognosis must incorporate molecular, clinical, histological, and other complementary factors.

Heavy water induces bundling in entangled actin networks.

Heavy water is known to affect many different biological systems, with the most striking effects observed at the cellular level. Many dynamic processes, such as migration or invasion, but also central processes of cell proliferation are measurably inhibited by the presence of deuterium oxide (D2O). Furthermore, individual cell deformabilities are significantly decreased upon D2O treatment. In order to understand the origin of these effects, we studied entangled filamentous actin networks, a commonly used model system for the cytoskeleton, which is considered a central functional element for dynamic cellular processes. Using bulk shear rheology to extract rheological signatures of reconstituted actin networks at varying concentrations of D2O, we found a non-monotonic behavior, which is explainable by a drastic change in the actin network architecture. Applying light scattering and fluorescence microscopy, we were able to demonstrate that the presence of deuterium oxide induces bundling in reconstituted entangled networks of filamentous actin. This constitutes an entirely novel and previously undescribed actin bundling mechanism.

Changes in Tissue Fluidity Predict Tumor Aggressiveness In Vivo.

Cancer progression is caused by genetic changes and associated with various alterations in cell properties, which also affect a tumor's mechanical state. While an increased stiffness has been well known for long for solid tumors, it has limited prognostic power. It is hypothesized that cancer progression is accompanied by tissue fluidization, where portions of the tissue can change position across different length scales. Supported by tabletop magnetic resonance elastography (MRE) on stroma mimicking collagen gels and microscopic analysis of live cells inside patient derived tumor explants, an overview is provided of how cancer associated mechanisms, including cellular unjamming, proliferation, microenvironment composition, and remodeling can alter a tissue's fluidity and stiffness. In vivo, state-of-the-art multifrequency MRE can distinguish tumors from their surrounding host tissue by their rheological fingerprints. Most importantly, a meta-analysis on the currently available clinical studies is conducted and universal trends are identified. The results and conclusions are condensed into a gedankenexperiment about how a tumor can grow and eventually metastasize into its environment from a physics perspective to deduce corresponding mechanical properties. Based on stiffness, fluidity, spatial heterogeneity, and texture of the tumor front a roadmap for a prognosis of a tumor's aggressiveness and metastatic potential is presented.

Systematic altering of semiflexible DNA-based polymer networks via tunable crosslinking.

In order to understand and predict the mechanical behaviours of complex, soft biomaterials such as cells or stimuli-responsive hydrogels, it is important to connect how the nanoscale properties of their constituent components impact those of the bulk material. Crosslinked networks of semiflexible polymers are particularly ubiquitous, being underlying mechanical components of biological systems such as cells or ECM, as well as many synthetic or biomimetic materials. Cell-derived components such as filamentous biopolymers or protein crosslinkers are readily available and well-studied model systems. However, as evolutionarily derived materials, they are constrained to a fixed set of structural parameters such as the rigidity and size of the filaments, or the valency and strength of binding of crosslinkers forming inter-filament connections. By implementing a synthetic model system based on the self-assembly of DNA oligonucleotides into nanometer-scale tubes and simple crosslinking constructs, we used the thermodynamic programmability of DNA hybridization to explore the impact of binding affinity on bulk mechanical response. Stepwise tuning the crosslinking affinity over a range from transient to thermodynamically stable shows an according change in viscoelastic behaviour from loosely entangled to elastic, consistent with models accounting for generalized inter-filament interactions. While characteristic signatures of concentration-dependent changes in network morphology found in some other natural and synthetic filament-crosslinker systems were not apparent, the presence of a distinct elasticity increase within a narrow range of conditions points towards potential subtle alterations of crosslink-filament architecture. Here, we demonstrate a new synthetic approach for gaining a deeper understanding of both biological as well as engineered hydrogel systems.

Mechanical properties of murine hippocampal subregions investigated by atomic force microscopy and in vivo magnetic resonance elastography.

