Interaction of imidazolium-based ionic liquids with supported phospholipid bilayers as model biomembranes.

The cytotoxicity of ionic liquids (ILs) has been receiving attention in the context of the biological and environmental impact of their vast field of applications. It has been ascertained that the cell membrane is the main target of ILs when they interact with microorganisms, cells and bacteria; nevertheless, studies at the micro- and nano-scale aiming at better understanding of the fundamental mechanisms of toxicity of ILs are lacking. In this work, we used atomic force microscopy (AFM) to investigate the impact of room-temperature ILs on the mechanical, morphological and electrostatic properties of solid-supported DOPC phospholipid bilayers, taken as models of biomembranes. In particular, we have characterized the concentration-dependent and time-dependent evolution of the morphological, structural and mechanical properties of DOPC lipid membranes in the presence of imidazolium-based ILs with different alkyl chain lengths and hydrophilic/hydrophobic characteristics. The majority of ILs investigated were found to possess the ability of restructuring the lipid bilayer, through the formation of new IL/lipid complexes, showing distinctive morphological features (increase of area and roughness). The nanomechanical analysis of the lipid membrane exposed to ILs revealed a progressive, concentration-dependent perturbation of the structural ordering and rigidity of the membrane, evidenced by a decrease in the breakthrough force, Young's modulus and area stretching modulus. AFM detected a modification of the electrostatic double-layer at the membrane surface, in terms of a reduction of the original negative surface charge density, suggesting a progressive stratification of cations on the exposed leaflet of the lipid membrane. Our findings may be helpful in designing novel ILs with tailored interaction with biological membranes.

The glycocalyx affects the mechanotransductive perception of the topographical microenvironment.

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of major glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon strong reduction of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features.

Force Sensing on Cells and Tissues by Atomic Force Microscopy.

Biosensors are aimed at detecting tiny physical and chemical stimuli in biological systems. Physical forces are ubiquitous, being implied in all cellular processes, including cell adhesion, migration, and differentiation. Given the strong interplay between cells and their microenvironment, the extracellular matrix (ECM) and the structural and mechanical properties of the ECM play an important role in the transmission of external stimuli to single cells within the tissue. Vice versa, cells themselves also use self-generated forces to probe the biophysical properties of the ECM. ECM mechanics influence cell fate, regulate tissue development, and show peculiar features in health and disease conditions of living organisms. Force sensing in biological systems is therefore crucial to dissecting and understanding complex biological processes, such as mechanotransduction. Atomic Force Microscopy (AFM), which can both sense and apply forces at the nanoscale, with sub-nanonewton sensitivity, represents an enabling technology and a crucial experimental tool in biophysics and mechanobiology. In this work, we report on the application of AFM to the study of biomechanical fingerprints of different components of biological systems, such as the ECM, the whole cell, and cellular components, such as the nucleus, lamellipodia and the glycocalyx. We show that physical observables such as the (spatially resolved) Young's Modulus (YM) of elasticity of ECMs or cells, and the effective thickness and stiffness of the glycocalyx, can be quantitatively characterized by AFM. Their modification can be correlated to changes in the microenvironment, physio-pathological conditions, or gene regulation.

Large colloidal probes for atomic force microscopy: Fabrication and calibration issues.

Atomic force microscopy (AFM) is a powerful tool to investigate interaction forces at the micro and nanoscale. Cantilever stiffness, dimensions and geometry of the tip can be chosen according to the requirements of the specific application, in terms of spatial resolution and force sensitivity. Colloidal probes (CPs), obtained by attaching a spherical particle to a tipless (TL) cantilever, offer several advantages for accurate force measurements: tunable and well-characterisable radius; higher averaging capabilities (at the expense of spatial resolution) and sensitivity to weak interactions; a well-defined interaction geometry (sphere on flat), which allows accurate and reliable data fitting by means of analytical models. The dynamics of standard AFM probes has been widely investigated, and protocols have been developed for the calibration of the cantilever spring constant. Nevertheless, the dynamics of CPs, and in particular of large CPs, with radius well above 10 µm and mass comparable, or larger, than the cantilever mass, is at present still poorly characterized. Here we describe the fabrication and calibration of (large) CPs. We describe and discuss the peculiar dynamical behaviour of CPs, and present an alternative protocol for the accurate calibration of the spring constant.

