Machine Learning Allows for Distinguishing Precancerous and Cancerous Human Epithelial Cervical Cells Using High-Resolution AFM Imaging of Adhesion Maps.

Previously, the analysis of atomic force microscopy (AFM) images allowed us to distinguish normal from cancerous/precancerous human epithelial cervical cells using only the fractal dimension parameter. High-resolution maps of adhesion between the AFM probe and the cell surface were used in that study. However, the separation of cancerous and precancerous cells was rather poor (the area under the curve (AUC) was only 0.79, whereas the accuracy, sensitivity, and specificity were 74%, 58%, and 84%, respectively). At the same time, the separation between premalignant and malignant cells is the most significant from a clinical point of view. Here, we show that the introduction of machine learning methods for the analysis of adhesion maps allows us to distinguish precancerous and cancerous cervical cells with rather good precision (AUC, accuracy, sensitivity, and specificity are 0.93, 83%, 92%, and 78%, respectively). Substantial improvement in sensitivity is significant because of the unmet need in clinical practice to improve the screening of cervical cancer (a relatively low specificity can be compensated by combining this approach with other currently existing screening methods). The random forest decision tree algorithm was utilized in this study. The analysis was carried out using the data of six precancerous primary cell lines and six cancerous primary cell lines, each derived from different humans. The robustness of the classification was verified using K-fold cross-validation (K = 500). The results are statistically significant at p < 0.0001. Statistical significance was determined using the random shuffle method as a control.

Mechanical Way To Study Molecular Structure of Pericellular Layer.

Atomic force microscopy (AFM) has been used to study the mechanical properties of cells, in particular, malignant cells. Softening of various cancer cells compared to their nonmalignant counterparts has been reported for various cell types. However, in most AFM studies, the pericellular layer was ignored. As was shown, it could substantially change the measured cell rigidity and miss important information on the physical properties of the pericellular layer. Here we take into account the pericellular layer by using the brush model to do the AFM indentation study of bladder epithelial bladder nonmalignant (HCV29) and cancerous (TCCSUP) cells. It allows us to measure not only the quasistatic Young's modulus of the cell body but also the physical properties of the pericellular layer (the equilibrium length and grafting density). We found that the inner pericellular brush was longer for cancer cells, but its grafting density was similar to that found for nonmalignant cells. The outer brush was much shorter and less dense for cancer cells. Furthermore, we demonstrate a method to convert the obtained physical properties of the pericellular layer into biochemical language better known to the cell biology community. It is done by using heparinase I and neuraminidase enzymatic treatments that remove specific molecular parts of the pericellular layer. The presented here approach can also be used to decipher the molecular composition of not only pericellular but also other molecular layers.

Identification of Geometrical Features of Cell Surface Responsible for Cancer Aggressiveness: Machine Learning Analysis of Atomic Force Microscopy Images of Human Colorectal Epithelial Cells.

It has been recently demonstrated that atomic force microscopy (AFM) allows for the rather precise identification of malignancy in bladder and cervical cells. Furthermore, an example of human colorectal epithelial cells imaged in AFM Ringing mode has demonstrated the ability to distinguish cells with varying cancer aggressiveness with the help of machine learning (ML). The previously used ML methods analyzed the entire cell image. The problem with such an approach is the lack of information about which features of the cell surface are associated with a high degree of aggressiveness of the cells. Here we suggest a machine-learning approach to overcome this problem. Our approach identifies specific geometrical regions on the cell surface that are critical for classifying cells as highly or lowly aggressive. Such localization gives a path to colocalize the newly identified features with possible clustering of specific molecules identified via standard bio-fluorescence imaging. The biological interpretation of the obtained information is discussed.

Loss of NECTIN1 triggers melanoma dissemination upon local IGF1 depletion.

Cancer genetics has uncovered many tumor-suppressor and oncogenic pathways, but few alterations have revealed mechanisms involved in tumor spreading. Here, we examined the role of the third most significant chromosomal deletion in human melanoma that inactivates the adherens junction gene NECTIN1 in 55% of cases. We found that NECTIN1 loss stimulates melanoma cell migration in vitro and spreading in vivo in both zebrafish and human tumors specifically in response to decreased IGF1 signaling. In human melanoma biopsy specimens, adherens junctions were seen exclusively in areas with low IGF1 levels, but not in NECTIN1-deficient tumors. Our study establishes NECTIN1 as a major determinant of melanoma dissemination and uncovers a genetic control of the response to microenvironmental signals.

