RU2530170C1 - Method of detecting stem cancer cells - Google Patents

Method of detecting stem cancer cells Download PDF

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RU2530170C1
RU2530170C1 RU2013113328/10A RU2013113328A RU2530170C1 RU 2530170 C1 RU2530170 C1 RU 2530170C1 RU 2013113328/10 A RU2013113328/10 A RU 2013113328/10A RU 2013113328 A RU2013113328 A RU 2013113328A RU 2530170 C1 RU2530170 C1 RU 2530170C1
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cells
dna
fragments
double
stranded dna
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RU2013113328A (en
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Михаил Аркадьевич Шурдов
Сергей Станиславович Богачев
Екатерина Анатольевна Алямкина
Евгения Владимировна Долгова
Владимир Алексеевич Рогачев
Валерий Петрович Николин
Нелли Александровна Попова
Елена Рэмовна Черных
Анастасия Сергеевна Проскурина
Александра Михайловна Минкевич
Сергей Васильевич Сидоров
Александр Анатольевич Останин
Сергей Иванович Байбородин
Ярослав Рейнгольдович Ефремов
Олег Михайлович Андрушкевич
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Михаил Аркадьевич Шурдов
Сергей Станиславович Богачев
Нелли Александровна Попова
Елена Рэмовна Черных
Евгения Владимировна Долгова
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Abstract

FIELD: biotechnology.
SUBSTANCE: method of detecting stem cancer cells is provided, based on incubation of cell samples with fluorescence dyes and subsequent identification of cancer cells in the ultraviolet light, where during the incubation of cell samples, which is carried out with the fluorescent dye, incorporated covalently into double-stranded DNA fragments, the internalization of extracellular exogenous fragments of double-stranded DNA into the intracellular space of stem cells included in the composition of the sample cells, is provided by incubation of cell samples in the solution of the preparation of the fragmented double-stranded DNA at a ratio of 0.5-1 mcg DNA per 1000000 cells in suspension or on a section of tissue for 60-120 minutes, and identification of cancer cells as stem is carried out in ultraviolet light with a wavelength corresponding to the absorption maximum of the fluorochrome inserted into the molecules of fragments of the double-stranded DNA, and the double-stranded DNA fragments are used as DNA fragments of Alu human repeat, enzymatically labelled by precursor comprising covalently sewn fluorochrome dye, or labelled by direct chemical introduction of fluorochrome.
EFFECT: due to increasing the efficiency of detecting stem cancer cells in the initiating condition the invention can be used in medicine.
2 cl, 11 dwg

Description

The invention relates to medicine, in particular to oncology and molecular biology, and can be used to detect (detect) a population of cancer cells with initiating properties (stem - tumorigenic cancer cells).

The idea of the existence of a stem (initiating) cancer cell raises the main question related to the possibility of a therapeutic change in the status of a cancer cell or its direct elimination. The first and necessary condition for targeting a stem (initiating) cancer cell is its individualization in the mass of tumor cells. There are several approaches and methods for identifying stem (initiating) cancer cells.

The most common way is the method of limiting dilutions (LDA) [1, 2]. Using numerous dilutions, the cell culture is titrated prior to dilution, when tumor development does not occur during transplant transplantation. Testing a large number of dilutions in numerous repetitions and statistical processing of the results allows you to select a sample containing one cell with the necessary trait. This method is time-consuming, time-consuming and has limited application, since not all tumors can be dissociated to an individual cell.

Also known is a method called the method of forming spheres [3, 4]. The method of forming spheres is a classic way of determining a pluripotent stem cell, which in a semi-fluid medium forms a sphere of descendant cells around itself. To evaluate the tumorigenic (initiating) properties of the obtained spheres, their subsequent transplantation and the cytological and biochemical characteristics of the tumor graft are used. The indicated method is also laborious, time consuming and has limited application, since it also cannot be applied to tumors that cannot be dissociated into an individual cell.

Another method that is widely used in experimental oncological practice is based on the use of biochemical markers of stem (initiating) cancer cells. The most common are CD133 (prominin-1), CD44 (receptor for hyoluronan), CD166, CD34, CD38 (markers of acute myelogenous leukemia), CD138 (marker for terminally differentiated B lymphocytes), CD24 (marker of epithelial cell adhesion factor EpCAM or ESA ), B5 (ABCB5 ATP binding protein) [5]. Unfortunately, to date, no universal tumor markers have been found. This shows a lack of effectiveness in the use of biochemical markers for the identification of initiating (stem) cancer cells.

Another way to identify stem (initiating) cancer cells is the approach implemented using two types of cells, MDA-MB-436 and MCF-7, which allow cancer cells to be sorted by their deformability. It is known that a cancer cell with initiating characteristics is capable of migration and has numerous abnormalities in the actin cytoskeleton formation systems. Deformed cancer cells have an increased metastatic ability and the ability to form mammospheres. Using the method of flow selection for the deformation ability of cancer cells, a cell population was obtained that has the characteristics of a stem (initiating) cancer cell [6]. The indicated method for determining stem (initiating) cancer cells is technically complicated, it is difficult to adapt to cells of other tumors having different physical sizes and ploidy, and has limited application, since it also cannot be applied to tumors that cannot be dissociated into an individual cell.

