rus
Issue #5/2020
I.V.Yaminskiy, А.I.Аkhmetova
Probe microscopy in the study of changes in growth, mobility, metabolism and secretion of cancer cells
Probe microscopy in the study of changes in growth, mobility, metabolism and secretion of cancer cells
DOI: 10.22184/1993-8578.2020.13.5.298.302
Modern methods of scanning probe microscopy make it possible to obtain a detailed pattern of the vital cells topology including cancer cells with a nanoscale spatial resolution during their growth. The development of high-speed atomic force microscopy enabled to produce images of cells with millisecond spatial resolution. Besides, it is possible to study a rough surface of vital cells by changing the ion current flow without force action on a cell using scanning capillary microscopy (ion-conducting microscopy). The use of scanning capillary microscopy in the study of cancer cells opens up new opportunities for screening of drugs in order to obtain new data on the influence of changes in external conditions on the kinetics of tumor growth and the new data on the vital activity of cells.
Modern methods of scanning probe microscopy make it possible to obtain a detailed pattern of the vital cells topology including cancer cells with a nanoscale spatial resolution during their growth. The development of high-speed atomic force microscopy enabled to produce images of cells with millisecond spatial resolution. Besides, it is possible to study a rough surface of vital cells by changing the ion current flow without force action on a cell using scanning capillary microscopy (ion-conducting microscopy). The use of scanning capillary microscopy in the study of cancer cells opens up new opportunities for screening of drugs in order to obtain new data on the influence of changes in external conditions on the kinetics of tumor growth and the new data on the vital activity of cells.
Теги: cancer cells high-speed atomic force microscopy scanning capillary microscopy (ion-conducting microscopy) screening of drugs высокоскоростная атомно-силовая микроскопия раковые клетки сканирующая капиллярная (ион-проводящая) микроскопия скрининг лекарств
PROBE MICROSCOPY IN THE STUDY OF CHANGES
IN GROWTH, MOBILITY, METABOLISM
AND SECRETION OF CANCER CELLS
I.V.Yaminskiy1, 2, 3, Doctor of Sc. (Physics and Mathematics), Prof. of Lomonosov Moscow State University, Physical and Chemical departments, Director of Advanced Technologies Center, Director of Energy Efficient Technologies, Leading Sci. of INEOS RAS, А.I.Аkhmetova1, 2, 3, Engineer of A.N. Belozersky Institute of Physico-Chemical Biology, Leading Specialist of Advanced Technologies Center and of Energy Efficient Technologies
DOI: 10.22184/1993-8578.2020.13.5.298.302
Получено: 21.08.2020 г.
Modern methods of scanning probe microscopy make it possible to obtain a detailed pattern of the vital cells topology including cancer cells with a nanoscale spatial resolution during their growth. The development of high-speed atomic force microscopy enabled to produce images of cells with millisecond spatial resolution. Besides, it is possible to study a rough surface of vital cells by changing the ion current flow without force action on a cell using scanning capillary microscopy (ion-conducting microscopy). The use of scanning capillary microscopy in the study of cancer cells opens up new opportunities for drugs screening in order to obtain new data on the influence of external conditions changes on the kinetics of tumor growth and the new data on the vital activity of cells.
Nowadays there exist a large number of problems in the field of anti-cancer medicine. In particular, the most important, but unsolved tasks are the following:
The emerging data indicate that many types of cancer cells have an elevated level of reactive oxygen species (ROS) compared to normal cells [1, 2]. Hydrogen peroxide (H2O2) is one of the most important ROS that is associated with various pathological and physiological processes. Besides, overstock amount of H2O2 can damage proteins, lipids and DNA in cells that causes various diseases such as cancer, Alzheimer’s, heart attacks and Parkinson’s disease [3, 4].
Moreover, cancer cells generate an excessive amount of H2O2 comparing to normal cells, so H2O2 can be used as a biomarker to estimate the ability of oxidative stress in various cells and to detect cancer cells [5]. Hence, an efficient and accurate detection of H2O2 is essential for monitoring its concentration in vital cells and understanding the corresponding biological processes.
Peculiarity of capillary microscopy consists in the contactless scanning method used to obtain the cell surface topography thereby practically eliminating influence of the measuring method on the experimental results. In contrast to the atomic force microscopy, the capillary microscopy uses a capillary filled with an electrolyte. The first Ag/AgCl electrode is inserted into it and the second one is placed in a buffer with cell samples. With the help of precision electronics, the capillary moves over the cell surface; the change in the ion current passing through the capillary tip makes it possible to control the position of the capillary over the sample. This eliminates the possibility of touching the cell with the capillary, because when the ion current is minimal, the capillary is located as close as possible to the cell. Thanks to this technique, it is possible to scan living objects in their natural environment and obtain images without fear of harming the sample, especially when it comes to soft samples such as living cells.
