Biology subjects

Lekka, M.

Publications and source records attributed to Lekka, M..

13 recordsLinked to original sources

bioRxiv2026

Differential effects of fenofibrate and fenofibric acid on the regulation of liver endothelial permeability

Background and AimsFenofibrate is widely prescribed for hyperlipidaemia and has been associated with rare but severe cases of drug-induced liver injury (DILI), yet its effects on liver sinusoidal endothelial cells (LSECs) remain to be investigated. LSECs maintain a highly permeable specialized sinusoidal barrier characterized by transcellular pores (fenestrations), regulating the bidirectional transfer of circulating compounds to and from the hepatocytes. As drug-induced alterations in fenestration architecture could influence xenobiotic access to hepatocytes, these changes may modulate pathways associated with DILI. Understanding the effects of fenofibrate on LSEC ultrastructure may therefore provide insights into previously underexplored endothelial contributions to hepatic drug responses. MethodsBoth fenofibrate and its active metabolite, fenofibric acid, were evaluated for their effects on LSEC ultrastructure, mechanical properties, and functional markers. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) and were used to quantify fenestration architecture. AFM was additionally used to measure cellular mechanical properties, which were interpreted in the context of fluorescence-based quantification of cytoskeletal organization. Gene expression, viability, and cytotoxicity were assessed using PCR-based and biochemical assays. ResultsFenofibrate reduced fenestration number and porosity at both tested concentration (10, and 25 {micro}M). It also decreased the apparent Youngs modulus of LSECs, accompanied by changes in tubulin and actin architecture, without detectable cytotoxicity. In contrast, treatment with fenofibric acid did not result in significant structural or mechanical effects on LSECs, even at higher concentrations. ConclusionsTogether, these data identify LSECs as a drug-responsive hepatic cell type for fenofibrate, suggesting that LSECs could represent an underrecognized contributor to the complex, multifactorial processes underlying DILI. This work provides a framework for evaluating endothelial contributions to fenofibrate-associated liver effects in more complex models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/718907v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f9ec6forg.highwire.dtl.DTLVardef@11174a1org.highwire.dtl.DTLVardef@1000e2borg.highwire.dtl.DTLVardef@a23b00_HPS_FORMAT_FIGEXP M_FIG Fenofibrate reduces LSEC fenestrations and metabolic activity at higher concentrations, while its metabolite, fenofibric acid, does not affect LSEC, regardless of its concentration. C_FIG

cell biology↗

bioRxiv2025

Matrix mechanics governs mechano-metabolic adaptation across cancer grades in bladder spheroids

Extracellular matrix (ECM) mechanics critically influence cancer progression, yet the interplay between ECM viscoelasticity, architecture, and tumor cell adaptation remains incompletely understood. Here, we engineered collagen-hyaluronan hydrogels with tunable stiffness to mimic soft and stiff tumor microenvironments and studied bladder cancer spheroids representing benign, low-invasive, and highly invasive stages. Using hydraulic force spectroscopy, rheometry, and molecular analyses, we found that matrix stiffness differentially modulates spheroid morphology, migration, and expression of adhesion and metabolic markers. Active ECM remodeling via metalloproteinase MMP-2 facilitated migration in compliant but not rigid matrices, while mechano-metabolic coupling varied with cancer progression stage. These findings reveal how bladder cancer cells adapt to mechanical cues through coordinated biomechanical and metabolic responses, underscoring the importance of integrating cellular and matrix mechanics in modeling tumor invasion and developing targeted therapies.

biophysics↗

bioRxiv2025

Offsetting ROS-mediated arrest of endothelial fenestration dynamics permits long-term optical super-resolution- and AFM-imaging