Nanoscale-Induced Formation of Silicide around Gold Nanoparticles Encapsulated in a-Si.

Decorating thin-film solar cells with plasmonic nanoparticles is being pursued in order to improve device efficiency through increased scattering and local field enhancement. Gold nanoparticles are particularly interesting due to their chemical inertness and plasmon resonance in the visible range of the spectrum. In this work, gold nanoparticles fabricated by a gas aggregation nanoparticle source and embedded in a-Si (a commercial solar cell material) are studied using X-ray photoelectron spectroscopy, transmission electron microscopy, electron energy-loss spectroscopy, and energy-dispersive X-ray spectroscopy. The formation of gold silicide around the nanoparticles is investigated, as it has important consequences for the optical and electronic properties of the structures. Different from previous studies, in which the silicide formation is observed for gold nanoparticles and thin films grown on top of crystalline silicon or silica, it is found that silicide formation is largely enhanced around the nanoparticles, owing to their increased surface/volume ratio. A detailed gold silicide formation mechanism is presented based on the results, and strategies for optimizing the design of plasmonically enhanced solar cells with gold nanoparticles encapsulated in a-Si are discussed.

Solid-Like Ordering of Imidazolium-Based Ionic Liquids at Rough Nanostructured Oxidized Silicon Surfaces.

The investigation of ionic liquids (ILs) confined in a solid porous matrix is of particular interest considering that these substances are increasingly used as an electrolyte in devices employing nanostructured nanoporous materials for the electrodes. Furthermore, the confinement of the ILs into a porous matrix would allow overcoming the difficulties of their packaging, leakage, and portability. In order to support the applications, a deeper understanding of the interaction of ILs with the nanoporous solid material and its increased interface is required. In this work, we report on the modification of morphological and mechanical properties of the imidazolium-based [Bmim][NTf2] IL upon surface spatial confinement on a cluster-assembled, nanostructured, rough, oxidized silicon (ns-SiOx) surface. An atomic force microscopy investigation revealed that upon the interaction with the ns-SiOx film, [Bmim][NTf2] locally rearranges into ordered, layered, stiff, and poorly conducting solid-like domains, coexisting with, and embedded into, the liquid IL film. The observed interfacial layering of [Bmim][NTf2] deposited on ns-SiOx suggests that the behavior of the IL-electrode interface in photoelectrochemical devices employing nanostructured nanoporous materials can be far more complex than expected under the hypothesis of an IL-based electrolyte in the stable liquid phase. The observed effects reported in this work could in principle also occur inside the bulk nanoporous matrix, where they could be further amplified by the extreme spatial confinement.

The interaction between uPAR and vitronectin triggers ligand-independent adhesion signalling by integrins.

The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure-function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin-matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non-canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.

Quantitative Control of Protein and Cell Interaction with Nanostructured Surfaces by Cluster Assembling.