Close to the proposed one is a method in which to detect stem (initiating) cancer cells, their ability to eject certain lipophilic fluorescent dyes from the cytoplasm, such as Hoechst 33342 or rhodamine-123, is used. This feature allows not only to identify stem cells during flow cytometry, but also to collect them for various studies as a fraction of unstained or slightly stained cells using cell sorting. However, a similar method also has limited application, since such detection loses the possibility of targeted exposure to a stem (initiating) cancer cell, since the method is based on the use, firstly, of single lipophilic molecules and, secondly, associated with their exclusion from the composition cells. Both characteristics suggest that there is no fundamental possibility of using these dyes as couriers that deliver active chemical molecules to the cancer cell that can destroy the stem (initiating) cancer cell.

These characteristics indicate that at the moment there is no universal way to detect the entire population or part of the population of initiating (cancerous) cells or they are laborious, take a lot of time and, therefore, are not applicable in real clinical conditions.

Closest to its essence, the proposed one is a method based on the use of a compound containing at least one directing module, which is capable of interacting with a specific molecular marker of cancer selected from the group consisting of DD3 and telomerase reverse transcriptase (TERT), and at least one detectable component selected from the group consisting of fluorescein or other related derivatives [RU 2450832, A61K 49/14, A61K 49/18, 05.20.2012].

The disadvantage of this method is the relatively narrow scope, since the method does not allow to identify cancer cells with initiating properties (stem - tumorigenic cancer cells).

The aim of the present invention is to expand the scope.

The required technical result is to expand the scope by introducing technical means that allow the detection of stem cancer cells.

The goal is realized, and the desired technical result is achieved by the fact that in a method for detecting stem cancer cells, based on incubation of cell samples with fluorescent lipophilic dyes and subsequent identification of cancer cells in ultraviolet light, during incubation of cell samples, which is carried out with a fluorescent dye covalently incorporated into fragments of double-stranded DNA, internalization of extracellular exogenous fragments of double-stranded DNA into intracellular the stem cells included in the cell samples by incubating cell samples in a solution of a fragmented double-stranded DNA preparation in a ratio of 0.5-1 μg DNA per 1,000,000 cells in suspension or on a tissue section for 60-120 minutes, and the identification of cancer cells as stem cells, they are carried out in ultraviolet light with a wavelength corresponding to the maximum absorption of fluorochrome obtained in molecules of double-stranded DNA fragments (in particular, TAMRA), moreover, as fragments of double-stranded D By using fragments enzymatically labeled precursor containing covalently sewn fluorochrome dye or direct chemical introduction labeled fluorochrome.

In addition, the desired technical result is achieved in that a fluorochrome dye is used as a fluorochrome dye, which can be covalently combined with the precursor of the polymeric form of DNA in places that do not interfere with the process of incorporation of the triphosphate into the nucleotide chain, or introduced chemically directly into the molecule of a double-stranded DNA fragment .

The proposed method is based on the natural ability of stem (initiating) cancer cells to internalize exogenous extracellular double-stranded DNA fragments and to jointly incubate individual tumor cells or a tumor tissue segment or a tumor or ascites cell culture with fluorochrome stained double-stranded DNA fragments.

The proposed method is characterized by the possibility of using double-stranded DNA fragments as a carrier, delivering biologically or chemically active molecular groups covalently attached to these fragments and interacting with the components of the cancer initiating cell, leading to its apoptotic, necrotic or other death.

There is no evidence in the modern literature on the proposed method for detecting, identifying, sorting and destroying or irreversibly reverse transforming a population of cancer cells with initiating properties (stem-tumorigenic cancer cells) in a population of freely existing cancer cells and in tumor cells growing in a mass of tissue.

Therefore, the proposal meets the criteria of novelty and inventive step.

The following are theoretical and experimental data confirming that the invention meets the criterion of practical (industrial) applicability.

The drawing shows:

figure 1 - Cytological analysis of the content of TAMRA labeled DNA Alu repeat human CD34 + bone marrow cells after co-incubation ex vivo. A) Two-dimensional image of CD34 + cells obtained with an Axioskop 2 plus microscope (Zeiss) and Axio VisionLE. 1 - DAPI, chromatin staining, 2 - FITC, specific marker of CD34 + cells, 3 - TAMRA, signal of exogenous double-stranded DNA fragments. Arrows indicate TAMRA signals indicating the presence of exogenous DNA in CD34 + cells. The scale shown is the same for all blocks. C) A 3D image of the nucleus of a CD34 + cell obtained using a LSM 510 META confocal microscope (Zeiss). DAPI (blue color) - color on chromatin, shows the boundaries of the nucleus; TAMRA (red color) - specific color of exogenous DNA fragments. As can be seen from the figure, the TAMRA signal is localized inside the core.

