Joanna Pabijan

The softening of human bladder cancer cells happens at an early stage of the malignancy process

Summary Various studies have demonstrated that alterations in the deformability of cancerous cells are strongly linked to the actin cytoskeleton. By using atomic force microscopy (AFM), it is possible to determine such changes in a quantitative way in order to distinguish cancerous from non-malignant cells. In the work presented here, the elastic properties of human bladder cells were determined by means of AFM. The measurements show that non-malignant bladder HCV29 cells are stiffer (higher Young’s modulus) than cancerous cells (HTB-9, HT1376, and T24 cell lines). However, independently of the histological grade of the studied bladder cancer cells, all cancerous cells possess a similar level of the deformability of about a few kilopascals, significantly lower than non-malignant cells. This underlines the diagnostic character of stiffness that can be used as a biomarker of bladder cancer. Similar stiffness levels, observed for cancerous cells, cannot be fully explained by the organization of the actin cytoskeleton since it is different in all malignant cells. Our results underline that it is neither the spatial organization of the actin filaments nor the presence of stress fibers, but the overall density and their 3D-organization in a probing volume play the dominant role in controlling the elastic response of the cancerous cell to an external force.

PDMS substrate stiffness affects the morphology and growth profiles of cancerous prostate and melanoma cells.

A deep understanding of the interaction between cancerous cells and surfaces is particularly important for the design of lab-on-chip devices involving the use of polydimethylsiloxane (PDMS). In our studies, the effect of PDMS substrate stiffness on mechanical properties of cancerous cells was investigated in conditions where the PDMS substrate is not covered with any of extracellular matrix proteins. Two human prostate cancer (Du145 and PC-3) and two melanoma (WM115 and WM266-4) cell lines were cultured on two groups of PDMS substrates that were characterized by distinct stiffness, i.e. 0.75 ± 0.06 MPa and 2.92 ± 0.12 MPa. The results showed the strong effect on cellular behavior and morphology. The detailed analysis of chemical and physical properties of substrates revealed that cellular behavior occurs only due to substrate elasticity.

Protocol of single cells preparation for time of flight secondary ion mass spectrometry.

There are several techniques like time of flight secondary ion mass spectrometry (ToF SIMS) that require a special protocol for preparation of biological samples, in particular, those containing single cells due to high vacuum conditions that must be kept during the experiment. Frequently, preparation methodology involves liquid nitrogen freezing what is not always convenient. In our studies, we propose and validate a protocol for preparation of single cells. It consists of four steps: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying under ambient conditions. The protocol was applied to samples with single melanoma cells i.e. WM115 and WM266-4 characterized by similar morphology. The surface and internal structures of cells were monitored using atomic force, scanning electron and fluorescent microscopes, used to follow any potential protocol-induced alterations. To validate the proposed methodology for sample preparation, ToF SIMS experiments were carried out using C60(+) cluster ion beam. The applied principal component analysis (PCA) revealed that chemical changes on cell surface of melanoma cells were large enough to differentiate between primary and secondary tumor sites. Subject category: Mass spectrometry.

Differentiation between Single Bladder Cancer Cells Using Principal Component Analysis of Time-of-Flight Secondary Ion Mass Spectrometry

Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) mass spectra measurements combined with an appropriate sample preparation protocol are the powerful tools to obtain unique information about the chemical composition of biological materials. In our studies, two questions were addressed, i.e., whether it is possible to develop a fixative-based sample preparation protocol and whether it allows one to distinguish between cells originating from various stages of cancer progression. Therefore, four human bladder cancer cell lines (with distinct malignancy degree) have been investigated. A chemical fixation protocol has been used for TOF-SIMS measurements, and mass spectra were obtained using a Bi3(+) primary ion beam. The principal component analysis (PCA) has been applied to analyze the whole range of mass spectra (without preselection of any particular masses) using two approaches of data preprocessing, namely, mean centering and autoscaling. The PC3 versus PC2 plot has showed significant differences between nonmalignant cancer cells and the cancerous ones for both of preprocessing approaches. The analysis of mass spectra of human bladder cells allows one to find a list of mass peaks with intensities significantly larger in cancerous bladder cells compared to nonmalignant cell cancer of the ureter (HCV29 cells). These findings show that TOF-SIMS in combination with PCA can be used to identify reference, human bladder cells from cancerous ones.

Nano-characterization of two closely related melanoma cell lines with different metastatic potential.

Cutaneous malignant melanoma is one of the most lethal types of skin cancer. Its progression passes through several steps, leading to the appearance of a new population of cells with aggressive biological potential. Here, we focused on the nano-characterization of two different melanoma cell lines with similar morphological appearance but different metastatic potential, namely, WM115 from vertical growth phase (VGP) and WM266-4 derived from metastasis to skin. The first cell line represents cells that progressed to the VGP, while the WM266-4 cell line denotes cells from the metastasis to skin. Exploring with a combination of atomic force and fluorescence microscopes, our goal was to identify cell surface characteristics in both cell lines that may determine differences in the cellular nano-mechanical properties. Cell elasticity was found to be affected by the presence of F-actin-based flexible ridges, rich in F-actin co-localized with β1 integrins in the studied cell lines. These results point out how progressive changes in the surface structure of melanoma cells can affect their bionanomechanical properties.