The development of smart prosthetics, scaffolds, and biomaterials for tissue engineering and organ-on-a-chip devices heavily depends on the understanding and control of biotic/abiotic interfaces. In recent years, the nanometer scale emerged as the predominant dimension for processes impacting on protein adsorption and cellular responses on surfaces. In this context, the extracellular matrix (ECM) can be seen as the prototype for an intricate natural structure assembled by nanoscale building blocks forming highly variable nanoscale configurations, dictating cellular behavior and fate. How exactly the ECM nanotopography influences mechanotransduction, that is, the cellular capacity to convert information received from the ECM into appropriate responses, remains partially understood due to the complexity of the involved biological structures, limiting also the attempts to artificially reproduce the nanoscale complexity of the ECM. In this Account, we describe and discuss our strategies for the development of an efficient and large-scale bottom-up approach to fabricate surfaces with multiscale controlled disorder as substrates to study quantitatively the effect of nanoscale topography on biological entities. Our method is based on the use of supersonic cluster beam deposition (SCBD) to assemble, on a substrate, neutral clusters produced in the gas phase and accelerated by a supersonic expansion. The assembling of clusters in the ballistic deposition regime follows simple scaling laws, allowing the quantitative control of surface roughness and asperity layout over large areas. Due to their biocompatibility, we focused on transition metal oxide nanostructured surfaces assembled by titania and zirconia clusters. We demonstrated the engineering of structural and functional properties of the cluster-assembled surfaces with high relevance for interactions at the biotic/abiotic interface. We observed that isoelectric point and wettability, crucial parameters for the adhesion of biological entities on surfaces, are strongly influenced and controlled by the nanoscale roughness. By developing a high-throughput method (protein surface interaction microarray, PSIM), we characterized quantitatively the capacity of the nanostructured surfaces to adsorb proteins, showing that with increasing roughness the adsorption rises beyond what could be expected by the increase in specific area, paralleled by an almost linear decrease in protein binding affinity. We also determined that the spatial layout of the surface asperities effectively perceived by the cells mimics at the nanoscale the topographical ECM characteristics. The interaction with these features consequently regulates parameters significant for cell adhesion and mechanotransductive signaling, such as integrin clustering, focal adhesion maturation, and the correlated cellular mechanobiology, eventually impacting the cellular program and differentiation, as we specifically showed for neuronal cells.

Electrostatic Double-Layer Interaction at the Surface of Rough Cluster-Assembled Films: The Case of Nanostructured Zirconia.

Here, we investigated the influence of the nanoscale surface morphology on the electrostatic double layer at corrugated surfaces in aqueous electrolytes. For this purpose, we have produced cluster-assembled nanostructured zirconium dioxide (ns-ZrO x, x ≈ 2) films with controlled morphological properties by supersonic cluster beam deposition (SCBD) and measured the double-layer interaction by atomic force microscopy with colloidal probes. SCBD allowed tuning the characteristic widths of the corrugated interface (root-mean-square roughness, correlation length) across a wide range of values, matching the width of the electrostatic double layer (Debye length) and the typical size of nanocolloids (proteins, enzymes, and catalytic nanoparticles). To accurately characterize the surface charge density in the high-roughness regime, we have developed a two-exponential model of the electrostatic force that explicitly includes roughness and better accounts for the roughness-induced amplification of the interaction. We were then able to observe a marked reduction of the isoelectric point of ns-ZrO x surfaces of increasing roughness. This result is in good agreement with our previous observations on cluster-assembled nanostructured titania films and demonstrates that the phenomenon is not limited to a specific material, but more generally depends on peculiar nanoscale morphological effects, related to the competition of the characteristic lengths of the system.

Imidazolium-Based Ionic Liquids Affect Morphology and Rigidity of Living Cells: An Atomic Force Microscopy Study.

The study of the toxicity, biocompatibility, and environmental sustainability of room-temperature ionic liquids (ILs) is still in its infancy. Understanding the impact of ILs on living organisms, especially from the aquatic ecosystem, is urgent, since large amounts of these substances are starting to be employed as solvents in industrial chemical processes, and on the other side, evidence of toxic effects of ILs on microorganisms and single cells have been observed. To date, the toxicity of ILs has been investigated by means of macroscopic assays aimed at characterizing the effective concentrations (like the EC50) that cause the death of a significant fraction of the population of microorganisms and cells. These studies allow us to identify the cell membrane as the first target of the IL interaction, whose effectiveness was correlated to the lipophilicity of the cation, i.e., to the length of the lateral alkyl chain. Our study aimed at investigating the molecular mechanisms underpinning the interaction of ILs with living cells. To this purpose, we carried out a combined topographic and mechanical analysis by atomic force microscopy of living breast metastatic cancer cells (MDA-MB-231) upon interaction with imidazolium-based ILs. We showed that ILs are able to induce modifications of the overall rigidity (effective Young's modulus) and morphology of the cells. Our results demonstrate that ILs act on the physical properties of the outer cell layer (the membrane linked to the actin cytoskeleton), already at concentrations below the EC50. These potentially toxic effects are stronger at higher IL concentrations, as well as with longer lateral chains in the cation.