Advances in cell biology demand methods that resolve the structure and dynamics of subcellular organelles in living cells. Live-cell super-resolution fluorescence microscopy meets this need but is constrained by phototoxicity, which disturbs cellular function and biases interpretation. Liver sinusoidal endothelial cells (LSECs), with their physiologically critical and highly dynamic fenestrations, represent a particularly challenging model system. We show that photoactivation-generated reactive oxygen species (ROS) are the primary cause of fenestration arrest during fluorescence imaging. Using three-dimensional structured illumination microscopy (3D SR-SIM), we systematically evaluated fluorophores and ROS scavengers to optimize imaging conditions. A combination of BioTracker staining and CO2-independent medium supplemented with N-acetylcysteine (NAC) preserved fenestration dynamics without altering fenestration number or size. Complementary atomic force microscopy (AFM) confirmed ROS-dependent impairment of fenestration dynamics and revealed nanomechanical changes upon illumination. These findings establish the mechanism underlying imaging-induced artefacts in LSECs and provide a broadly applicable strategy to extend live-cell super-resolution microscopy.

cell biology↗

bioRxiv2025

Indenting multi-cellular spheroids with various cantilever tip geometry

Spheroids are of great interest in the study of cancer as they can partially mimic the tumour microenvironment, thus allowing to investigate several aspects of cell - microenvironment interactions in healthy and diseased conditions, including those pertaining to mechanobiology. Atomic Force Microscopy (AFM) is a versatile tool for studying biological samples and their mechanobiological properties. In AFM, the tip shape and dimensions determine the contact geometry between the tip and the sample and the length scales at which the mechanical properties are probed. Given the complex multiscale structure of spheroids, the choice of tip geometry and size would allow, in principle, to dissect the mechanical response of the overall system into the contributions of the constituents, from the single cell level to the cellular aggregate. In this work, we studied the mechanical properties of spheroids derived from four cell lines (A549, NHLF, HT-29, CCD-18Co). Our studies revealed that using different contact geometries in the fitting procedure results in significantly different Youngs modulus values, highlighting the multiscale response of these complex cellular systems and the importance of a precise experiment design and choice of the AFM probe for the nano-mechanical measurements. We observed that the location of F-actin filaments is correlated to the rigidity of the spheroids.

biophysics↗

bioRxiv2024

Tubulin-Targeted Therapy in Melanoma Increases Cell Invasive Potential by Activation of Actomyosin Cytoskeleton - an in vitro study

One of the most dangerous aspects of cancers is their ability to metastasize, which is the leading cause of death. Hence, it holds significance to develop therapies targeting the eradication of cancer cells, in parallel, inhibiting metastases in cells surviving the applied therapy. Here, we focused on two melanoma cell lines - WM35 and WM266-4 - representing the less and more invasive melanomas. We investigated mechanisms of cellular processes regulating the activation of actomyosin being the effect of colchicine treatment. Additionally, we investigated biophysical aspects of supplement therapy using Rho-associated protein kinase (ROCK) inhibitor (Y-27632), and myosin II inhibitor ((-)-blebbistatin), focusing on the microtubules and actin filaments. We analyzed their effect on the proliferation, migration, and invasiveness of melanoma cells, supported by studies on cytoskeletal architecture using confocal fluorescence microscopy and nanomechanics using atomic force microscopy and novel method based on constriction channels. Our results showed that colchicine inhibits the migration of most melanoma cells, while for a small cell population, it paradoxically increases their migration and invasiveness. These changes are also accompanied by the formation of stress fibers compensating for the loss of microtubules. Simultaneous administration of selected agents led to the inhibition of this compensatory effect. Collectively, our results highlighted that colchicine led to actomyosin activation and increased cancer cell invasiveness. We emphasized a cellular pathway of Rho-ROCK-dependent actomyosin contraction to be responsible for the increased invasive potential of melanoma cells in tubulin-targeted therapy. HighlightsO_LIColchicine strongly increased (trans)migration in the subpopulation of melanoma cells. C_LIO_LIColchicine disrupted tubulin but compensated for the activation of the actomyosin network. C_LIO_LIRho-ROCK-targeted or myosin-targeted therapies vanished the side-effect of colchicine. C_LIO_LIIncreased actin polymerisation correlated with the transit time in the constriction channel. C_LIO_LIElastic modulus variations reflected observed alterations in the actomyosin network. C_LI

biophysics↗

bioRxiv2024

Migration, proliferation, and elasticity of bladder cancer cells on lectin-coated surfaces