figure 2 - Cytological analysis of the content of TAMRA-labeled DNA in the fraction of bone marrow cells that do not contain CD34 + cells (CD34-bone marrow cells). A) Two-dimensional image of CD34 cells obtained with an Axioskop 2 plus microscope and Axio VisionLE; 1 - TAMRA signal, indicating the presence of exogenous DNA fragments in the bone marrow cells, 2 - DAPI signal, chromatin staining. C) Image of orthogonal projections of the nuclei of bone marrow CD34 cells (X is the region on the right, Y is the region on the top, Z is the central image) obtained with the LSM 510 META confocal microscope (Zeiss). The figure shows that the TAMRA signal, indicating the presence of exogenous Alu repeat DNA, is located inside the nuclear space of the cell.

figure 3 - Analysis of genomic DNA isolated from bone marrow cells after co-incubation with exogenous DNA of plasmid pEGFP-N1. A) Fractionated genomic DNA isolated from mouse bone marrow cells after co-incubation with exogenous DNA of plasmid pEGFP-N1 and centrifugation in a NaCl gradient; 6-10, 23 - numbers of fractions; M is a molecular weight marker of 1 kb. C) Plasmids obtained as a result of transformation of competent cells of the DNA of the 8 fraction isolated from bone marrow cells after co-incubation with the DNA of plasmid pEGFP-N1 and subsequent ligation on itself; plasmids 8lig1 and 8lig7 are smaller in size than the initial form of plasmid pEGFP-N1. C) Analysis of the nucleotide sequence of truncated plasmids 8lig1 and 8lig7. Map of the original plasmid pEGFP-N1, as well as two plasmids isolated from the genomic DNA of bone marrow cells, after co-incubation of the cells with the initial form of the indicated plasmid linearized at the HindIII restriction site.

figure 4 - Cytological analysis of the content of TAMRA-labeled DNA in ascites cells of the Krebs-2 tumor. A) Two-dimensional image of ascites cells obtained with an Axioskop 2 plus microscope and Axio VisionLE; 1 - TAMRA signal, indicating the presence in the nuclear space of ascites cells of exogenous DNA fragments, 2 - control cells. Right blocks - DAPI signal, chromatin staining. C) Image of orthogonal projections of the nuclei of ascites cells (X is the region on the right, Y is the region on the top, Z is the central image) obtained with the LSM 510 META confocal microscope (Zeiss). The figure shows that the TAMRA signal, indicating the presence of exogenous Alu repeat DNA, is located inside the nuclear space of the cell.

figure 5 - Internalization of fragments of exogenous, extracellular double-stranded DNA into Krebs-2 tumor cells growing in the form of ascites. A) Radio autographs demonstrating the internalization of αP 32 PCR of the labeled GFP gene in Krebs-2 ascites cells. Two left blocks are molecular weight markers: gGFP - PCR-labeled GFP gene (1) and HindIII phage λ DNA hydrolyzate (2). (3) - (5): the left blocks are the electrophoregram, the right blocks are the radio-autographs of the same blocks after drying and exposure on the phosphoimager. For electrophoresis, DNA was isolated from ascites cells taken from the ascites fluid of mice 18 hours after treatment with cytostatic cyclophosphamide (3), isolated from ascites cells taken from the ascites fluid of mice without treatment with the cytostatic cyclophosphamide (4) and isolated from bone marrow cells of mice without treatment with cytostatic cyclophosphamide (5). Cells were incubated for different times (indicated at the top of the blocks) with αP 32 PCR labeled GFP gene. Everywhere you can see the obvious presence of the labeled DNA fragment in its original form. The signal strength in cancer cells is much more intense than in bone marrow cells. B) Blots demonstrating the internalization of HindIII linearized plasmid pGFP into ascites cells of the Krebs-2 tumor. 1) molecular weight marker HindIII phage λ DNA hydrolyzate. (2) - (4): the left blocks are the electrophoregram, the right blocks are the autobiography of the blots after hybridization of the fractionated DNA of ascites cells (2, 3) and bone marrow cells (4) with αP 32 HindIII-labeled end of the DNA of the pGFP plasmid after its restriction by specified restrictase. For electrophoresis, we used DNA isolated from ascites cells obtained from mouse ascites fluid 18 hours after injection of cyclophosphamide cytostatic (2), isolated from ascites cells obtained from mouse ascites fluid without cytostatic treatment (3) and isolated from mouse bone marrow cells ( four). C) Charts showing the amount (%) of exogenous double-stranded DNA delivered to the internal compartments of ascites and bone marrow cells based on the total number of cells in the sample.

6 - A) Rafts showing the dependence of the number of cells internalizing a labeled double-stranded DNA probe on the amount of extracellular DNA present in the culture medium. C) A graph reflecting this relationship. With a logarithmic increase in the amount of DNA, a linear increase in% of cells with TAMRA content occurs.

Fig.7 - A) the Cell cycle of the cells of the Krebs-2 acyte. C) Rafts showing the presence in ascitic cells of two populations of cells containing a TAMRA signal (fragments of double-stranded DNA) and bearing the CD34 + marker, characteristic of mouse hematopoietic stem cells. C) Cytological analysis demonstrating the joint localization of the CD34 + signal and the TAMRA signal.