The Incorporation of Ribonucleotides Induces Structural and Conformational Changes in DNA.

Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition.

Tuning the Extent and Depth of Penetration of Flexible Liposomes in Human Skin.

In this work we made an attempt to assess the effect of drug-induced changes of flexibility on the penetration of deformable vesicles into the human skin. Eight cationic liposomes with different degrees of flexibility were obtained by entrapping unfractionated heparin, enoxaparin, and nadroparin. The deformability was studied by a novel, facile, and reliable extrusion assay appositely developed and validated by means of quantitative nanoscale mechanical AFM measurements of vesicle elastic modulus (log10(YM)). The proposed extrusion assay, determining the forces involved in vesicles deformation, resulted very sensitive to evidence of minimal changes in bilayer rigidity (σ) and vesicle deformation (K). The drug loading caused a reduction of liposome flexibility with respect to the reference plain liposomes and in accordance to the heparin type, drug to cationic lipid (DOTAP) ratio, and drug distribution within the vesicles. Interestingly, the σ and log10(YM) values perfectly correlated (R2 = 0.935), demonstrating the reliability of the deformability data obtained with both approaches. The combination of TEM and LC-MS/MS spectrometry allowed the pattern of the penetration of the entire vesicles into the skin to be followed. In all cases, intact liposomes in the epidermis layers were observed and a relationship between the depth of penetration and the liposome flexibility was found, supporting the hypothesis of the whole vesicle penetration mechanism. Moreover, the results of the extent (R24) of vesicle penetration in the human skin samples showed a direct relation to the flexibility values (σ1 = 0.65 ± 0.10 MPa → R24 = 3.33 ± 0.02 µg/mg; σ2 = 0.95 ± 0.04 MPa → R24 = 1.18 ± 0.26 µg/mg; σ3 = 1.89 ± 0.30 MPa → R24 = 0.53 ± 0.33 µg/mg).

Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation.

BACKGROUND: Thanks to mechanotransductive components cells are competent to perceive nanoscale topographical features of their environment and to convert the immanent information into corresponding physiological responses. Due to its complex configuration, unraveling the role of the extracellular matrix is particularly challenging. Cell substrates with simplified topographical cues, fabricated by top-down micro- and nanofabrication approaches, have been useful in order to identify basic principles. However, the underlying molecular mechanisms of this conversion remain only partially understood. RESULTS: Here we present the results of a broad, systematic and quantitative approach aimed at understanding how the surface nanoscale information is converted into cell response providing a profound causal link between mechanotransductive events, proceeding from the cell/nanostructure interface to the nucleus. We produced nanostructured ZrO2 substrates with disordered yet controlled topographic features by the bottom-up technique supersonic cluster beam deposition, i.e. the assembling of zirconia nanoparticles from the gas phase on a flat substrate through a supersonic expansion. We used PC12 cells, a well-established model in the context of neuronal differentiation. We found that the cell/nanotopography interaction enforces a nanoscopic architecture of the adhesion regions that affects the focal adhesion dynamics and the cytoskeletal organization, which thereby modulates the general biomechanical properties by decreasing the rigidity of the cell. The mechanotransduction impacts furthermore on transcription factors relevant for neuronal differentiation (e.g. CREB), and eventually the protein expression profile. Detailed proteomic data validated the observed differentiation. In particular, the abundance of proteins that are involved in adhesome and/or cytoskeletal organization is striking, and their up- or downregulation is in line with their demonstrated functions in neuronal differentiation processes. CONCLUSION: Our work provides a deep insight into the molecular mechanotransductive mechanisms that realize the conversion of the nanoscale topographical information of SCBD-fabricated surfaces into cellular responses, in this case neuronal differentiation. The results lay a profound cell biological foundation indicating the strong potential of these surfaces in promoting neuronal differentiation events which could be exploited for the development of prospective research and/or biomedical applications. These applications could be e.g. tools to study mechanotransductive processes, improved neural interfaces and circuits, or cell culture devices supporting neurogenic processes.