In English literature, this method is called scanning ion-conductance microscopy (SICM). When using two-channel capillaries, not only topography data can be obtained, but also electrochemical analysis can be performed. The second channel can be used as a sensor or as a delivery vehicle. In this case we are dealing with scanning electrochemical microscopy (SECM). One channel is filled with electrolyte and delivered molecules; through this channel, the capillary is also positioned over the sample. The second channel is a carbon electrode that measures the local concentration and flow of molecules supplied through the channel [6]. Local delivery is carried out by controlling the applied voltage, which allows increasing or decreasing the number of molecules supplied through the capillary. The work [7] presents a technique for the manufacture of carbon nanoelectrodes for intracellular electrochemical studies, in particular, the modification of the nanoelectrode with platinum made it possible to register the concentration of reactive oxygen species near and inside hippocampal cells. Changes in reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels are indicators of cancer cells in the population.
Obviously, this technique holds great promise for drug delivery and screening. There is the use of carbon capillaries, where the current arising during the electrochemical oxidation / reduction of redox molecules on the carbon surface reacts to the movement of particles [8].
Ozel and colleagues [9] used capillaries as glucose sensors by covalent immobilization of glucose oxidase (GOx) at the capillary tip. Interaction of glucose with GOx leads to catalytic oxidation of β-D-glucose to D-gluconic acid, which can be measured as a change in impedance due to a drop in the pH of the medium at the capillary tip. A glucose sensor quantified intracellular glucose levels in human fibroblasts as well as in metastatic breast cancer lines MDA-MB-231 and MCF7.
Cancer cells were found to show reproducible and accurate increases in glucose levels compared to non-cancerous cells. With the aid of capillary microscopy, a pH sensor was developed to study the oxidation of the extracellular space by normal and cancer cells [10].
Capillary microscopy can be used as a platform for cancer research and clinical screening, to help explain the role of heterogeneity in primary tumor tissues, and to systematically identify critical parameters in disease progression and potential metastatic conditions [11, 12].
Capillary microscopy can be used to measure surface charge density and electrochemical activity, as well as to deliver substances. The AC phase is sensitive to surface charge, so the phase shift can provide useful information for visualizing charge at the same time as topography. In vitro visualization of the cancer cell morphology during their growth, motility, metabolism and secretion provides direct information on the development of cancer. Force mapping of the surface using mechanical action from the probe of an atomic force microscope or induced mechanical pressure on the cell created by the fluid flow through the capillary of the ion-conducting microscope provides detailed information on the change in the local mechanical properties of cells under the influence of various external factors, including chemical and biomedical drugs.
The successes of capillary microscopy in the study of living cells and their metabolism confirm the need to continue research on the effect of drugs on single cancer cells in order to develop the most promising treatment therapy. A new-to-date and highly informative method for studying cancer cells appears thanks to three-dimensional in vitro visualization of the morphology of cancer cells with nanometer precision and simultaneous measurement of local properties – rigidity, adhesion ability, metabolic activity and others. ■
This work was supported by the Russian Science Foundation, project No. 20-12-00389, and the Russian Foundation for Basic Research, project No. 20-32-90036.
IN GROWTH, MOBILITY, METABOLISM
AND SECRETION OF CANCER CELLS
I.V.Yaminskiy1, 2, 3, Doctor of Sc. (Physics and Mathematics), Prof. of Lomonosov Moscow State University, Physical and Chemical departments, Director of Advanced Technologies Center, Director of Energy Efficient Technologies, Leading Sci. of INEOS RAS, А.I.Аkhmetova1, 2, 3, Engineer of A.N. Belozersky Institute of Physico-Chemical Biology, Leading Specialist of Advanced Technologies Center and of Energy Efficient Technologies
DOI: 10.22184/1993-8578.2020.13.5.298.302
Получено: 21.08.2020 г.
Modern methods of scanning probe microscopy make it possible to obtain a detailed pattern of the vital cells topology including cancer cells with a nanoscale spatial resolution during their growth. The development of high-speed atomic force microscopy enabled to produce images of cells with millisecond spatial resolution. Besides, it is possible to study a rough surface of vital cells by changing the ion current flow without force action on a cell using scanning capillary microscopy (ion-conducting microscopy). The use of scanning capillary microscopy in the study of cancer cells opens up new opportunities for drugs screening in order to obtain new data on the influence of external conditions changes on the kinetics of tumor growth and the new data on the vital activity of cells.
Nowadays there exist a large number of problems in the field of anti-cancer medicine. In particular, the most important, but unsolved tasks are the following:
- detection of tumor markers in cells or in fluid, such as microRNA in serum;
- identification of the phenotypes of cancer cells in response to microenvironmental stimuli in vitro, such as cell growth, cell mobility, cell metabolism, cell secretion, etc.;
- determination of the cellular response to small molecule drugs that have recently been developed in accordance with pathological mechanisms;
- studying the mechanism of cancer development, such as drug resistance in cancer treatment.