The alterations in migration, proliferation, and mechanics of cells observed during cancer progression can potentially be linked to enhanced tumor invasiveness. These properties are frequently attributed to the ability to form distant metastasis; however, the direct mutual connection between these properties is not always proven. Here, we studied the migratory, proliferative, and mechanical phenotype of three bladder cancer cells originating from various stages of cancer progression, i.e., non-malignant cell cancer of ureter (HCV29 cells), bladder carcinoma (HT1376 cells) and transitional bladder carcinoma (T24 cells). The results were linked with the organization of actin filaments because of their major role in cell migration. The results classified cells into non-malignant, non-invasive, and invasive, revealing the significant impact of actin filaments in bladder cancer invasion. Based on the reported changes in cancer cell glycosylation, the potential applicability of the observed cancer-related changes to identify invasive cells was demonstrated for the lectin-coated surfaces, which is the potential surface modification for biosensors.

biophysics↗

bioRxiv2023

Changes in Secondary Structure and Properties of Bovine Serum Albumin as a Result of Interactions with Gold Surface

Proteins can alter their shape when interacting with a surface. This study explores how bovine serum albumin (BSA) modifies structurally when it adheres to a gold surface, depending on the protein concentration and pH. We verified that the gold surface induces significant structural modifications to the BSA molecule using circular dichroism, infrared spectroscopy, and atomic force microscopy. Specifically, adsorbed molecules displayed increased levels of disordered structures and {beta}-turns, with fewer -helices than the native structure. MP-SPR spectroscopy demonstrated that the protein molecules preferred a planar orientation during adsorption. Molecular dynamics simulations revealed that the interaction between cysteines exposed to the outside of the molecule and the gold surface was vital, especially at pH = 3.5. The macroscopic properties of the protein film observed by AFM and contact angles confirm the flexible nature of the protein itself. Notably, structural transformation is joined with the degree of hydration of protein layers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/567678v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@525beborg.highwire.dtl.DTLVardef@110d1adorg.highwire.dtl.DTLVardef@135eed9org.highwire.dtl.DTLVardef@1d3ec41_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Multiscale Rheology of Aging Cancer Spheroids

Cancer spheroids offer a valuable experimental model that mimics the complexity and heterogeneity of solid tumors. Characterizing their mechanical response is crucial for understanding tumor development, progression, and drug response. Currently, whole live spheroids are analyzed primarily using image analysis, which is challenging, requires extended incubation times, and has limited imaging depth. Here, we present a new label-free approach for characterizing sub-superficial structures of bladder cancer spheroids and measuring their mechanical response at three distinct stages of cancer progression. We study the microrheological changes induced by aging at the cellular and cluster levels by conducting a multi-physics characterization and modeling approach. We find that spheroids exhibit viscoelastic behavior that can be described by fractional models. We show that spheroids are mechanically heterogeneous, with strong depth and time-dependent variations associated with evolving structural features. Our approach opens new possibilities to study 3D in vitro models, paving the way for the discovery of novel and more precise procedure in cancer diagnosis based on the use of mechanomarkers.

biophysics↗

bioRxiv2023

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 (Youngs 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.

biophysics↗

bioRxiv2022

Nanomechanical and microrheological properties of bladder cancer cells at cellular and spheroid levels