Nanoscale roughness affects the activity of enzymes adsorbed on cluster-assembled titania films.

In this study, we investigated how the adsorption properties governed by the nanometer-scale surface morphology of cluster-assembled titanium oxide films influence the catalytic activity of immobilized serine-protease trypsin. We developed an activity assay for the parallel detection of physisorbed enzyme activity and mass density of the adsorbed proteins in microarray format. The method combines a microarray-based technique and advanced quantitative confocal microscopy approaches based on fluorescent labeling of enzymes and covalent labeling of active sites of surface-bound enzymes. The observed diminishing trypsin binding affinity with increasing roughness, as opposed to the steep rise in its saturation uptake, was interpreted as heterogeneous nucleation-driven adsorption of trypsin at the rough nanoporous titania surface. The increase in relative activity of adsorbed trypsin is proportional to the fractional saturation of titania surfaces, expressed as percentage of saturation uptake. In turn, the specific activity, that is, the ratio of active proteins to the absolute number of adsorbed proteins, drops with growing saturation uptake and surface roughness, witnessing a reduction in the accessibility of enzyme active sites. Both geometrical constraints of titania nanopores and the clusterwise adsorption of trypsin were identified as the key factors underpinning the steric hindrance of the immobilized enzyme. These findings are relevant for the optimization of rough nanoporous surfaces as carriers of immobilized enzymes. The proposed activity assay is particularly advantageous in the screening of candidate materials for enzyme immobilization.

Ferritin at different iron loading: From biological to nanotechnological applications.

The characterization of the structure of ferritin in solution and the arrangement of iron stored in its cavity are intriguing subjects for both cell biology and applied science, since the protein structure, stability, and easiness of production make it an ideal tool for biomedical applications. We characterized the ferritin structure over a wide range of iron loadings by visible light, X-ray, and neutron scattering techniques. We found that the arrangement of iron ions inside the protein cage resulted in a more disposable arrangement at lower loading factors and then in a crystalline structure. At very high iron content the inner core is composed of magnetite more than ferrihydrite, and the shell of the protein is elastically deformed by the iron crystal growth in an ellipsoidal arrangement. The application of an external radiofrequency (RF) magnetic field affected ferritins at low iron loading factors. Notably the RF modified the iron disposition towards a more dispersed arrangement. The structural characterization of the ferritin at different LFs and in presence of magnetic fields provides useful insights into their physiological behaviour and can help in the design and fine-tuning of ferritin-based nanosystems for biotechnological applications.

Progressive alteration of murine bladder elasticity in actinic cystitis detected by Brillouin microscopy.

Bladder mechanical properties are critical for organ function and tissue homeostasis. Therefore, alterations of tissue mechanics are linked to disease onset and progression. This study aims to characterize the tissue elasticity of the murine bladder wall considering its different anatomical components, both in healthy conditions and in actinic cystitis, a state characterized by tissue fibrosis. Here, we exploit Brillouin microscopy, an emerging technique in the mechanobiology field that allows mapping tissue mechanics at the microscale, in non-contact mode and free of labeling. We show that Brillouin imaging of bladder tissues is able to recognize the different anatomical components of the bladder wall, confirmed by histopathological analysis, showing different tissue mechanical properties of the physiological bladder, as well as a significant alteration in the presence of tissue fibrosis. Our results point out the potential use of Brillouin imaging on clinically relevant samples as a complementary technique to histopathological analysis, deciphering complex mechanical alteration of each tissue layer of an organ that strongly relies on mechanical properties to perform its function.