Fig. 8 shows the results of assessing the probability of the existence of a period of time in the cell cycle during which ascites cells acquire, retain and lose the property of internalization of fragments of extracellular exogenous dsDNA. It has been repeatedly shown that during the first hour from 1 to 2% of ascites cells become TAMRA positive. If the internalization of DNA fragments is associated with a temporary, transitional state of the cells, then during incubation with a labeled DNA probe for several hours, one could observe a linear (or close to that) increase in the number of cells internalizing the TAMRA DNA probe, since through the “temporary gate” hourly in the development of the cell cycle, the number of cells equal to the number receiving the TAMRA marker in the first hour would pass. From the results of the analysis it follows that with prolonged incubation (1-4 hours) of ascites cells with a DNA probe, the number of TAMRA positive cells increases by approximately 10% -30%. Such an increase is more likely associated with the completion of marker internalization in all competent cells than with the passage of ascites cells through a “competent” period of time in the cell cycle and their acquisition of the ability to internalize. A) Flow cytometry results of TAMRA-treated Krebs-2 ascites cells. The rafts show the dynamics of changes in the population of TAMRA positive cells during 1-4 hours of incubation. B) A graphical representation of the results of flow cytometry of Krebs-2 ascites cells treated for 1-4 hours with a TAMRA-labeled dsDNA fragment.

figure 9 - The initiating ability of two populations of ascitic tumor cells Krebs-2 to initiate the growth of a new tumor. A) Rots characterizing the distribution of two cell populations and the percentage of cells after sorting containing a TAMRA signal (fragments of double-stranded DNA) and not containing a TAMRA signal. C) The time of appearance of transplants in mice after transplantation of two different populations of cells containing the TAMRA signal (fragments of double-stranded DNA) and not containing the TAMRA signal.

figure 10 - The initiating ability of CD34 + populations of ascitic tumor cells Krebs-2 to initiate the growth of a new tumor. A) Rots characterizing the number of CD34 positive cells in a population of ascites cells of the Krebs-2 tumor. C) The time of the appearance of grafts in mice after inoculation of a sorted CB34 + cell population compared with inoculation of the total ascites cell population.

11 - Internalization of TAMRA-labeled DNA probe into cells of the solid form of the Krebs-2 tumor. The figure shows a series of sections of a tumor capsule containing an island of cells internalizing a TAMRA DNA probe. The arrows indicate the accumulation of cancer cells internalizing TAMRA DNA. The scale is indicated in bars. To the left of the panels are fluorochromes DAPI, TAMRA, DAPI + TAMRA.

The relevance of this invention is confirmed by a generalized, most complete and at the same time concise review, characterizing the general provisions of the concept of cancer stem cells, describing methods for identifying and characterizing cancer stem cells from various tumors and, finally, determining the significance of cancer stem cells in biomedical research [7]. The main points of the review can be summarized in the following theses.

Currently, there are two models that characterize (describe) a tumor as a hierarchical system of organization of a population of non-transformed cells. The stochastic model assumes that all tumor cells have the same potential in inducing the development of a new tumor [8-11]. The second model assumes a hierarchical organization of the tumor, where stem cancer cells are located at the top of the hierarchical ladder [12, 13].

The assumption of the existence of a stem or initiating cancer cell was first put forward in the work of Dyke and colleagues [12, 13], where it was shown that only a small number of CD34-CD38 cells isolated from the blood of patients with leukemia have leno potential. The remaining cells were not able to induce the development of cancer. In the future, this assumption was confirmed in studies conducted on tumors of various etiologies [14-26].

The main properties characteristic of stem cancer cells are self-maintenance in the series of an unlimited number of divisions, the ability to produce a committed daughter cell with high proliferative potential, but not capable of initiating a new tumor, resistance to multiple transplantation while maintaining the histological characteristics of the tumor.

The proposed method for the detection, identification, sorting and destruction or irreversible reverse transformation of a population of cancer cells with initiating properties (stem-tumorigenic cancer cells) in a population of freely existing cancer cells and in tumor cells growing in a mass of tissue is implemented as follows.

Based on the discovered property of stem (initiating) cancer cells to internalize exogenous extracellular fragments of double-stranded DNA, a joint incubation of individual tumor cells, or a segment of tumor tissue, or a culture of tumor or ascites cells with fluorochrome-labeled dye (e.g., TAMRA) fragments of double-stranded DNA is performed. Stem-initiating cancer cells internalize fragments of double-stranded DNA containing a dye covalently linked to them. Cells internalizing the labeled DNA substrate can be identified, isolated as a population of cells or as an individual cell by any known method, where fluorochrome is used as a selection marker (cytological manipulations, flow cytometry, cell sorting). Stem (initiating) cells can be detected in tissue after incubation of a tumor segment with a DNA probe, a standard procedure for obtaining frozen tissue sections 1-10 cells thick and analysis using a confocal or fluorescence microscope. For the purpose of influencing an initiating (stem) cancer cell, fragments of double-stranded DNA are covalently attached to an active chemical group or molecule that is in an inactive state as a result of blocking of its active centers with blocking reagents. After delivery to the cell space, the blocking groups dissociate either under the influence of the metabolic processes of the cancer cell, or as a result of additional processing. Also, as a result of additional treatments, activation of active chemical groups associated with fragments of double-stranded DNA directly in the stem (initiating) cancer cell after their internalization can be performed.

The following is experimental evidence for the detection, identification, sorting of stem (initiating-tumorigenic) cancer cells in a population of freely existing cancer cells and in tumor cells growing in a mass of tissue.