Atomic force microscopy as a tool for assessing the cellular elasticity and adhesiveness to identify cancer cells and tissues

From the first experiments of the atomic force microscopy (AFM) with biological samples, the range of its potential applications grows extensively. One of them is the use of AFM to characterize biophysical fingerprints of cancer progression in search of non-labelled biomarkers of the disease. The technique offers various functionalities, starting from surface imaging to detection of interaction forces, delivering quantitative parameters that can describe changes characteristic for various diseases, including cancer. In this review, the special emphasis was laid on these studies that compare the AFM-derived properties of reference and cancerous cells using all functionalities from cellular deformability measurements to quantification of the interaction forces at the single-molecule and single-cell levels. Despite the large effort and evidence of the microscope applicability to detect pathologically altered cells, there are still practical challenges remained to be solved before AFM can be implemented for routine cancer tracking and diagnosis. To-date, the AFM can be used to achieve a better understanding of cancer-related processes and mechanisms that could be further employed to design high-resolution clinical assays in a quantitative way.

Fibroblasts change spreading capability and mechanical properties in a direct interaction with keratinocytes in conditions mimicking wound healing

Keratinocytes are predominant in the uppermost layer of the skin, while fibroblasts dominate in the dermal layer. These cells interact with each other directly when fibroblasts migrate to a region of the wound where they induce keratinocytes proliferation through double paracrine signalling. Since a response from both keratinocytes and fibroblasts dominates during the inflammatory and proliferative phases, the exact knowledge how these two types of cells interact with each other is crucial for deeper understanding of mechanisms involved in the wound healing process. The aim of this study was to quantify alterations in mechanical properties of cells, i.e. fibroblasts and keratinocytes, in conditions mimicking direct cellular interactions observed in wound healing. Single cell elasticity was measured using atomic force microscope. To verify the influence of keratinocyte neighbors on fibroblasts elasticity (and vice versa), the effect of cellular confluency was studied in parallel. Our results enabled us to distinguish cellular density-related effects from intercellular interactions occurring between fibroblasts and keratinocytes. While the presence of keratinocytes affects fibroblasts spreading capability and mechanical properties, the keratinocytes remain unaffected by the fibroblasts. These results highlight the importance of the cellular deformability in understanding of the role of biomechanics in double paracrine signalling as fibroblast-keratinocyte interaction can change the potential of the wound healing.

Modification of Polymer Substrates with Extreme Ultraviolet - Potential Application in Cancer Cell Identification

Conformal Invariance and Conserved Quantities for Lagrange Equation of Thin Elastic Rod Peng Wanga,∗, Hui-Rong Feng and Zhi-Mei Lou School of Civil Engineering and Architecture, University of Jinan, Jinan, Shangdong, 250022, P.R. China College of Transportation and Civil Engineering, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, P.R. China Department of Physics, Shaoxing University, Shaoxing, Zhejiang, 312000, P.R. China

Data on step-by-step atomic force microscopy monitoring of changes occurring in single melanoma cells undergoing ToF SIMS specialized sample preparation protocol.

Data included in this article are associated with the research article entitled ‘Protocol of single cells preparation for time-of-flight secondary ion mass spectrometry’ (Bobrowska et al., 2016 in press) [1]. This data file contains topography images of single melanoma cells recorded using atomic force microscopy (AFM). Single cells cultured on glass surface were subjected to the proposed sample preparation protocol applied to prepare biological samples for time-of-flight secondary ion mass spectrometry (ToF SIMS) measurements. AFM images were collected step-by-step for the single cell, after each step of the proposed preparation protocol. It consists of four main parts: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying. In total 13 steps are required, starting from imaging of a living cell in a culture medium and ending up at images of a dried cell in the air. The protocol was applied to melanoma cells from two cell lines, namely, WM115 melanoma cells originated from primary melanoma site and WM266-4 ones being the metastasis of WM115 cells to skin.

Comparing surface properties of melanoma cells using time of flight secondary ions mass spectrometry

Various techniques have been already reported to differentiate between normal (non-malignant) and cancerous cells based on their physico-chemical properties. This is relatively simple when studied cancerous cells originate from distant stages of cancer progression. Here, studies on chemical properties of two closely related human melanoma cell lines are presented: WM115 melanoma cells were taken from the vertical growth phase while WM266-4 from the skin metastatic site of the same patient. Their chemical properties were studied by two techniques, namely time-of-flight secondary ion mass spectra (ToF SIMS) and photothermal microspectroscopy (PTMS), used to record mass and photothermal spectra of cells, respectively. In our approach, independently of the spectra type, its full range, i.e. masses and wavenumbers within the range 0-500 kDa and 500-4000 cm-1, underwent a similar methodology for principal component analysis (PCA). PCA outcome shows results groupped depending on the sample type (either WM115 or WM266-4 cells). The results are independent of the method applied to study chemical properties of melanoma cells, indicating that cancer-related changes are large enough to be identified with these techniques and to differentiate between cells originating from vertical growth phase and skin metastatis.