Epigallocatechin-gallate tailors the cell adhesivity of fibronectin coatings in oxidation and concentration-dependent manner

Fibronectin is an extracellular matrix component that plays a significant role in many physiological processes, such as cell adhesion, growth, differentiation, and migration. In this study, we revealed the interaction between this important protein and the widely studied natural active substance green tea polyphenol epigallocatechin-gallate (EGCG) and its oxidized form. Furthermore, we investigated the kinetics of cancer cell adhesion on the polyphenol-treated fibronectin coatings. We applied a high- throughput, label-free optical biosensor capable of monitoring the above processes in real time with an excellent signal-to-noise ratio. Our results show that EGCG and its oxidized form bind to fibronectin in a concentration-dependent manner and can form multilayers as well. Furthermore, both polyphenol forms inhibited cellular adhesion, however, the effect was more pronounced in the case of the oxidized form. The results were compared to the measurements performed on bare biosensor surfaces without fibronectin, and the roles of oxidation were investigated. It is suggested that the polyphenols can interact and block important cell adhesion protein motifs and affect the rigidity of the layers as well. Moreover, a novel molecular scale active mechanism involving the disulfide bridges of fibronectin was proposed to explain the recorded kinetic signals and highlight that these proteins can be active participants in the molecular scale transformations affecting adhesion

Population distributions of single-cell adhesion parameters during the cell cycle from high-throughput robotic fluidic force microscopy

Single-cell adhesion plays an essential role in biological and biomedical sciences, but its precise measurement for a large number of cells is still a challenging task. At present, typical force measuring techniques usually offer low throughput, a few cells per day, and therefore are unable to uncover phenomena emerging at the population level. In this work, robotic fluidic force microscopy (FluidFM) was utilized to measure the adhesion parameters of cells in a high-throughput manner to study their population distributions in-depth. The investigated cell type was the genetically engineered HeLa Fucci construct with cell cycle-dependent expression of fluorescent proteins. This feature, combined with the high-throughput measurement made it possible for the first time to characterize the single-cell adhesion distributions at various stages of the cell cycle. It was found that parameters such as single-cell adhesion force and energy follow a lognormal population distribution. Therefore, conclusions based on adhesion data of a low number of cells or treating the population as normally distributed can be misleading. Moreover, we found that the cell area was significantly the smallest, and the area normalized maximal adhesion force was significantly the largest for the colorless cells (the mitotic (M) and early G1 phases). Notably, the parameter characterizing the elongation of the cells until the maximum level of force between the cell and its substratum was also dependent on the cell cycle, which quantity was the smallest for the colorless cells. A novel parameter, named the spring coefficient of the cell, was introduced as the fraction of maximal adhesion force and maximal cell elongation during the mechanical detachment, which was found to be significantly the largest for the colorless cells. Cells in the M phase adhere in atypical way, with so-called reticular adhesions, which are different from canonical focal adhesions. We first revealed that reticular adhesion can exert a higher force per unit area than canonical focal adhesions, and cells in this phase are significantly stiffer. The possible biological consequences of these findings were also discussed, together with the practical relevance of the observed population-level adhesion phenomena

Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data

The glycocalyx is thought to perform a potent, but not yet defined function in cellular adhesion and signaling. Since 95% of cancer cells have altered glycocalyx structure, this role can be especially important in cancer development and metastasis. The glycocalyx layer of cancer cells directly influences cancer progression, involving the complicated kinetic process of cellular adhesion at various levels. In the present work, we investigated the effect of enzymatic digestion of specific glycocalyx components on cancer cell adhesion to RGD (arginine–glycine–aspartic acid) peptide motif displaying surfaces. High resolution kinetic data of cell adhesion was recorded by the surface sensitive label-free resonant waveguide grating (RWG) biosensor, supported by fluorescent staining of the cells and cell surface charge measurements. We found that intense removal of chondroitin sulfate (CS) and dermatan sulfate chains by chondroitinase ABC reduced the speed and decreased the strength of adhesion of HeLa cells. In contrast, mild digestion of glycocalyx resulted in faster and stronger adhesion. Control experiments on a healthy and another cancer cell line were also conducted, and the discrepancies were analysed. We developed a biophysical model which was fitted to the kinetic data of HeLa cells. Our analysis suggests that the rate of integrin receptor transport to the adhesion zone and integrin-RGD binding is strongly influenced by the presence of glycocalyx components, but the integrin-RGD dissociation is not. Moreover, based on the kinetic data we calculated the dependence of the dissociation constant of integrin-RGD binding on the enzyme concentration. We also determined the dissociation constant using a 2D receptor binding model based on saturation level static data recorded at surfaces with tuned RGD densities. We analyzed the discrepancies of the kinetic and static dissociation constants, further illuminating the role of cancer cell glycocalyx during the adhesion process. Altogether, our experimental results and modelling demonstrated that the chondroitin sulfate and dermatan sulfate chains of glycocalyx have an important regulatory function during the cellular adhesion process, mainly controlling the kinetics of integrin transport and integrin assembly into mature adhesion sites. Our results potentially open the way for novel type of cancer treatments affecting these regulatory mechanisms of cellular glycocalyx

Single-cell adhesivity distribution of glycocalyx digested cancer cells from high spatial resolution label-free biosensor measurements

The glycocalyx is a cell surface sugar layer of most cell types that greatly influences the interaction of cells with their environment. Its components are glycolipids, glycoproteins, and oligosaccharides. Interestingly, cancer cells have a thicker glycocalyx layer compared to healthy cells, but to date, there has been no con- sensus in the literature on the exact role of cell surface polysaccharides and their derivatives in cellular adhesion and signaling. In our previous work we discovered that specific glycocalyx components of cancer cells regulate the kinetics and strength of adhesion on RGD (arginine-glycine-aspartic acid) peptide- coated surfaces [1]. Depending on the employed enzyme concentration digesting specific components both adhesion strengthening and weakening could be observed by monitoring the averaged behavior of thousands of cells. The enzyme chondroitinase ABC (ChrABC) was used to digest the chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate components in the glycocalyx of cancer cells. In the present work, a high spatial resolution label-free optical biosensor was employed to monitor the adhesivity of cancer cells both at the single-cell and population level. Population-level distributions of single-cell adhesivity were first recorded and analyzed when ChrABC was added to the adhering cells. At relatively low and high ChrABC concentrations subpopulations with remarkably large and weak adhesivity were identified. The changes in the adhesivity distribution due to the enzyme treatment were analyzed and the subpopulations most affected by the enzyme treatment were highlighted. The presented results open up new directions in glycocalyx related cell adhesion research and in the development of more meaningful targeted cancer treatments affecting adhesion

Single-Cell Classification based on Label-Free High-Resolutio Optical Data of Cell Adhesion Kinetics

Selecting and isolating various cell types is a critical procedure in many applications, including immune therapy, regenerative medicine, and cancer research. Usually, these selection processes involve some labeling or another invasive step potentially affecting cellular functionality or damaging the cell. In the current proof of principle study, we first introduce an optical biosensor-based method capable of classification between healthy and numerous cancerous cell types in a label-free setup. We present high classification accuracy based on the monitored single-cell adhesion kinetic signals. We developed a high-throughput data processing pipeline to build a benchmark database of ~ 4500 single-cell adhesion measurements of a normal preosteoblast (MC3T3-E1) and various cancer (HeLa, LCLC-103H, MDA-MB-231, MCF-7) cell types. Several datasets were used with different cell-type selections to test the performance of deep learning-based classification models, reaching above 70–80% depending on the classification task. Beyond testing these models, we aimed to draw interpretable biological insights from their results; thus, we applied a deep neural network visualization method (grad-CAM) to reveal the basis on which these complex models made their decisions. Our proof-of-concept work demonstrated the success of a deep neural network using merely label-free adhesion kinetic data to classify single mammalian cells into different cell types. We propose our method for label-free single-cell profiling and in vitro cancer research involving adhesion. The employed label-free measurement is noninvasive and does not affect cellular functionality. Therefore, it could also be adapted for applications where the selected cells need further processing, such as immune therapy and regenerative medicine