Used techniques.

TAMRA-labeled human Alu repeat DNA was prepared by PCR [27]. The sequence of the Alu-repeat of a person cloned in plasmid pUC19 was used as a matrix, which consists of the final and initial parts of two tandemly located repeats: AluJ and AluY (AC002400.1, 53494-53767). Standard M13 primers were used as primers: M13 for: GTAAAACGACGGCCAGT; M13 rev: CAGGAAACAGCTATGAC. Not included nucleotides were disposed of by reprecipitation of DNA.

To clean the PCR product from unincorporated nucleotides, the volume of the reaction mixture was brought to 100 μl, an equal volume of phenol-chloroform was added, and after shaking, it was centrifuged at room temperature for 2 min, 3000 g. The upper phase was transferred to a new tube. ¼ volume of 10M ammonium acetate was added and mixed. An equal volume of isopropanol was added and incubated for 10 min at room temperature. DNA was precipitated by centrifugation at room temperature, 12,000 g, for 15 minutes. The precipitate was washed with 70% ethanol and dissolved in 200 μl of water [27]. DNA concentration and dUTP-TAMRA incorporation were checked on a Nanodrop spectrophotometer (Eppendorf, USA), calculating the percentage of signal before and after reprecipitation of the preparation.

To analyze the internalization of TAMRA-labeled exogenous DNA in CD34 + and CD34 cells, bone marrow cells were washed from the tubular bones of intact mice with RPMI-1640 medium, precipitated at 400 g, 4 ° C for 5 min and washed in 1 ml of RPMI-1640 medium once. After the destruction of red blood cells using a lysis buffer (0.15 M NH 4 Cl; 10 mm Tris-HCl pH 7.5; 0.5 mm EDTA) 10 million cells were incubated with 0.8-1 μg of TAMRA-labeled Alu repeat DNA person in 0.5 ml of medium for 3 hours. At the end of the incubation, the cells were washed 3-4 times with medium, as described above. Then the cells were resuspended in PBS with 0.1% NaN 3 , 1% FBS. Antibodies (FITC Rat anti-Mouse CD34, BD Pharmingen) and isotype control (FITC Rat IgG2a, to Isotype Control, BD Pharmingen) were added to the samples at the rate of 3 μg per 3 million cells in 1 ml and incubated for 40 min at 4 ° C. Then the cells were selected on CD34 + and CD34- using a BD FACSAria flow cytophotometer. The separation boundary between CD34 + and CD34- was established taking into account the measurement results when staining the isotype control. After sorting, the cells were besieged at 400 g, 4 ° С for 5 min, methanol: acetic acid (3: 1) was fixed for one hour. The cell suspension was applied to wet defatted glass, the preparation was dried in air, a drop of Antifade DABCO containing 0.4 μg / ml DAPI was added, and analyzed using an Axioskop 2 plus fluorescence microscope using the Axio VisionLE program or an LSM 510 META laser scanning microscope.

To perform an incubation procedure of bone marrow cells with pEGFP-N1, plasmid DNA pEGFP-N1 (200 μg) was opened at the HindIII restriction site located in the polylinker. The mice received an intraperitoneal injection of a cyclophosphamide cytostatic at a dose of 200 mg / kg. 18 hours after cytostatic injection, bone marrow cells were washed with stem cell medium (D-MEM + Glu + NEAA + LIF + antibiotics). Cells were washed twice with the same medium and resuspended in 4 ml of medium. Then the cells were incubated with 100 μg of plasmid DNA for 4 hours in a CO 2 incubator, at 95% air humidity, 37 ° C (Memmert, Germany).

Isolation and fractionation of DNA was carried out as follows. After incubation, the cells were washed 3 times with PBS and resuspended in 1 ml of buffer A [28], supplemented with 0.1% Triton X-100. Cells were incubated for 5 min on ice and carefully layered on 3V 10% sucrose. The gradient was centrifuged for 20 min at 500 g, 4 ° C. The core pellet was washed once with buffer A, supplemented with 0.1% Triton-X100, and centrifuged at 1000 g, 4 ° C for 5 minutes. Cell pellet was resuspended in lysis buffer (10 mM Tris-HCl pH 7.4; 50 mM EDTA, 1% SDS, 200 μg / ml Proteinase K) and incubated for 2 hours at 58 ° C. DNA was purified with phenol, chloroform, precipitated with ethanol and dissolved in water. The resulting genomic DNA was fractionated in a NaCl gradient prepared with 50 mM EDTA. A gradient was prepared by layering 1 ml of each fraction: 30%, 25%, 20%, 15%, 10% (by weight), then DNA was carefully layered onto the gradient and centrifuged at 43,000 rpm for 2.5 hours on an ultracentrifuge. Fractions of 250 μl were collected, DNA was reprecipitated and analyzed by electrophoresis.

XLBlue-MRF 'electrocompetent cells were transformed with various fractions of DNA: isolated from bone marrow cells after co-incubation with exogenous DNA or preliminarily ligated onto T4 DNA ligase itself. The entire cell suspension was plated on a Petri dish with LB agar and a selective antibiotic kanamycin (25 μg / ml). Plasmid DNA was isolated from all grown colonies by alkaline lysis [29] and analyzed by gel electrophoresis.