The hippocampus is a very heterogeneous brain structure with different mechanical properties reflecting its functional variety. In particular, adult neurogenesis in rodent hippocampus has been associated with specific viscoelastic properties in vivo and ex vivo. Here, we study the microscopic mechanical properties of hippocampal subregions using ex vivo atomic force microscopy (AFM) in correlation with the expression of GFP in presence of the nestin promoter, providing a marker of neurogenic activity. We further use magnetic resonance elastography (MRE) to investigate whether in vivo mechanical properties reveal similar spatial patterns, however, on a much coarser scale. AFM showed that tissue stiffness increases with increasing distance from the subgranular zone (p = 0.0069), and that stiffness is 39% lower in GFP than non-GFP regions (p = 0.0004). Consistently, MRE showed that dentate gyrus is, on average, softer than Ammon´s horn (shear wave speed = 3.2 ± 0.2 m/s versus 4.4 ± 0.3 m/s, p = 0.01) with another 3.4% decrease towards the subgranular zone (p = 0.0001). The marked reduction in stiffness measured by AFM in areas of high neurogenic activity is consistent with softer MRE values, indicating the sensitivity of macroscopic mechanical properties in vivo to micromechanical structures as formed by the neurogenic niche of the hippocampus.

Electron beam treated injectable agarose/alginate beads prepared by electrospraying.

Granular hydrogels have evolved into an innovative technology for biomedicine. Unlike conventional hydrogels, granular hydrogels display dynamic properties like injectability and porosity, making them feasible for applications in 3D bioprinting and tissue engineering. High-energy electron irradiation combines sterilization and tuning of hydrogel properties without adding potentially cytotoxic chemicals. In this study, granular agarose/alginate hydrogels are prepared by electrospraying. Utilizing 10 MeV electron irradiation, the granular hydrogels are treated in a dose range of 0 kGy-30 kGy relevant for sterilization. Herein, a size reduction of the microparticles is observed. Mechanical properties of individual agarose/alginate beads are examined using AFM measurements revealing a gel softening attributed to radiation induced chain scission. Shear-thinning and self-healing characteristics of the entire granular hydrogel are studied employing rheology. Although viscoelasticity changes under irradiation, shear-thinning and self-healing prevails. These dynamic properties enable injection, which is demonstrated for 27 G needles. This study presents a mechanical characterization of high-energy electron irradiated granular agarose/alginate hydrogels that extends the diversity of available injectable hydrogels and provides a basis for biomedical applications of this scaffold.

Microfluidic Lab-on-a-Chip for Studies of Cell Migration under Spatial Confinement.

Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists' analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic devices.

Comparative analysis of molecular signatures reveals a hybrid approach in breast cancer: Combining the Nottingham Prognostic Index with gene expressions into a hybrid signature.

The diagnosis of breast cancer-including determination of prognosis and prediction-has been traditionally based on clinical and pathological characteristics such as tumor size, nodal status, and tumor grade. The decision-making process has been expanded by the recent introduction of molecular signatures. These signatures, however, have not reached the highest levels of evidence thus far. Yet they have been brought to clinical practice based on statistical significance in prospective as well as retrospective studies. Intriguingly, it has also been reported that most random sets of genes are significantly associated with disease outcome. These facts raise two highly relevant questions: What information gain do these signatures procure? How can one find a signature that is substantially better than a random set of genes? Our study addresses these questions. To address the latter question, we present a hybrid signature that joins the traditional approach with the molecular one by combining the Nottingham Prognostic Index with gene expressions in a data-driven fashion. To address the issue of information gain, we perform careful statistical analysis and comparison of the hybrid signature, gene expression lists of two commercially available tests as well as signatures selected at random, and introduce the Signature Skill Score-a simple measure to assess improvement on random signatures. Despite being based on in silico data, our research is designed to be useful for the decision-making process of oncologists and strongly supports association of random signatures with outcome. Although our study shows that none of these signatures can be considered as the main candidate for providing prognostic information, it also demonstrates that both the hybrid signature and the gene expression list of the OncotypeDx signature identify patients who may not require adjuvant chemotherapy. More importantly, we show that combining signatures substantially improves the identification of patients who do not need adjuvant chemotherapy.

Whole tissue and single cell mechanics are correlated in human brain tumors.