Influence of Hydrogen Plasma on the Surface Structure of Beryllium.

This paper presents the research results of hydrogen plasma effect on the surface structure of the TGP-56 beryllium. In the linear simulator, the operating conditions of the first wall of ITER are simulated. Beryllium was irradiated with hydrogen plasma at surface temperatures of ~360 °C, ~800 °C, and ~1200 °C, depending on its location in the ITER chamber; with a different number of pulses with a duration of each pulse of 500 s. Samples of irradiated beryllium were subjected to a set of material studies. Experimental data were obtained on the change in the structure of the surface and edges of the beryllium samples after the plasma effect. It was found that at normal (2 MW/m2) and increased (4.7 MW/m2) heat fluxes on the first wall of the ITER, the edges and beryllium surface have good resistance to erosion. Under critical conditions close to the melting point, beryllium strongly erodes and evaporates. It has been established that this material has a high resource resistance to hydrogen plasma effect in the ITER under operating conditions.

Correlation of cell mechanics with the resistance to malignant transformation in naked mole rat fibroblasts.

Naked mole rats (NMRs) demonstrate exceptional longevity and resistance to cancer. Using a biochemical approach, it was previously shown that the treatment of mouse fibroblast cells with RasV12 oncogene and SV40 Large T antigen (viral oncoprotein) led to malignant transformations of cells. In contrast, NMR fibroblasts were resistant to malignant transformations upon this treatment. Here we demonstrate that atomic force microscopy (AFM) can provide information which is in agreement with the above finding, and further, adds unique information about the physical properties of cells that is impossible to obtain by other existing techniques. AFM indentation data were collected from individual cells and subsequently processed through the brush model to obtain information about the mechanics of the cell body (absolute values of the effective Young's moduli). Furthermore, information about the physical properties of the pericellular layer surrounding the cells was obtained. We found a statistically significant decrease in the rigidity of mouse cells after the treatment, whereas there was no significant change found in the rigidity of NMR cells upon the treatment. We also found that the treatment caused a substantial increase in a long part of the pericellular layer in NMR cells only (the long brush was defined as having a size of >10 microns). The mouse cells and smaller brush did not show statistically significant changes upon treatment. The observed change in cell mechanics is in agreement with the frequently observed decrease in cell rigidity during progression towards cancer. The change in the pericellular layer due to the malignant transformation of fibroblast cells has practically not been studied, though it was shown that the removal of part of the pericellular layer of NMR fibroblasts made the cells susceptible to malignant transformation. Although it is plausible to speculate that the observed increase in the long part of the brush layer of NMR cells might help cells to resist malignant transformations, the significance of the observed change in the pericellular layer is yet to be understood. As of now, we can conclude that changes in cell mechanics might be used as an indication of the resistance of NMR cells to malignant transformations.

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish.

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Ultrabright fluorescent silica nanoparticles for in vivo targeting of xenografted human tumors and cancer cells in zebrafish.

New ultrabright fluorescent silica nanoparticles capable of the fast targeting of epithelial tumors in vivo are presented. The as-synthesized folate-functionalized ultrabright particles of 30-40 nm are 230 times brighter than quantum dots (QD450) and 50% brighter than the polymer dots with similar spectra (excitation 365 nm and emission 486 nm). To decrease non-specific targeting, particles are coated with polyethylene glycol (PEG). We demonstrate the in vivo targeting of xenographic human cervical epithelial tumors (HeLa cells) using zebrafish as a model system. The particles target tumors (and probably even individual HeLa cells) as small as 10-20 microns within 20-30 minutes after blood injection. To demonstrate the advantages of ultrabrightness, we repeated the experiments with similar but 200× less bright particles. Compared to those, ultrabright particles showed ∼3× faster tumor detection and ∼2× higher relative fluorescent contrast of tumors/cancer cells.

Ultrabright fluorescent cellulose acetate nanoparticles for imaging tumors through systemic and topical applications.