To analyze the internalization of TAMRA-labeled exogenous DNA in the ascites form of Krebs-2 tumor cells, tumor cells were taken from the abdominal cavity of mice with a syringe, washed twice with PBS, and then incubated with TAMRA-labeled Alu human repeat DNA, similar to the method described for bone marrow cells . The content of labeled material in Krebs-2 cells was also evaluated in a similar manner.

To analyze the delivery of labeled DNA to ascites cells of a mouse tumor, the Krebs-2 tumor cells were taken from the abdominal cavity in one mouse without treatment and in one mouse after injection of cyclophosphamide cytostatic (200 mg / kg) using a syringe, washed twice with PBS at 400 g , 4 ° C. Then, the cells were counted in a Goryaev’s chamber and incubated for a certain time interval (1, 2, 4, and 8 hours) in the amount of 10 million cells in 500 μl of RPMI-1640 medium with pre-prepared DNA — 32 P-labeled GPR gene 1162 p in size. N., as well as the linear form of pEGFP-N1, hydrolyzed by restriction enzyme HindIII. At the end of the incubation, the cells were harvested, washed twice with the same medium, poured into blocks of low-melting agarose, and, if 32 P labeled DNA was added, the blocks were counted on a 1209 Rackbeta counter (Wallac, Finland). Then the material in the blocks was fractionated by electrophoresis and, depending on the experiment, either gel was dried and flashed on a Molecular Imager FX Pro + (with the addition of labeled material), or transferred through a Southern membrane to a Zeta-ProbeGenomicTestedBlottingMembrane (Bio-Rad) (when plasmid hydrolyzate was added ) and hybridized with labeled DNA. In order to control DNA delivery in this experiment, similar procedures were performed with mouse bone marrow cells.

When analyzing the amount of material delivered to the cells of the ascites form of the Krebs-2 tumor, the QuantityOne program evaluated the percentage of exposure to the size of the added fragment with respect to all labeled material that entered the gel or remained at the start. With known strengths of the radioactive signal added to the cells of labeled DNA and a block containing a certain number of these cells, the number of pure fragments delivered to the cell was calculated.

The analysis of the number of Krebs-2 cells containing TAMRA-labeled DNA after co-incubation in the culture medium was carried out using a BD FACSAria flow cytometer (Becton Dickinson, USA).

To analyze the distribution of cells of the ascites form of the Krebs-2 mouse tumor over the cell cycle, tumor cells were taken from the abdominal cavity of mice with a syringe, washed twice with PBS at 400 g, 4 ° C for 5 min. Next, the cells were fixed in 60% methanol for 1 hour at 4 ° C. Cells were besieged, washed with PBS and treated with RNase (200 μg / ml) for 30 min at 37 ° C. Then the cells were besieged, the pellet was resuspended in PBS with 0.1% NaN 3 , 1% FBS (Fetal Bovine Serum) and stained with anti-CD34 + antibodies as described above. Then, 20 μl of propidium iodide (5 mg / ml) was added to the cell suspension and the cells were stained for 10 min at room temperature. The cell cycle of CD34 + cells was determined using a BD FACSAria flow cytometer (Becton Dickinson, USA).

To transplant cells of the ascites form of the Krebs-2 mouse tumor after co-incubation of Krebs-2 cells with TAMRA-labeled DNA or to process the total population of ascites CD34 cells, AT cells were sorted with and without TAMRA-labeled DNA or with cells labeled CD34 + with BD FACSAria flow cytometer (Becton Dickinson, USA). Then the cells were besieged by centrifugation for 5 minutes at 400 g, 4 ° C and resuspended in a small volume of medium. The number of cells was counted in the Goryaev chamber. Next, the two obtained cell types were inoculated into mice in an amount of 170 thousand (1 experiment) or 60 thousand cells (2 experiment) for TAMRA DNA and 60 thousand cells for CD34 + cells. Tumor volume was measured using a caliper. Tumor dimensions were calculated using the standard formula: length × height × width.

To analyze the internalization of TAMRA-labeled exogenous DNA into cells of a solid tumor, a Krebs-2 capsule about 1 cm 3 in size was prepared. The tumor was cut into two parts, after which one half was divided into two more parts. Tumor segments were incubated with TAMRA-labeled human Alu repeat DNA in the same manner as described for bone marrow cells. After incubation, tissue segments were washed three times with PBS. The treated tumor tissue was frozen, after which, using a microtome, it was cut into plates with a thickness of 7 μm. Sections were poured into an antipheid containing 0.4 μg / ml DAPI and analyzed using a confocal microscope.

Let us consider in more detail the phenomenon of internalization of exogenous DNA fragments into the internal compartments of stem cells of higher organisms.

Numerous studies have been performed previously that characterize the delivery of exogenous DNA to a eukaryotic cell [30–34]. In particular, the ability to internalize exogenous extracellular fragments of double-stranded DNA mouse hematopoietic stem cells, human embryonic stem cells, human and mouse dendritic cells, MCF-7 human breast adenocarcinoma cell culture cells was studied [30-34]. The cited sources show that double-stranded DNA can be internalized in the cell space, which includes various additional molecular groups - biotin, various fluorochromes, and a radioactively labeled precursor. It was also found that DNA molecules reaching a size of 10,000 bp can be delivered to the cell.