Biomechanical changes are critical for cancer progression. However, the relationship between the rheology of single cells measured ex-vivo and the living tumor is not yet understood. Here, we combined single-cell rheology of cells isolated from primary tumors with in vivo bulk tumor rheology in patients with brain tumors. Eight brain tumors (3 glioblastoma, 3 meningioma, 1 astrocytoma, 1 metastasis) were investigated in vivo by magnetic resonance elastography (MRE), and after surgery by the optical stretcher (OS). MRE was performed in a 3-Tesla clinical MRI scanner and magnitude modulus |G*|, loss angle φ, storage modulus G', and loss modulus G'' were derived. OS experiments measured cellular creep deformation in response to laser-induced step stresses. We used a Kelvin-Voigt model to deduce two parameters related to cellular stiffness (µKV) and cellular viscosity (ηKV) from OS measurements in a time regimen that overlaps with that of MRE. We found that single-cell µKV was correlated with |G*| (R = 0.962, p < 0.001) and G'' (R = 0.883, p = 0.004) but not G' of the bulk tissue. These results suggest that single-cell stiffness affects tissue viscosity in brain tumors. The observation that viscosity parameters of individual cells and bulk tissue were not correlated suggests that collective mechanical interactions (i.e. emergent effects or cellular unjamming) of many cancer cells, which depend on cellular stiffness, influence the mechanical dissipation behavior of the bulk tissue. Our results are important to understand the emergent rheology of active multiscale compound materials such as brain tumors and its role in disease progression.

Cells in Slow Motion: Apparent Undercooling Increases Glassy Behavior at Physiological Temperatures.

Solvent conditions are unexpectedly sufficient to drastically and reversibly slow down cells. In vitro on the molecular level, protein-solvent interactions drastically change in the presence of heavy water (D2 O) and its stronger hydrogen bonds. Adding D2 O to the cell medium of living cells increases the molecular intracellular viscosity. While cell morphology and phenotype remain unchanged, cellular dynamics transform into slow motion in a changeable manner. This is exemplified in the slowdown of cell proliferation and migration, which is caused by a reversible gelation of the cytoplasm. In analogy to the time-temperature superposition principle, where temperature is replaced by D2 O, an increase in viscosity slows down the effective time. Actin networks, crucial structures in the cytoplasm, switch from a power-law-like viscoelastic to a more rubber-like elastic behavior. The resulting intracellular resistance and dissipation impair cell movement. Since cells are highly adaptive non-equilibrium systems, they usually respond irreversibly from a thermodynamic perspective. D2 O induced changes, however, are fully reversible and their effects are independent of signaling as well as expression. The stronger hydrogen bonds lead to glass-like, drawn-out intramolecular dynamics, which may facilitate longer storage times of biological matter, for instance, during transport of organ transplants.

The Mechanical Fingerprint of Circulating Tumor Cells (CTCs) in Breast Cancer Patients.

Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells-possible CTCs-were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin-Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization.

The study of cellular migration dynamics and strategies plays a relevant role in the understanding of both physiological and pathological processes. An important example could be the link between cancer cell motility and tumor evolution into metastatic stage. These strategies can be strongly influenced by the extracellular environment and the consequent mechanical constrains. In this framework, the possibility to study the behavior of single cells when subject to specific topological constraints could be an important tool in the hands of biologists. Two-photon polymerization is a sub-micrometric additive manufacturing technique that allows the fabrication of 3D structures in biocompatible resins, enabling the realization of ad hoc biochips for cell motility analyses, providing different types of mechanical stimuli. In our work, we present a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs for the study of cell motility which guarantees complete optical accessibility and the possibility to freely shape the migration area, to tailor it to the requirements of the specific cell type or experiment. The device includes a series of micro-constrictions that induce different types of mechanical stress on the cells during their migration. We show the realization of different possible geometries, in order to prove the versatility of the technique. As a proof of concept, we present the use of one of these devices for the study of the motility of murine neuronal cancer cells under high physical confinement, highlighting their peculiar migration mechanisms.

Cell-cell adhesion and 3D matrix confinement determine jamming transitions in breast cancer invasion.