Optical Sensor Reveals the Hidden Influence of Cell Dissociation on Adhesion Measurements

Cell adhesion experiments are important in tissue engineering and for testing new biologically active surfaces, prostheses, and medical devices. Additionally, the initial state of adhesion (referred to as nascent adhesion) plays a key role and is currently being intensively researched. A critical step in handling all adherent cell types is their dissociation from their substrates for further processing. Various cell dissociation methods and reagents are used in most tissue culture laboratories (here, cell dissociation from the culture surface, cell harvesting, and cell detachment are used interchangeably). Typically, the dissociated cells are re-adhered for specific measurements or applications. However, the impact of the choice of dissociation method on cell adhesion in subsequent measurements, especially when comparing the adhesivity of various surfaces, is not well clarified. In this study, we demonstrate that the application of a label-free optical sensor can precisely quantify the effect of cell dissociation methods on cell adhesivity, both at the single-cell and population levels. The optical measurements allow for high-resolution monitoring of cellular adhesion without interfering with the physiological state of the cells. We found that the choice of reagent significantly alters cell adhesion on various surfaces. Our results clearly demonstrate that biological conclusions about cellular adhesion when comparing various surfaces are highly dependent on the employed dissociation method. Neglecting the choice of cellular dissociation can lead to misleading conclusions when evaluating cell adhesion data from various sources and comparing the adhesivity of two different surfaces (i.e., determining which surface is more or less adhesive)

Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data

The glycocalyx is thought to perform a potent, but not yet defined function in cellular adhesion and signaling. Since 95% of cancer cells have altered glycocalyx structure, this role can be especially important in cancer development and metastasis. The glycocalyx layer of cancer cells directly influences cancer progression, involving the complicated kinetic process of cellular adhesion at various levels. In the present work, we investigated the effect of enzymatic digestion of specific glycocalyx components on cancer cell adhesion to RGD (arginine–glycine–aspartic acid) peptide motif displaying surfaces. High resolution kinetic data of cell adhesion was recorded by the surface sensitive label-free resonant waveguide grating (RWG) biosensor, supported by fluorescent staining of the cells and cell surface charge measurements. We found that intense removal of chondroitin sulfate (CS) and dermatan sulfate chains by chondroitinase ABC reduced the speed and decreased the strength of adhesion of HeLa cells. In contrast, mild digestion of glycocalyx resulted in faster and stronger adhesion. Control experiments on a healthy and another cancer cell line were also conducted, and the discrepancies were analysed. We developed a biophysical model which was fitted to the kinetic data of HeLa cells. Our analysis suggests that the rate of integrin receptor transport to the adhesion zone and integrin-RGD binding is strongly influenced by the presence of glycocalyx components, but the integrin-RGD dissociation is not. Moreover, based on the kinetic data we calculated the dependence of the dissociation constant of integrin-RGD binding on the enzyme concentration. We also determined the dissociation constant using a 2D receptor binding model based on saturation level static data recorded at surfaces with tuned RGD densities. We analyzed the discrepancies of the kinetic and static dissociation constants, further illuminating the role of cancer cell glycocalyx during the adhesion process. Altogether, our experimental results and modelling demonstrated that the chondroitin sulfate and dermatan sulfate chains of glycocalyx have an important regulatory function during the cellular adhesion process, mainly controlling the kinetics of integrin transport and integrin assembly into mature adhesion sites. Our results potentially open the way for novel type of cancer treatments affecting these regulatory mechanisms of cellular glycocalyx