In the study of the ability of stem hematopoietic cells to internalize fragments of exogenous double-stranded DNA, the interaction of double-stranded DNA was analyzed both with intact bone marrow cells and with bone marrow cells under the influence of either cytostatic or gamma radiation. It has been shown that bone marrow cells interact with double-stranded DNA fragments, and that such fragments are internalized into the internal compartments of the cells. The effect of exogenous DNA fragments on a cell was different depending on the type of treatments and the time that DNA entered the cell after exposure to a damaging factor. In the case of irradiation, injections of the double-stranded DNA preparation saved the blood stem cells from damage caused by radiation. In the case of treatment with cross-linking cytostatic cyclophosphamide, stem cells changed their genetic mode and became incapable for a long time to develop into a lymphoid hematopoietic sprout, which was accompanied by the occurrence of immunodeficiency and the death of experimental animals. Numerous data obtained showed that fragments of double-stranded DNA reach blood stem cells and take part in the molecular processes occurring in the cell. Since the action of exogenous DNA was clearly manifested against the background of damaging agents leading to the formation of double-stranded breaks, an assumption was made that was experimentally justified that fragments of double-stranded DNA take part in repair processes during the repair of double-strand breaks [33].

Investigating the property of bone marrow cells to internalize fragments of extracellular exogenous DNA, it was found that of the entire population of bone marrow cells, only 2% of the cells demonstrated the internalization of labeled fragments. Of the defined two percent, 1% were CD34 cells, and 1% were CD34 +. The total content of CD34 + cells was 2.2%. This meant that 40% of hematopoietic stem cells internalize fragments of extracellular exogenous DNA. The share of CD34-cells accounts for 1% (figure 1, 2).

To prove that the labeled intracellular material is specifically internalized fragments, and not a consequence of the incorporation of labeled precursors formed as a result of degradation of DNA fragments into the chromosomes of the cell as a result of synthetic processes occurring in it, a series of experiments were carried out, the results of which indicate that whole fragments of extracellular double-stranded DNA are delivered to the cell and deposited in the nuclear space [33] (Fig. 5).

When analyzing the fate of double-stranded DNA fragments internalized in the internal compartments of blood stem cells, it was found that linear fragments of double-stranded DNA undergo processing associated with the activity of the cell's reparative systems. This was direct evidence that fragments of exogenous DNA delivered into the intranuclear space participate in reparative processes as a substrate for the reparative molecular recombinant machine of the cell (Fig. 3) and that due to this participation in the hematopoietic stem cell, changes occur that disrupt the mechanisms of the cellular differentiation, as was shown in [33]. The nature of the processing products suggested that activated molecular repair factors could disrupt the structure of the double-stranded ends of the chromosomes, which exist as intermediates for the repair of inter-chain cross-links in the case of treatment with cyclophosphamide, so that the functional topology of the chromatin will be irreversibly disturbed.

As noted above, the analysis of bone marrow cells for the ability of various cell populations to internalize exogenous fragments of double-stranded DNA showed that the main type of cells internalizing DNA fragments are CD34 + stem cells. This led us to speculate that this trait, namely the ability to internalize exogenous fragments of double-stranded DNA, is a characteristic trait for stem cells of various etiologies. This suggested that stem (initiating) cancer cells also possess this ability. An allogeneic model of the aggressive Krebs-2 tumor was chosen to prove this assumption.

Analyzing the ability of Krebs-2 mouse tumor cells growing in the form of ascites to capture fragments of extracellular exogenous DNA, it was found that only about 2% of the cells internalize the labeled DNA material (Fig. 4).

To prove that the labeled intracellular material is specifically internalized fragments, and not a consequence of the incorporation of labeled precursors formed as a result of degradation of DNA fragments into the chromosomes of the cell as a result of synthetic processes occurring in it, a series of experiments were carried out, the results of which indicate that whole fragments of extracellular double-stranded DNA are delivered to the cell and deposited in the nuclear space (Fig. 5).

As a result of the discovered phenomenon, the following questions were posed:

How does the number of cells capable of interpolating fragments of exogenous DNA depend on the concentration of exogenous DNA in the pericellular environment? It turned out that with a progressive increase in the concentration of exogenous DNA, a linear increase in the number of cells internalizing exogenous fragments occurs. This suggested that the ability of cells to internalize fragments of exogenous DNA is finite and that the population of cells with this property is limited (Fig.6).

What is the relationship between the proliferating portion of cancer cells and the number of cancer cells internalizing exogenous DNA fragments? It is known that actively dividing cells are able to capture extracellular nucleic acid molecules. In this regard, it could be assumed that it is the division process that causes the internalization of extracellular DNA fragments. And thus, we compared the number of cells in the S + G2 phases and the number of cells internalizing extracellular DNA fragments. It turned out that about 50% of ascitic cells are in the S + G2 phase and only ~ 2% of the cells internalize exogenous DNA fragments. This meant that the signs are not connected by a direct connection or during the division there is a certain period of time when ascites cells are able to internalize exogenous fragments (Fig. 7).