The emerging data indicate that many types of cancer cells have an elevated level of reactive oxygen species (ROS) compared to normal cells [1, 2]. Hydrogen peroxide (H2O2) is one of the most important ROS that is associated with various pathological and physiological processes. Besides, overstock amount of H2O2 can damage proteins, lipids and DNA in cells that causes various diseases such as cancer, Alzheimer’s, heart attacks and Parkinson’s disease [3, 4].
Moreover, cancer cells generate an excessive amount of H2O2 comparing to normal cells, so H2O2 can be used as a biomarker to estimate the ability of oxidative stress in various cells and to detect cancer cells [5]. Hence, an efficient and accurate detection of H2O2 is essential for monitoring its concentration in vital cells and understanding the corresponding biological processes.
Peculiarity of capillary microscopy consists in the contactless scanning method used to obtain the cell surface topography thereby practically eliminating influence of the measuring method on the experimental results. In contrast to the atomic force microscopy, the capillary microscopy uses a capillary filled with an electrolyte. The first Ag/AgCl electrode is inserted into it and the second one is placed in a buffer with cell samples. With the help of precision electronics, the capillary moves over the cell surface; the change in the ion current passing through the capillary tip makes it possible to control the position of the capillary over the sample. This eliminates the possibility of touching the cell with the capillary, because when the ion current is minimal, the capillary is located as close as possible to the cell. Thanks to this technique, it is possible to scan living objects in their natural environment and obtain images without fear of harming the sample, especially when it comes to soft samples such as living cells.
In English literature, this method is called scanning ion-conductance microscopy (SICM). When using two-channel capillaries, not only topography data can be obtained, but also electrochemical analysis can be performed. The second channel can be used as a sensor or as a delivery vehicle. In this case we are dealing with scanning electrochemical microscopy (SECM). One channel is filled with electrolyte and delivered molecules; through this channel, the capillary is also positioned over the sample. The second channel is a carbon electrode that measures the local concentration and flow of molecules supplied through the channel [6]. Local delivery is carried out by controlling the applied voltage, which allows increasing or decreasing the number of molecules supplied through the capillary. The work [7] presents a technique for the manufacture of carbon nanoelectrodes for intracellular electrochemical studies, in particular, the modification of the nanoelectrode with platinum made it possible to register the concentration of reactive oxygen species near and inside hippocampal cells. Changes in reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels are indicators of cancer cells in the population.
Obviously, this technique holds great promise for drug delivery and screening. There is the use of carbon capillaries, where the current arising during the electrochemical oxidation / reduction of redox molecules on the carbon surface reacts to the movement of particles [8].
Ozel and colleagues [9] used capillaries as glucose sensors by covalent immobilization of glucose oxidase (GOx) at the capillary tip. Interaction of glucose with GOx leads to catalytic oxidation of β-D-glucose to D-gluconic acid, which can be measured as a change in impedance due to a drop in the pH of the medium at the capillary tip. A glucose sensor quantified intracellular glucose levels in human fibroblasts as well as in metastatic breast cancer lines MDA-MB-231 and MCF7.
Cancer cells were found to show reproducible and accurate increases in glucose levels compared to non-cancerous cells. With the aid of capillary microscopy, a pH sensor was developed to study the oxidation of the extracellular space by normal and cancer cells [10].
Capillary microscopy can be used as a platform for cancer research and clinical screening, to help explain the role of heterogeneity in primary tumor tissues, and to systematically identify critical parameters in disease progression and potential metastatic conditions [11, 12].
Capillary microscopy can be used to measure surface charge density and electrochemical activity, as well as to deliver substances. The AC phase is sensitive to surface charge, so the phase shift can provide useful information for visualizing charge at the same time as topography. In vitro visualization of the cancer cell morphology during their growth, motility, metabolism and secretion provides direct information on the development of cancer. Force mapping of the surface using mechanical action from the probe of an atomic force microscope or induced mechanical pressure on the cell created by the fluid flow through the capillary of the ion-conducting microscope provides detailed information on the change in the local mechanical properties of cells under the influence of various external factors, including chemical and biomedical drugs.
The successes of capillary microscopy in the study of living cells and their metabolism confirm the need to continue research on the effect of drugs on single cancer cells in order to develop the most promising treatment therapy. A new-to-date and highly informative method for studying cancer cells appears thanks to three-dimensional in vitro visualization of the morphology of cancer cells with nanometer precision and simultaneous measurement of local properties – rigidity, adhesion ability, metabolic activity and others. ■
This work was supported by the Russian Science Foundation, project No. 20-12-00389, and the Russian Foundation for Basic Research, project No. 20-32-90036.
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