Cancer progression is associated with changes in cell mechanical and rheological properties that could be probed by atomic force microscopy (AFM). In this study, we applied AFM to measure elastic (by compressing the cells) and viscoelastic (by applying shear stress) properties of bladder cancers in relation to their culture morphology (in single cells, cell monolayers, and spheroids). Three different cell lines, HCV29 (non-malignant cell cancer of ureter), HT1376 (grade III bladder carcinoma), T24 (grade IV transitional cell carcinoma), were investigated. Nanoindentation measurements only differentiate between non-malignant and cancer cells, but it is difficult to distinguish between specific bladder cancers. By applying microrheological measurements, we confirm that non-malignant cells are more rigid than cancer cells but more importantly, it was possible to differentiate between two cancerous cell lines, regardless of the culture conditions. As each of them is characterized by a distinct actin filament network inside the cell, we showed that actin filaments are a key element in defining the rheological properties of spheroids originating from cells having thick actin bundles. Our results showed that HCV29 cells are more rigid than the studied cancer cells, indicating that normal cells are resistant to compressive and shear forces. Therefore, we conclude that cell mechanical and rheological properties may serve as a biophysical marker to distinguish normal and cancer cells of different malignancies.

biophysics↗

bioRxiv2022

Changes in nanomechanical properties of single neuroblastoma cells as a model for oxygen and glucose deprivation (OGD)

The biological processes underlying ischemic stroke, although complex, are better known than those related to biomechanical alterations of single cells. Mechanisms of biomechanical changes and their relations to the molecular processes are crucial for understanding the function and dysfunction of the brain. In our study, we applied atomic force microscopy (AFM) to quantify the alterations in biomechanical properties in neuroblastoma SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) and reoxygenation (RO). Obtained results reveal several characteristics. Cell viability remained at the same level, regardless of the OGD and RO conditions, but, in parallel, the metabolic activity of cells decreased with OGD duration. 24h RO did not recover the metabolic activity fully. Cells subjected to OGD appeared softer than control cells. Cell softening was strongly present in cells after 1h of OGD and, with longer OGD duration and in RO conditions, cells recovered their mechanical properties. Changes in the nanomechanical properties of cells were attributed to the remodelling of actin filaments, which was related to cofilin-based regulation and impaired metabolic activity of cells. The presented study shows the importance of nanomechanics in research on ischemic-related pathological processes such as stroke.

biophysics↗

bioRxiv2022

PPMO-mediated exon skipping induces uniform sarcolemmal dystrophin rescue with dose-dependent restoration of circulating microRNA biomarkers and muscle biophysical properties

Duchenne muscular dystrophy (DMD) is a paediatric muscle-wasting disorder caused by genetic loss of the gene encoding the dystrophin protein. Therapies that restore dystrophin expression are presumed to correct the disease, with antisense-mediated exon skipping being the leading approach. In this study, we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. To this end, dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping and dystrophin rescue at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed two weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed. Importantly, dystrophin expression was uniformly distributed at the sarcolemma of corrected myofibers at all doses. The abundance of serum microRNA biomarkers (i.e. miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase were restored towards wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e. Youngs modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression, and that both serum miRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in the context of DMD.

molecular biology↗

bioRxiv2020

Stiffening of prostate cancer cells driven by actin filaments - microtubules crosstalk confers resistance to microtubule-targeting drugs

The crucial role of microtubules in the mitotic-related segregation of chromosomes makes them an excellent target for anticancer microtubule targeting drugs (MTDs) such as vinflunine, colchicine, and docetaxel. MTDs affect mitosis by directly perturbing the structural organization of microtubules. By a direct assessment of the biomechanical properties of prostate cancer cells exposed to different MTDs using atomic force microscopy, we show that cell stiffening is a candidate mechanism through which cancer cells preserve the original phenotype in response to the application of MTDs. While changes in cellular rigidity are typically mainly attributed to remodeling of the actin filaments in the cytoskeleton, here we provide evidence that cell stiffening can be driven by a crosstalk between actin filaments and microtubules in drug-treated cells. Our findings improve the interpretation of biomechanical data obtained for living cells in studies of various physiological and pathological processes.

biophysics↗