Interaction of imidazolium-based room-temperature ionic liquids with DOPC phospholipid monolayers: electrochemical study.

To test the biocompatible character of room-temperature ionic liquids (ILs), the interaction of various ILs with biological membrane (biomembrane) models was studied in this work. Dioleoyl phosphatidylcholine (DOPC) adsorbed on a mercury (Hg) electrode forms an impermeable defect-free monolayer which is a well established biomembrane model, prone to be studied by electrochemical techniques. We have monitored the modifications of the Hg supported monolayer caused by ILs using rapid cyclic voltammetry (RCV), alternating current voltammetry (ACV), and electrochemical impedance spectroscopy (EIS). A series of imidazolium-based ILs were investigated whose interaction highlighted the role of anion and lateral side chain of cation during the interaction with DOPC monolayers. It was shown that the hydrophobic and lipophilic character of the IL cations is a primary factor responsible for this interaction. Hg-supported monolayers provide an accurate analysis of the behavior of ILs at the interface of a biomembrane leading to a comprehensive understanding of the interaction mechanisms involved. At the same time, these experiments show that the Hg-phospholipid model is an effective toxicity sensing technique as shown by the correlation between literature in vivo toxicity data and the data from this study.

Interaction of bacterial cells with cluster-assembled nanostructured titania surfaces: an atomic force microscopy study.

The nanoscale interaction of bacterial cells with solid surfaces is a key issue in biomedicine because it constitutes the first pathogenic event in the complex series of biofilm development on prosthetic devices. We report on an Atomic Force Microscopy study of the interaction of Escherichia coli and Pseudomonas aeruginosa bacterial cells with nanostructured titania thin films with controlled and reproducible nanometer-scale morphology, produced by assembling Ti clusters from the gas phase in a Supersonic Cluster Beam Deposition apparatus. The results demonstrate that bacterial adhesion and biofilm formation are significantly influenced by a pure physical stimulus, that is, the nanoscale variation of surface topography. The increase of nanoscale film roughness promotes bacterial adhesion with respect to flat substrates; remarkably, Pseudomonas aeruginosa cells lose their flagella on nanostructured TiO2 thin films upon adhesion, as opposed to same bacteria onto reference smooth glass substrates. Further, we have observed increased cell biovolume and other biofilm properties on nanostructured substrates in comparison with smooth glasses. These findings suggest that the design of innovative biomaterials with a suitable patterning of biomaterials surfaces can be an effective approach to control the adhesion of microorganisms to in vivo implant surfaces with active biological functionalities.

Correlation between biological and mechanical properties of extracellular matrix from colorectal peritoneal metastases in human tissues.

Peritoneal metastases (PM) are common routes of dissemination for colorectal cancer (CRC) and remain a lethal disease with a poor prognosis. The properties of the extracellular matrix (ECM) are important in cancer development; studying their changes is crucial to understand CRC-PM development. We studied the elastic properties of ECMs derived from human samples of normal and neoplastic PM by atomic force microscopy (AFM); results were correlated with patient clinical data and expression of ECM components related to metastatic spread. We show that PM progression is accompanied by stiffening of the ECM, increased cancer associated fibroblasts (CAF) activity and increased deposition and crosslinking in neoplastic matrices; on the other hand, softer regions are also found in neoplastic ECMs on the same scales. Our results support the hypothesis that local changes in the normal ECM can create the ground for growth and spread from the tumour of invading metastatic cells. We have found correlations between the mechanical properties (relative stiffening between normal and neoplastic ECM) of the ECM and patients' clinical data, like age, sex, presence of protein activating mutations in BRAF and KRAS genes and tumour grade. Our findings suggest that the mechanical phenotyping of PM-ECM has the potential to predict tumour development.

Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.