Plasticity of cancer invasion and metastasis depends on the ability of cancer cells to switch between collective and single-cell dissemination, controlled by cadherin-mediated cell-cell junctions. In clinical samples, E-cadherin-expressing and -deficient tumours both invade collectively and metastasize equally, implicating additional mechanisms controlling cell-cell cooperation and individualization. Here, using spatially defined organotypic culture, intravital microscopy of mammary tumours in mice and in silico modelling, we identify cell density regulation by three-dimensional tissue boundaries to physically control collective movement irrespective of the composition and stability of cell-cell junctions. Deregulation of adherens junctions by downregulation of E-cadherin and p120-catenin resulted in a transition from coordinated to uncoordinated collective movement along extracellular boundaries, whereas single-cell escape depended on locally free tissue space. These results indicate that cadherins and extracellular matrix confinement cooperate to determine unjamming transitions and stepwise epithelial fluidization towards, ultimately, cell individualization.

Influence of hyaluronic acid binding on the actin cortex measured by optical forces.

Melanoma cells are often surrounded by hyaluronic acid (HA) rich environments, which are considered to promote tumor progression and metastasis. Induced effects in compound materials consisting of cells embedded in an extracellular matrix have been studied, however, alterations of the single cells have never been addressed. Here, we explicitly addressed single cell properties and measured HA-induced biomechanical changes via deformations induced solely by optical forces. With the optical stretcher setup, cells were deformed after culturing them in either the presence or absence of HA revealing the crucial interplay of HA with the CD44 receptor. To assess the role of CD44 in transducing effects of HA, we compared a CD44 expressing variant of the melanoma cell line RPM-MC to its natural CD44-negative counterpart. Our measurements revealed a significant stiffness change, which we attribute to changes of the actin cytoskeleton.

Detecting heterogeneity in and between breast cancer cell lines.

BACKGROUND: Cellular heterogeneity in tumor cells is a well-established phenomenon. Genetic and phenotypic cell-to-cell variability have been observed in numerous studies both within the same type of cancer cells and across different types of cancers. Another known fact for metastatic tumor cells is that they tend to be softer than their normal or non-metastatic counterparts. However, the heterogeneity of mechanical properties in tumor cells are not widely studied. RESULTS: Here we analyzed single-cell optical stretcher data with machine learning algorithms on three different breast tumor cell lines and show that similar heterogeneity can also be seen in mechanical properties of cells both within and between breast tumor cell lines. We identified two clusters within MDA-MB-231 cells, with cells in one cluster being softer than in the other. In addition, we show that MDA-MB-231 cells and MDA-MB-436 cells which are both epithelial breast cancer cell lines with a mesenchymal-like phenotype derived from metastatic cancers are mechanically more different from each other than from non-malignant epithelial MCF-10A cells. CONCLUSION: Since stiffness of tumor cells can be an indicator of metastatic potential, this result suggests that metastatic abilities could vary within the same monoclonal tumor cell line.

How tissue fluidity influences brain tumor progression.

Mechanical properties of biological tissues and, above all, their solid or fluid behavior influence the spread of malignant tumors. While it is known that solid tumors tend to have higher mechanical rigidity, allowing them to aggressively invade and spread in solid surrounding healthy tissue, it is unknown how softer tumors can grow within a more rigid environment such as the brain. Here, we use in vivo magnetic resonance elastography (MRE) to elucidate the role of anomalous fluidity for the invasive growth of soft brain tumors, showing that aggressive glioblastomas (GBMs) have higher water content while behaving like solids. Conversely, our data show that benign meningiomas (MENs), which contain less water than brain tissue, are characterized by fluid-like behavior. The fact that the 2 tumor entities do not differ in their soft properties suggests that fluidity plays an important role for a tumor's aggressiveness and infiltrative potential. Using tissue-mimicking phantoms, we show that the anomalous fluidity of neurotumors physically enables GBMs to penetrate surrounding tissue, a phenomenon similar to Saffman-Taylor viscous-fingering instabilities, which occur at moving interfaces between fluids of different viscosity. Thus, targeting tissue fluidity of malignant tumors might open horizons for the diagnosis and treatment of cancer.