Cellulose acetate (CA), viscose, or artificial silk are biocompatible human-benign derivatives of cellulose, one of the most abundant biopolymers on earth. While various optical materials have been developed from CA, optical CA nanomaterials are nonexistent. Here we report on the assembly of a new family of extremely bright fluorescent CA nanoparticles (CA-dots), which are fully suitable for in vivo imaging / targeting applications. CA-dots can encapsulate a variety of molecular fluorophores. Using various commercially available fluorophores, we demonstrate that the fluorescence of CA-dots can be tuned within the entire UV-VIS-NIR spectrum. We also demonstrate excellent specific targeting of tumors in vivo, when injected in blood in zebrafish (xenograft model of human cervical epithelial cancer), and unusually strong ex-vivo topical labeling of colon cancer in mice utilizing CA folate-functionalized nanoparticles.

Data on ultrabright fluorescent cellulose acetate nanoparticles for imaging tumors through systemic and topical applications.

Characterization data of fluorescent nanoparticles made of cellulose acetate (CA-dots) are shown. The data in this article accompanies the research article "Ultrabright fluorescent cellulose acetate nanoparticles for imaging tumors through systemic and topical applications" [1]. The measurements and calculation of brightness of individual CA-dots are presented. The description of conjugation procedure Pluronic F127-Folic Acid copolymer and folic acid is shown. Identification of composition of CA dots using Raman and absorbance spectroscopy is demonstrated. The methods for image analysis of efficiency of CA-dot targeting of epithelial tumors xenografted in zebrafish is presented.

AFM Indentation Analysis of Cells to Study Cell Mechanics and Pericellular Coat.

Atomic force microscopy (AFM) indentation analysis of cells is a unique method of measuring stiffness of the cell body and physical properties of its pericellular coat. These cell parameters correlate with cells of abnormality and diseases. Viable biological cells can be studied with this method directly in a culture dish with no special preparation. Here we describe a step-by-step method to analyze the AFM force-indentation curves to derive cell mechanics (the modulus of elasticity of the cell body) and the parameters of the pericellular coat (density and the thickness of the coat layer). Technical details, potential difficulties, and points of special attention are described.

A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

EXOPLANET MODULATION OF STELLAR CORONAL RADIO EMISSION.

The search for exoplanets in the radio bands has been focused on detecting radio emissions produced by the interaction between magnetized planets and the stellar wind (auroral emission). Here we introduce a new tool, which is part of our MHD stellar corona model, to predict the ambient coronal radio emission and its modulations induced by a close planet. For simplicity, the present work assumes that the exoplanet is stationary in the frame rotating with the stellar rotation. We explore the radio flux modulations using a limited parameter space of idealized cases by changing the magnitude of the planetary field, its polarity, the planetary orbital separation, and the strength of the stellar field. We find that the modulations induced by the planet could be significant and observable in the case of hot Jupiter planets - above 100% modulation with respect to the ambient flux in the 10 - 100 MHz range in some cases, and 2-10% in the frequency bands above 250 MHz for some cases. Thus, our work indicates that radio signature of exoplanets might not be limited to low-frequency radio range. We find that the intensity modulations are sensitive to the planetary magnetic field polarity for short-orbit planets, and to the stellar magnetic field strength for all cases. The new radio tool, when applied to real systems, could provide predictions for the frequency range at which the modulations can be observed by current facilities.

Fractal Analysis of Cancer Cell Surface.

Fractal analysis of the cell surface is a rather sensitive method which has been recently introduced to characterize cell progression toward cancer. The surface of fixed and freeze-dried cells is imaged with atomic force microscopy (AFM) modality in ambient conditions. Here we describe the method to perform the fractal analysis specifically developed for the AFM images. Technical details, potential difficulties, points of special attention are described.

AFM study shows prominent physical changes in elasticity and pericellular layer in human acute leukemic cells due to inadequate cell-cell communication.