To verify this assumption, ascites cells were incubated with TAMRA-labeled DNA for 4 hours. It was shown that within one hour, about 1-2% of ascites cells demonstrate the internalization of dsDNA fragments. If there is a cell cycle gap where the cells are able to internalize dsDNA, then a sequential, rectilinear increase in the number of cells that capture TAMRA during 4 hours of incubation could be observed. The result of the experiment indicates that during 4 hours of incubation there is no significant increase in the number of cells internalizing dc DNA fragments. This means that in the population initially there are cells with the ability to internalize dsDNA and their number is about 2% (Fig. 8).

Are the Krebs-2 cancer ascites cells positive for the CD34 and TAMRA DNA markers the same cells? CD34 glycoprotein is a stem marker for hematopoietic mouse bone marrow precursors. It turned out that in the ascites of the Krebs 2 tumor there is a population of cells that carries the CD34 mouse hematopoietic cell marker. We hypothesized that the population of such cells may overlap with the population of cells internalizing fragments of extracellular double-stranded DNA. It turned out that both cell populations overlap when analyzed in flow cytometry. In a cytological analysis of the distribution of the two fluorochromes, it was found that more than 90% of CD34 + positive cells at the same time are TAMRA positive cells. It was also shown that about half of the TAMRA positive cells carry the CD34 marker, and the remaining TAMRA positive CD34 cells are negative (Fig. 7).

The consequence of the experiments was the assumption that the Krebs-2 ascites cancerous cells that capture extracellular exogenous DNA also possess stem cell characteristics. The experiments on transplantation of sorted populations of cells containing TAMRA-labeled DNA and not containing labeled DNA fragments confirmed the ability of cells internalizing dsDNA to initiate the development of a tumor graft (Fig. 9).

Analyzing the described, initiating the development of a new tumor, property of cells in which fragments of exogenous dsDNA were internalized, the question arises whether this property is associated with changes in the cellular molecular mechanisms of a certain population of ascites cells due to the indirect appearance of DNA fragments in the cell. To answer this question, the described property of a population of initiating ascites cells internalizing TAMRA was simultaneously used to carry the CDK marker CD34 (Fig. 7). It has been suggested that if the sign of TAMRA DNA internalization is not associated with the initiating properties of a certain population of ascites cells, then cells sorted by the presence of a CD34 marker should have a similar initiating property. To test this hypothesis, CD34 + ascites cells were transplanted in an equivalent amount. It turned out that the appearance and development of a tumor graft is practically no different from that defined for TAMRA - DNA cells. This indicated that the internalization of dsDNA is a sign of IBS, and not the cause of this property (Fig. 10).

The most intriguing was the analysis of the internalization of the TAMRA DNA probe into tumor cells growing in solid form. To carry out such an analysis, a 1 cm 3 tumor capsule was excreted from the body (the femoral part of the paw) and was prepared and processed according to the procedure described in the procedures used. It was found that the internal contents of the tumor transplant is a mixed mass of cell arrays, trabeculae, voids. Labeled islets of cells are found in the interior of the tumor capsule. The size of the islands is about 200/100 μm with a total number of cells in one island of about 500. For the analyzed thickness (about 20 sections of 7 μm each), 3 distinct colonies were found (when comparing the number of cells labeled with TAMRA with the total number of cells of one section, the ratio is approximately is 1/100, that is, 1% of the total cell mass (Fig. 11).

Thus, due to the improvement of the known method, the required technical result is achieved, which consists in expanding the scope, since due to the introduction of additional operations of the method and their specific implementation, effective detection of stem cancer cells is provided.

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Claims (2)

1. A method for detecting stem cancer cells, based on the incubation of cell samples with fluorescent dyes and the subsequent identification of cancer cells in ultraviolet light, characterized in that, when the cell samples are incubated, which is carried out with a fluorescent dye covalently incorporated into fragments of double-stranded DNA, internalization is provided extracellular exogenous fragments of double-stranded DNA into the intracellular space of stem cells that make up the cell samples by incubation of cell samples in a solution of a fragmented double-stranded DNA preparation in a ratio of 0.5-1 μg of DNA per 1,000,000 cells that are in suspension or on a tissue section for 60-120 minutes, and the identification of cancer cells as stem cells is carried out in ultraviolet light with a wavelength corresponding to the maximum absorption introduced into the molecules of the fragments of double-stranded DNA of fluorochrome, and, as fragments of double-stranded DNA, fragments of human Alu DNA are used, enzymatically labeled with the precursor, containing a covalently sewn fluorochrome dye, or labeled with direct chemical introduction of fluorochrome.
2. The method according to claim 1, characterized in that a fluorochrome dye is used as a fluorochrome dye, which can be covalently combined with the precursor of the polymeric form of DNA in places that do not interfere with the process of incorporation of the triphosphate into the nucleotide chain, or introduced chemically directly into the fragment molecule double stranded DNA.
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US20050226812A1 (en) * 2003-04-14 2005-10-13 Nuvelo, Inc. Methods of therapy and diagnosis using targeting of cells that express killer cell immunoglobulin-like receptor-like proteins
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