Biomechanical properties of single cells in vitro or ex vivo and their pericellular interfaces have recently attracted a lot of attention as a potential biophysical (and possibly prognostic) marker of various diseases and cell abnormalities. At the same time, the influence of the cell environment on the biomechanical properties of cells is not well studied. Here we use atomic force microscopy to demonstrate that cell-cell communication can have a profound effect on both cell elasticity and its pericellular coat. A human pre-B p190BCR/ABL acute lymphoblastic leukemia cell line (ALL3) was used in this study. Assuming that cell-cell communication is inversely proportional to the distance between cells, we study ALL3 cells in vitro growing at different cell densities. ALL3 cells demonstrate a clear density dependent behavior. These cells grow very well if started at a relatively high cell density (HD, >2 × 105 cells ml-1) and are poised to grow at low cell density (LD, <1 × 104 cells ml-1). Here we observe ∼6× increase in the elastic (Young's) modulus of the cell body and ∼3.6× decrease in the pericellular brush length of LD cells compared to HD ALL3 cells. The difference observed in the elastic modulus is much larger than typically reported for pathologically transformed cells. Thus, cell-cell communication must be taken into account when studying biomechanics of cells, in particular, correlating cell phenotype and its biophysical properties.

Biophysical differences between chronic myelogenous leukemic quiescent and proliferating stem/progenitor cells.

The treatment of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder has improved recently, but most patients have not yet been cured. Some patients develop resistance to the available tyrosine kinase treatments. Persistence of residual quiescent CML stem cells (LSCs) that later resume proliferation is another common cause of recurrence or relapse of CML. Eradication of quiescent LSCs is a promising approach to prevent recurrence of CML. Here we report on new biophysical differences between quiescent and proliferating CD34+ LSCs, and speculate how this information could be of use to eradicate quiescent LSCs. Using AFM measurements on cells collected from four untreated CML patients, substantial differences are observed between quiescent and proliferating cells in the elastic modulus, pericellular brush length and its grafting density at the single cell level. The higher pericellular brush densities of quiescent LSCs are common for all samples. The significance of these observations is discussed.

Towards early detection of cervical cancer: Fractal dimension of AFM images of human cervical epithelial cells at different stages of progression to cancer.

We used AFM HarmoniX modality to analyse the surface of individual human cervical epithelial cells at three stages of progression to cancer, normal, immortal (pre-malignant) and carcinoma cells. Primary cells from 6 normal strains, 6 cancer, and 6 immortalized lines (derived by plasmid DNA-HPV-16 transfection of cells from 6 healthy individuals) were tested. This cell model allowed for good control of the cell phenotype down to the single cell level, which is impractical to attain in clinical screening tests (ex-vivo). AFM maps of physical (nonspecific) adhesion are collected on fixed dried cells. We show that a surface parameter called fractal dimension can be used to segregate normal from both immortal pre-malignant and malignant cells with sensitivity and specificity of more than 99%. The reported method of analysis can be directly applied to cells collected in liquid cytology screening tests and identified as abnormal with regular optical methods to increase sensitivity. FROM THE CLINICAL EDITOR: Despite cervical smear screening, sometimes it is very difficult to differentiate cancers cells from pre-malignant cells. By using AFM to analyze the surface properties of human cervical epithelial cells, the authors were able to accurately identify normal from abnormal cells. This method could augment existing protocols to increase diagnostic accuracy.

Recovery of aging-related size increase of skin epithelial cells: in vivo mouse and in vitro human study.

The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment). An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8). A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20-40% for cells of older passage (6-8 passages) whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.

If cell mechanics can be described by elastic modulus: study of different models and probes used in indentation experiments.

Here we investigated the question whether cells, being highly heterogeneous objects, could be described with the elastic modulus (effective Young's modulus) in a self-consistent way. We performed a comparative analysis of the elastic modulus derived from the indentation data obtained with atomic force microscopy (AFM) on human cervical epithelial cells (both normal and cancerous). Both sharp (cone) and dull (2500-nm radius sphere) AFM probes were used. The indentation data were processed through different elastic models. The cell was approximated as a homogeneous elastic medium that had either 1), smooth hemispherical boundary (Hertz/Sneddon models) or 2), the boundary covered with a layer of glycocalyx and membrane protrusions ("brush" models). Consistency of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrated that only one model showed consistency in treating cells as a homogeneous elastic medium, namely, the brush model, when processing the indentation data collected with the dull AFM probe. The elastic modulus demonstrated strong depth dependence in all models: Hertz/Sneddon models (no brush taken into account), and when the brush model was applied to the data collected with sharp conical probes. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus, used in a self-consistent way, when using the brush model to analyze data collected with a dull AFM probe. The nature of these results is discussed.